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posterior reversible encephalopathy syndrome pres is a clinical syndrome that can include headache altered consciousnessvisual disturbances and seizures usually related to autoregulatory cerebral failure and hypertension the neuroimaging isessential to diagnosis showing white matter vasogenic edema in posterior areas we present a case of a 66yearold womanwith severe pneumonia by sarscov2 who developed a posterior reversible encephalopathy syndrome with a typical clinicaland radiological presentation after being treated with antiinterleukin treatment anakinra and tocilizumab following localguidelines we report a case of posterior reversible encephalopathy syndrome in a patient with covid19 disease possiblyrelated to antiil1 or antiil6 suggesting that antiinterleukin treatments may cause this syndrome at least in patients withpredisposing conditions such as infections and hydroelectrolytic disorderskeywords posterior reversible encephalopathy syndrome pres covid19 anakinra tocilizumab immunomodulatorsintroductiona 66yearold woman with covid19 presented with adultrespiratory distress syndrome ards besides bilateralpneumonia she developed multiple complications suchas cardiorespiratory arrest bacterial superinfectionhyponatremia massive hemoptysis requiring embolizationand acute renal injury she was started on lopinavirritonavirhydroxychloroquine and azithromycin after radiological pulmonary progression antiil1 daily anakinra and antiil6single dose of tocilizumab were started following local andhospital guidelines these drugs are recommended incovid19 when there is clinical blood test or radiologicalprogression to avoid an excessive immunological systemicthis is part of the topical collection on covid19 laura llansllansocliniccatxabi urraxurracliniccat department of neurology hospital clnic c villarroel barcelona catalonia spain comprehensive stroke center department of neurosciencehospital clnic august pi i sunyer biomedical research instituteidibaps barcelona catalonia spainresponse to the virus which is thought to worsen pulmonaryinfiltrates and disease prognosis ten days after the initiationof immunodepressants she developed altered mental statuswithout fever previous headache or visual disturbancescase presentationat the examination the patient opened eyes to painful stimulihad no verbal response and showed withdrawal response topain glasgow coma scale the blood tests showed stablehyponatremia meql and leukocytosis without any other significant findings her vitals were within normal limitsand blood pressure had been mildly increased during the previous h with a maximum systolic pressure of mmhgelectrocardiogram showed sinus rhythm and had not atrioventricular node blocks a ct scan with angiography was performed there was no large vessel occlusion no perfusionalterations and the baseline ct fig showed temporooccipital white matter hypodensity with symmetric obliteration of the sulci in that regionconsidering the infectious the immunomodulatory treatments modest hypertension in the hours before thesymptoms and the distribution of the lesions on the ct scanthe most likely diagnosis is posterior reversible encephalopathy syndrome pres [] hypertension plays a vital role in 0cfig ct baseline scan showedtemporooccipital symmetricwhite matter hypodensitysn compr clin medthe disease due to a failure in cerebral blood flow autoregulation and in this case rapid rise or fluctuations in blood pressure from baseline may have been harmful despite not beingseverely high the electroencephalogram showed focalslowing and epileptiform discharges in both occipital areasand ruled out nonconvulsive status epilepticus the symptoms improved over the following days after tight controlof blood pressure with labetalol infusion and discontinuinganakinraafter week radiological infiltrates worsened and a bloodtest showed increased acute phase reactants in this context adiffuse alveolar hemorrhage was diagnosed by bronchoalveolar lavage with suspicion of hemophagocytic syndrome sherequired red blood cell transfusions and immunosupressantswere restarted as well as mechanical ventilation the ct pulmonary scan showed worsening of infiltrates and presence ofan intrapulmonary hematoma finally the patient had a torpidevolution to multian failure and deathsthe absence of fever and radiological findings did not suggestan encephalitic cause of the symptoms the serum sodiumlevels were only slightly decreased and had been stable forthe previous days without intravenous infusion of sodiumthus hyponatremia was not assumed to be the cause of thesudden loss of consciousness the cardiorespiratory arresthappened a month before the event while intubated and itwas secondary to a mucus plug after a period of desaturationand bronchospasm it lasted less than min and ended as themucus plug was removed by fibrobronchoscopy the postanoxic cerebral damage was prevented by treating feveravoiding systemic hypotension hypoxemia or glycemicdisbalance and continuing renal replacement therapy withhemodiafiltration instead of hemodialysis to prevent largechanges in volemia one week later sedation was stoppedand a tracheostomy was placed and the patient progressivelyawakened up to a normal state of consciousness without focalneurologic signs the timeline and posterior completerecovery from the respiratory arrest cannot explain the currentepisode as hypoxicischemic encephalopathypres has been associated with immunosuppressive andcytotoxic therapies such as platinumcontaining drugs rchop regimens gemcitabine cyclosporine tacrolimussirolimus and interferon therapies also agents that targetangiogenesis such as bevacizumab antivegf and tyrosinekinase inhibitors tki against vegf receptor pazopanibsorafenib sunitinib have been described as risk factors prior exposure to the predisposing drug does not appearto be protective and patients can develop pres even afterseveral months after exposure furthermore the disorderhas been associated with both acute and chronic renal diseaseas was the case in our patient and medical conditions such ashyponatremia or pulmonary infection could exacerbate theneurological findingsdespite not being described yet the occurrence of pres afew days after antiinterleukin il6 or il1 treatments whichwere given in this patient raises the possibility that these kindsof immunomodulatory agents may also favor prescompliance with ethical standardsconflict of interest the authors declare that they have no conflict ofinterestethical approval and informed consent consent for publication wasobtained from the next of kin daughter approval from the hospitalsirb was provided for this studyreferences fugate je claassen do cloft hj kallmes df kozak osrabinstein aa posterior reversible encephalopathy syndrome associated clinical and radiologic findings mayo clin proc gao b lyu c lerner a mckinney am controversy of posteriorreversible encephalopathy syndrome what have we learnt in the last years j neurol neurosurg psychiatry 0csn compr clin med allen ja adlakha a bergethon pr reversible posteriorleukoencephalopathy syndrome after bevacizumabfolfiriregimen for metastatic colon cancer arch neurol ito y arahata y goto y cisplatin neurotoxicity presenting asreversible posterior leukoencephalopathy syndrome ajnr am jneuroradiol schwartz rb jones km kalina p bajakian rl mantello mtgarada b hypertensive encephalopathy findings on ctmr imaging and spect imaging in cases ajr am jroentgenol publishers note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c' | Colon_Cancer |
" tumor microenvironment tme plays an important role in malignant tumors our study aimed toinvestigate the effect of the tme and related genes in osteosarcoma patientsmethods gene expression profiles and clinical data of osteosarcoma patients were downloaded from the targetdataset estimate algorithm was used to quantify the immune score then the association between immune scoreand prognosis was studied afterward a differential analysis was performed based on the high and lowimmunescores to determine tmerelated genes additionally cox analyses were performed to construct two prognosticsignatures for overall survival os and diseasefree survival dfs respectively two datasets obtained from the geodatabase were used to validate signaturesresults eightyfive patients were included in our research the survival analysis indicated that patients with higherimmune score have a favorable os and dfs moreover genes were determined as tmerelated genes theunsupervised clustering analysis revealed two clusters were significantly related to immune score and t cells cd4memory fraction in addition two signatures were generated based on three and two tmerelated genesrespectively both two signatures can significantly divide patients into low and highrisk groups and were validatedin two geo datasets afterward the risk score and metastatic status were identified as independent prognosticfactors for both os and dfs and two nomograms were generated the cindexes of os nomogram and dfsnomogram were and respectively tme was associated with the prognosis of osteosarcoma patients prognostic models based on tmerelated genes can effectively predict os and dfs of osteosarcoma patientskeywords tumor microenvironment osteosarcoma prognosis immune features nomogram osteosarcoma is the most common bone tumor especiallyin children and adolescents it was reported that approximately of patients are between and yearsold and osteosarcoma is considered as the second leadingcause of death in this age group currently surgery and correspondence 407404159qqcom4wenzhou medical university wenzhou chinafull list of author information is available at the end of the chemotherapy are still major treatments for osteosarcomapatients and these therapies are constantly improving inrecent years however due to the susceptibility of localaggressiveness and lung metastasis in osteosarcoma patients the prognosis of osteosarcoma remains unfavorable previous studies indicated that the 5years survivalrates were and in metastatic and nonmetastaticpatients respectively thereforeit is necessary to the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0chu bmc cancer page of investigate the mechanism of pathogenesis and progressionof osteosarcoma and accurately classify the risk of patientsrecently an increasing number of diagnostic and prognostic biomarkers of osteosarcoma patients have beenidentified for example chen reported that tumorsuppressor p27 is a novel biomarker for the metastasis andsurvival status in osteosarcoma patients moreover huang discovered that dysregulated circrnas serve asprognostic and diagnostic biomarkers in osteosarcomapatients and the relative potential mechanism mainly attributes to the regulation of downstream signaling pathwaysby sponging microrna in addition lncrna microrna and many clinical data were also identified asprognostic biomarkers for osteosarcoma patients however osteosarcoma is one of the malignant cancers entitiescharacterized by the high level of heterogeneity in humanstherefore it is necessary to find accurate biomarkers forosteosarcomain recent years researchers have paid more and moreattention to the role of the tumor microenvironmenttme in malignant tumors the function of tme inthe tumorigenesis progression and therapy of tumorshave been initially understood [ ] more importantly estimation of stromal and immune cells in malignant tumor tissues using expression data estimate an algorithm to quantify the score of immune cellsand stromal cells by analyzing the gene expression datawas developed in based on the algorithm theprognostic value of immune and stromal cells in bladdercancer acute myeloid leukemia gastric cancer cervicalsquamous cell carcinoma adrenocortical carcinomaclear cell renal cell carcinoma hepatocellular carcinomathyroid cancer and cutaneous melanoma have beenreported [] generally the above research indicatedthat tme can serve as the prognostic biomarker in tumorsand many tmerelated genes were determined as the prognostic genes however the role of tme and tmerelatedgenes in osteosarcoma patients remains unclearin the present study gene expression data and corresponding clinicopathologic data were obtained from thetherapeutically applicable research to generate effectivetreatments target dataset then the estimatealgorithm was performed to quantify the immune score ofosteosarcoma and the tmerelated genes were identifiedby the differential expression analysis subsequently theprognostic value of tme and tmerelated genes weredetermined by a series of bioinformatics methodsmethodsgene expression datasetslevel data of gene expression profiles and correspondingclinical data of osteosarcoma patients were downloadedfrom target dataset ocgcancergovprogramstarget accessed on oct the correspondingclinicopathologic data included in the present study wereage gender race ethnicity tumor site and metastaticstatus after data were extracted from the public domainthe estimate an algorithm inferring tumor puritystromal score and immune cell admixture from expression data was performed to evaluate the immune score byusing the estimate package in r software version meanwhile the messenger rnamrna expressionprofiles and clinical data ofincludinggse21257 and gse39055 were obtained fromthe gene expression omnibus as external validationcohortstwo cohortssurvival analysis and correlation analysisafter scores were obtained patients were divided intohighscore group and lowscore group according to themedian of the immune score the kaplanmeier survivalanalysis with logrank test was performed to estimatethe differences of overall survival os and diseasefreesurvival dfs between high and lowscore cohorts inaddition the association between clinicopathologic dataand tme score was also studied mannwhitney signedrank test was performed to compare the differences ofimmune score between each clinical group all statisticalanalyses in the present study were performed using rsoftware except for the special instructions p value twoside was identified as statistically significantin the present studydegexpressed genedifferentially expressed gene analysisdifferentiallyanalysis wasperformed by comparing the proteincoding genesexpression between the lowimmune score group andthe highimmune score group the limma package in rsoftware was used to perform the differential analysisand genes with log fc and adjusted pvalue qvalue were identified as degs to further understand the function of degs identifiedin the present study gene ontology goincludingbiological processes bp molecular functions mf andcellular componentscc and kyoto encyclopedia ofgenes and genomes kegg analysis were performedby clusterprofiler package in r software evaluation of association with immune cellsto further investigate the association between degs andimmune cells the cibersort package was used toestimate the relative proportions of types of immunecells meanwhile the consensusclusterplus package was used to cluster in an unbiased and unsupervisedmanner based on the overlapping degs cumulative distribution function cdf and relative change inarea under the cdf curve were used to determine theoptimal number of clusters k then mannwhitney 0chu bmc cancer page of signedrank test was performed to study the differenceof immune cells proportion between the clusters and theviolin plot was established to show the differences ofimmune cells among clusters survival analysis of degsbased on the degs the univariate cox analysis was performed to determine the prognostic value of immunerelated genes then the osrelated genes were validatedin the gse21257 dataset while the dfsrelated geneswere validated in the gse39055 dataset only genes successfully validated were selected for further analysis afterward based on the validated genes the multivariate coxanalysis was performed to establish the prognostic signature for predicting the prognosis of osteosarcoma patientsthe risk score for each patient was calculated based onthe coefficient from the multivariate cox analysis and thecorresponding gene expression meanwhile all patientswere divided into the high and lowrisk groups accordingto the median of the risk score the survival receiver operating characteristic roc curve was used to show the discrimination of signatures and the kaplanmeier survivalcurve with the logrank test was generated to show thedifferences of os and dfs between high and lowriskgroups in addition the risk score of patients in the validation cohort was also calculated according to the aforementioned risk signature the kaplanmeier survivalcurve and survival roc curve were generated to show thepredictive ability of the signature in the validation cohortdevelopment of a nomogram for osteosarcoma patientsnomogram is a tool to visualize the predictive model andconvenient for clinical practice therefore we attemptedto develop a nomogram based on the tmerelated genessignature and clinicopathologic data to predict the prognosis of osteosarcoma patients firstlythe univariatecox analysis was performed to filter prognostic variableswhich will be further included in the multivariate coxanalysis secondly based on independent prognostic variables two nomograms were established for predicting theos and dfs respectively the cindex was used to assessthe discriminatory performance of the nomogram whichrange from to a cindex of means agreement by chance and a cindex of represents perfectdiscriminatory performance the higher value of the cindex the better performance of the nomogram is furthermore the calibration curves of and 3year weredeveloped to evaluate the effectiveness of nomogramsresultsimmune significantly associated with the prognosis ofosteosarcoma patients osteosarcoma patients were included in the presentstudy including males and females the immunescore of the cohort range from to tostudy the relationship between the immune score and theprognosis of osteosarcoma patients patients wereincorporated into the lowimmune score group while theremaining patients were incorporated into the highimmune score group the survival analysis indicated thatpatients with higher immune score had a favorable osand dfs fig 1a and b after adjusted age tumor siteand metastatic status the immune score still was a prognostic variable for both os and dfsfig 1a and b inaddition the relationship between immune score and clinical features was also investigated however there was nosignificant relationship between immune score and clinicalvariables supplementary figure 1a1cdifferential expression analysisaccording to the median of the immune score patients were divided into highscore n and lowfig association between immune score and prognosis in osteosarcoma patients a kaplanmeier survival analysis of overall survival for patientswith high vs low immune score b kaplanmeier survival analysis of diseasefree survival for patients with high vs low immune score 0chu bmc cancer page of score group n there were differentiallyexpressed genes between two groups which include upregulated genes and downregulated genesfig 2a b and supplementary table to furtherunderstand the function of degs go analysisand kegg analysis were performed the top significant results of go analysis among three types wereillustrated in fig 2c interestingly we can find that theresults of go analysis are mostly associated with immunity which further verify that the immunerelated degsare associated with immune features in addition the results of kegg also confirmed it such as phagosomeautoimmune thyroid disease antigen processing andpresentation b cell receptor signaling pathway intestinal immune network for iga production inflammatorybowel disease primary immunodeficiency th1 andth2 cell differentiation th17 cell differentiation natural killer cell mediated cytotoxicity and nfkappa bsignaling pathway fig 2dconsensusunsupervisedevaluation of degs and immune cellsto further understand the molecular heterogeneity ofosteosarcomaanalysis wasperformed to divide patients into subgroups to explorewhether immunerelated genes presented discernable patterns based on the consensus matrix heat map patientswere clearly divided into two clustersfig 3a in additionby comprehensively analyzing the relative change in areaunder the cumulative distribution function two clusterswere determined fig 3bc the immune score betweentwo clusters was significantly different fig 3d in additionthe proportion of types of immune cells in osteosarcomapatients was illustrated in a barplot fig 3e interestinglywe can see that the t cells cd4 memory activated ofcluster is significantly higher than cluster fig 5fprognostic value of tmerelated genesprevious studies indicated that tmerelated genes canserve as the prognostic biomarker for tumor patientsfig differentially expressed genes with the immune score in osteosarcoma patients a heatmap of significantly differentially expressed genesbased on immune score b the volcano figure to show the upregulated and downregulated genes c go analysis of differentially expressedgenes d kegg of differentially expressed genes go gene ontology kegg kyoto encyclopedia of genes and genomes 0chu bmc cancer page of fig the immune landscape of the tumor microenvironment ac unsupervised clustering of all samples based on the overlapping degs dcomparison of immune score between two clusters e the distribution of types of immune cells in osteosarcoma patients f the comparisonof types of immune cells between clusters deg differentially expressed genehence we performed the univariate cox analysis toidentify prognostic degs the results showed that and genes were identified as os and dfsrelateddegs respectively supplementary table and afterward five osrelated genes were successfully validated inthe gse21257 data set and five dfsrelated genes were successfully validated in the gse39055 cohort furthermoremultivariate cox analysis was performed and two prognostic signatures were generated for predicting the os anddfs respectively the risk score for predicting the os wasasrisk score fcgr2b0766 gfap0702 mpp70387 in addition the risk score for predicting thedfs was as follows risk score cyp2s10574 icam3 the auc values of osrelated signature were follows 0chu bmc cancer page of and in and 3year respectively fig 4aand the auc values of dfsrelated signature were and in and 3year respectively fig 5amoreover survival curves showed that patients in the highrisk group had worse os and dfs compared with the lowrisk patients figs 4b and 5b heat maps risk score plotsand survival status were generated to show the distinctionbetween highrisk patients and lowrisk patients figs 4ceand 5ce then both signatures were validated in independent cohorts for os signature the auc values ofvalidation cohort were and at and3year fig 4f for dfs signature the auc values ofvalidation cohort were and at and3year fig 5f additionallyin both validation cohortssurvival curves showed that lowrisk patients were favorableprognosis than highrisk patients figs 4g and 5gheat maps risk score plots and survival status of validation cohorts were also generated to show the distinction between highrisk patients and lowrisk patientsfigs 4hj and f 5hjdevelopment of a nomogram for osteosarcoma patientsto generate a nomogram for clinical use the cox analysiswas performed to select the clinical prognostic variables infig establishment and validation of the prognostic model for overall survival based on significant degs a receiver operating characteristiccurves of prognostic signature in the training cohort b the survival curve showed the different overall survival status between high and lowriskpatients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each sample reordered by riskscore e the scatter plot showed the overall survival status of osteosarcoma patients in the training cohort f receiver operating characteristiccurves of prognostic signature in validation cohort g the survival curve showed the different overall survival status between high and lowriskpatients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reordered by riskscore j the scatter plot showed the overall survival status of osteosarcoma patients in the validation cohort 0chu bmc cancer page of fig establishment and validation of the prognostic model for diseasefree survival based on significant degs a receiver operatingcharacteristic curves of prognostic signature in the training cohort b the survival curve showed the different diseasefree status between highand lowrisk patients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each samplereordered by risk score e the scatter plot showed the diseasefree status of osteosarcoma patients in the training cohort f receiver operatingcharacteristic curves of prognostic signature in validation cohort g the survival curve showed the different diseasefree status between high andlowrisk patients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reorderedby risk score j the scatter plot showed the diseasefree status of osteosarcoma patients in the validation cohortthe univariate cox analysis risk score and metastatic status were identified as both os and dfsrelated variablesfig 6a and e afterward risk score and metastatic statuswere determined as both independent os and dfsrelated variables in the multivariate cox analysis fig 6band f based on independent variables two nomogramswere established for predicting the os and dfs in osteosarcoma patients respectively fig 6c and g the cindexvalues were and in os nomogram and dfsnomogram respectively the results of cindex mean thatboth two nomograms have good discrimination meanwhile to evaluate the calibration of nomograms six calibration curves were generated and the results showed thatthe predictive curves were close to the ideal curve fig 6dand h which indicated a good calibrationdiscussionthe relationship between tme and tumor have beenwidely studied in recent years in the present study estimate algorithm was utilized to quantify the immunescore based on gene expression profiles in osteosarcomapatients from target database we confirmed that thetme is significantly associated with the prognosis ofosteosarcoma patientsinadditionfunctional enrichment analyses of tmerelated genes indicated that immunerelated processesincluding os and dfs 0chu bmc cancer page of fig nomograms based on the tumor microenvironment related genes for osteosarcoma patients a univariate cox analysis of overall survivalrelated variables b multivariate cox analysis of overall survivalrelated variables c nomogram for predicting the overall survival in osteosarcomapatients d1 and 3year calibration curves of overall survival nomogram e univariate cox analysis of diseasefree survivalrelated variables fmultivariate cox analysis of diseasefree survivalrelated variables g nomogram for predicting the diseasefree survival in osteosarcoma patientsh1 and 3year calibration curves of diseasefree survival nomogramknown to contribute to tumor progression more importantly degs based on the tme were identified asimportant prognostic biomarkers for osteosarcoma patients and two nomograms were developed for predicting the os and dfs of osteosarcoma patientsrespectivelyin recent years an increasing number of studiesfocused on the carcinogenesis and progression of tumorsbased on the tme and the estimate algorithm is oneof the most important quantitative tools for this researchfield based on the estimate algorithm the association between the prognosis and tme has been initially 0chu bmc cancer page of elucidated in some tumors such as cervical squamouscell carcinoma gastric cancer cutaneous melanomaacute myeloid leukemia bladder cancer and clear cellrenal carcinoma [ ] however previousstudies indicated that tme scores serve as a differentrole in different tumors for example for hepatocellularcarcinoma gastric cancer acute myeloid leukemiabladder cancer and clear cell renal carcinoma patientswith high immune score have a worse prognosis [ ] however for cervical squamous cell carcinoma adrenocortical carcinoma and cutaneous melanoma patients with high immune score have a favorableprognosis [ ] therefore we can find great heterogeneity among different tumors from the perspectiveof tme for osteosarcoma patients the present studyindicated that patients with higher immune score had abetter os and dfs hence the present study indicatedthat immune cells infiltrating tumor tissue may play animportant role in suppressing tumor progressionin our research tmerelated genes were identified by comparing the highscore and lowscore osteosarcoma patients the functional enrichment includinggo and kegg analyses showed that tmerelated geneswere mainly involved in the immune features such asregulation of leukocyte activation mhc protein complex mhc protein and complex binding more importantly the unsupervised cluster analysis based on degswas performed and all patients were divided into twoclusters immune score and t cell cd4 memory activated fraction were significant difference between twoclusters which further elucidated the relationship between degs and immune featuresdue to the poor prognosis of osteosarcoma patientsidentifying robust prognostic biomarker is very importantthe tumor immune microenvironment is closely relatedto the prognosis of bone tumor patients emilie etal performed the first genomewide study to describe therole of immune cells in osteosarcoma and found thattumorassociated macrophages are associated with reduced metastasis and improved survivalin highgradeosteosarcoma recently the prognostic signature based ontmerelated genes have been established for many tumors [ ] but only one study focused on osteosarcoma patients compared with the study performedby zhang we think that our research have someadvantages firstly our signatures were established basedon several validated genes and both two signatures weresuccessfully validated in independent cohorts secondlythe outcome of dfs was not reported in the previousstudy as reported in published studies tumor recurrenceis a terrible medical problem for osteosarcoma patientsand the 5year survival rate for osteosarcoma patients withmetastasis or relapse remains disappointing [ ]hence the dfs nomogram can improve the managementof osteosarcoma patients finally two nomograms incorporated tmerelated signature and clinical variables wereestablished in our research which further facilitated theclinical application of our findingsin our research five genes were incorporated into thefinal prognostic signatures fcgr2b gfap and mpp7were identified and validated as osrelated biomarkerswhile cyp2s1 and icam3 were dfsrelated biomarkersthe role of these genes in tumor prognosis had beenwidely reported in previous studies [] fcgr2bhas been confirmed as an immunerelated gene previously although the relationship between fcgr2band prognosis in sarcoma patients had not been reported the prognostic value of fcgr2b had been widelyconfirmed in other cancerssuch as hepatocellularcarcinoma and glioblastoma [ ] in addition newm etal demonstrated that mpp7 is novel regulatorsof autophagy which was thought to be responsible forthe prognosis of pancreatic ductal adenocarcinomacyp2s1 described as cytochrome p450 family subfamily s member was reported significantly associatedwith colorectal cancer in primary colorectal cancercyp2s1 was present at a significantly higher level ofintensity compared with normal colon more importantly the presence of strong cyp2s1 immunoreactivity was associated with poor prognosis the roleof icam3 in cancer was also widely reported in published studies and the akt pathway plays an importantrole in the impact of icam3 on tumors yg kim etal reported that icam3 can induce the proliferationof cancer cells through the pi3kakt pathway additionally jk park etal showed that the icam3 can enhancethe migratory and invasive potential of human nonsmall celllung cancer cells by inducing mmp2 andmmp9 via akt pathway showed that the icam3can enhance the migratory and invasive potential ofhuman nonsmall celllung cancer cells by inducingmmp2 and mmp9 via akt pathwayalthough the role of tme and tmerelated genes inosteosarcoma patients have been initially studied by bioinformatic and statistical analyses in our research somelimitations should be elucidated firstly the treatmentinformation cannot be obtained from the target database which may influence the prognosis of osteosarcomapatients secondly two nomograms were generated andshowed good performance in our study however externalvalidation by a large cohort is needed thirdly many independent prognostic genes for osteosarcoma patients wereidentified in the present study but the potential mechanism to influence osteosarcoma remains unclear finally inthe training cohort and degs were identified asos and dfsrelated degs respectively however onlyfive os and five dfsrelated genes were identified in thevalidation cohort the different age structures smaller 0chu bmc cancer page of sample sizes and the platform covering only part of thegenes may contribute to this resultreceived february accepted july in tme plays an important role in osteosarcoma patients and related with the progression of thetumor moreover tmerelated genes can serve as prognostic biomarkers in osteosarcoma patients howeverfurther researches are needed to study the potentialmechanism and validate the nomogram that developedin our present studysupplementary informationsupplementary information accompanies this paper at doi101186s12885020072162additional file additional file additional file additional file abbreviationstme tumor microenvironment deg differentially expressed genesos overall survival dfs diseasesfree survival roc receiver characteristiccurve estimate estimation of stromal and immune cells in malignanttumor tissues using expression data target therapeutically applicableresearch to generate effective treatments go gene ontology bp biologicalprocesses mf molecular functions cc cellular components kegg kyotoencyclopedia of genes and genomes cdf cumulative distribution functionacknowledgementsnoneauthors contributionsc h l y sq t c l and yh w conceived of and designed the study c h r sand c l performed literature search r s l y and b c generated the figuresand tables l y hl r x y and jy l analyzed the data c h wrote themanuscript and sq t and l y critically reviewed the manuscript l ysupervised the research all authors have read and approved the manuscriptfundingwe received no external funding for this studyavailability of data and materialsthe data of this study are from target and geo databaseethics approval and consent to participatethe research didnt involve animal experiments and human specimens noethics related issuesconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of joint surgery the affiliated hospital of qingdao universityqingdao china 2department of medical oncology the first hospital ofchina medical university shenyang china 3department of nursing sir runrun shaw hospital affiliated to zhejiang university hangzhou china4wenzhou medical university wenzhou chinareferencesjaffe n bruland os bielack s pediatric and adolescent osteosarcoma vol new york springer science business media vander rg osteosarcoma and its variants orthopedic clin north am biermann js adkins d benjamin r brigman b chow w conrad eu 3rdfrassica d frassica fj gee s healey jh bone cancer j natl comprcancer netw simpson s dunning md de brot s grauroma l mongan np rutland cscomparative review of human and canine osteosarcoma morphologyepidemiology prognosis treatment and genetics acta vet scand chen x cates jm du yc jain a jung sy li xn hicks jm man tkmislocalized cytoplasmic p27 activates pak1mediated metastasis and is aprognostic factor in osteosarcoma mol oncol huang x yang w zhang z shao z dysregulated circrnas serve as prognosticand diagnostic markers in osteosarcoma by sponging microrna to regulatethe downstream signaling pathway j cell biochem liu m yang p mao g deng j peng g ning x yang h sun h long noncoding rna malat1 as a valuable biomarker for prognosis in osteosarcoma asystematic review and metaanalysis int j surg xu k xiong w zhao s wang b microrna106b serves as a prognosticbiomarker and is associated with cell proliferation migration and invasionin osteosarcoma oncol lett zheng w huang y chen h wang n xiao w liang y jiang x su w wens nomogram application to predict overall and cancerspecific survival inosteosarcoma cancer manag res kahlert c kalluri r exosomes in tumor microenvironment influence cancerprogression and metastasis j mol med binnewies m roberts ew kersten k chan v fearon df merad m coussenslm gabrilovich di ostrandrosenberg s hedrick cc understanding thetumor immune microenvironment time for effective therapy nat med yoshihara k shahmoradgoli m martÃnez e vegesna r kim h torresgarcia wtreviño v shen h laird pw levine da inferring tumour purity and stromaland immune cell admixture from expression data nat commun yang s liu t nan h wang y chen h zhang x zhang y shen b qian pxu s comprehensive analysis of prognostic immunerelated genes inthe tumor microenvironment of cutaneous melanoma j cell physiol deng z wang j xu b jin z wu g zeng j peng m guo y wen z miningtcga database for tumor microenvironmentrelated genes of prognosticvalue in hepatocellular carcinoma biomed res int zhao k yang h kang h wu a identification of key genes in thyroid cancermicroenvironment med sci monit xu wh xu y wang j wan fn wang hk cao dl shi gh qu yyzhang hl ye dw prognostic value and immune infiltration of novelsignatures in clear cell renal cell carcinoma microenvironment agingalbany ny chen b chen w jin j wang x cao y he y data mining of prognosticmicroenvironmentrelated genes in clear cell renal cell carcinoma a studywith tcga database dis markers li x gao y xu z zhang z zheng y qi f identification of prognostic genesin adrenocortical carcinoma microenvironment based on bioinformaticmethods cancer med pan xb lu y huang jl long y yao ds prognostic genes in the tumormicroenvironment in cervical squamous cell carcinoma aging albany ny wang h wu x chen y stromalimmune scorebased gene signature aprognosis stratification tool in gastric cancer front oncol huang s zhang b fan w zhao q yang l xin w fu d identification ofprognostic genes in the acute myeloid leukemia microenvironment agingalbany ny yan h qu j cao w liu y zheng g zhang e cai z identification ofprognostic genes in the acute myeloid leukemia immunemicroenvironment based on tcga data a | Colon_Cancer |
" bridge to surgery bts using a selfexpandable metallic stent sems for the treatment of obstructivecolorectal cancer improves the patients quality of life this study aimed to examine prognostic factors ofobstructive colorectal cancermethods we analyzed stage iiiii resectable colon cancer cases cur a retrospectively registered between january and december overall patients with cur a obstructive colorectal cancer were evaluated ofthem underwent emergency surgery es group and of them after bts with sems placement bts group wecompared surgical results and prognoses between the two groupsresults a total of patients underwent endoscopic sems placement which technical success of andmorbidity rate of primary anastomosis rates were in es and in bts p postoperativecomplication in es and in bts p pathological findings of lymphatic invasion in es and in bts p venous invasion were in es and in bts p and recurrence of in esand in bts the 3year overall survival was significantly different between two groups es 868bts bts is worse than es logrank test p venous invasion independently predicted worsened recurrencefreeand overall survivals the vascular invasiveness was correlated with tumor progression after sems placement and thesurvival rate was lower in bts sems potentially worsens prognostic outcomes in stage iiiii obstructive colorectalcancerkeywords bowel obstruction colorectal cancer selfexpandable metallic stent colorectal cancer crc remains the leading cause ofcancerrelated deaths worldwide because several patientsare initially diagnosed during advanced stages correspondence ohtakmedkindaiacjp1gastroenterological surgery higashiosaka city medical center osaka japan2department of gastroenterological surgery kindai university nara hospital otodacho ikomacity nara japanfull list of author information is available at the end of the approximately of patients with crc were diagnosed with acute colonic obstruction [] severe malignancy with bowel obstruction needs urgent surgicalintervention which includes primary lesion resectionand stoma creation leading to increased morbidity andmortality and a potential failure to achieve completeoncological resection [ ]an endoscopic procedure with selfexpandable metallic stent sems is an acceptable bridge to surgery bts 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 surgery page of treatment for acute colonic obstruction [] preoperative sems placement provides an opportunity to perform medical resuscitation comorbidity optimizationbowel preparation tumor staging and observation ofproximal lesions the procedure prevents highriskemergency surgeries and increase oncological resectionand primary anastomosis rates [ ] after the inclusion of colonic sems placement as bts in the coverageof the national health insurance in japan several physicians joined the colonic stent safety procedure researchgroup and developed skills to provide safe treatmentthe largest multicenter prospective study demonstratedthe feasibility and safety of sems placement as bts inpatients with malignant colorectal obstruction the oncological safety and minimalinvasiveness ofthis procedure have confirmed that sems placement asa bridge to elective surgery is not recommended as astandard treatment for symptomatic leftsided malignantcolonic obstruction [ ] several studies reportedthat prognostic factors of malignant colonic obstructionin sems placement had oncological disadvantages compared with those in emergency surgery es [ ] incontrast several trials showed that sems placement as abridge to elective surgery did not improve the survivalrates [] how sems placement worsens prognosticoutcomes remains unclear [ ]this study aimed to evaluate the induction of curativesurgery in patients with malignant colorectal obstructionafter a sems placement and its longterm results andprognostic factors postoperatively compared to patientswithout sems placement we demonstrated prognosticfactors and overall survival os and recurrencefreesurvival rfs rates for curative surgery after a semsplacementmethodspatientsmedical records of patients who underwent primarycolorectal resection at higashiosaka city medical centerbetween january and december werereviewed all participants provided written informedconsent oralintake and symptoms before and aftersems placement were assessed in table using thecolorectal obstruction scoring system cross from to we recruited patients with all class oftable the colorectal obstruction scoring system crosspatients symptom and their condition of an oral intakesolid meal low residue and full diet without symptomcrosscross as stent insertion candidate from we excluded patients with cross and based on updatedstent insertion guideline malignant colorectal obstruction was diagnosed through clinical examinationcross radiography and computed tomography surgery was performed using three approaches es comprised laparotomylymph node dissection as possibleand primary anastomosis on the same day between and bts after sems placement comprised standbylaparoscopy d3 lymph node dissection and primaryanastomosis since january overall patientswith stage iiiii cur a obstructive colorectal cancerwere evaluated of them underwent emergency surgery as es group and of them after bts with semsplacement as bts group we compared surgical resultsand prognoses between the two groupssems devices and the procedurepatients were endoscopically treated with placement ofan uncovered wallflex enteral colonic stent boston scientific corporation natick ma usa or nitis enteralcolonic uncovered stent taewoong inc gimpo southkorea placements were performed as presented in thepreintroduction publicity announcement placement details were mentioned on the website as a brief guideline obstruction structures were determined using aguide wire and a contrast tube was inserted into theproximal colorectal lumen obstructions were measuredusing contrast agents and then the endoscopist determined the number size and type of stent pathologicalbiopsies were recommended after sems locations andintraluminal or extraluminal marking using an endoscopic clip were recommended via visual recognition ofthe endoscopist dilatation of the colonic obstructionbefore sems placement was generally not allowedhistological findingsparaffinembedded specimens were obtained from a cohort of patients diagnosed by the union for international cancer control stage iiiiisurvival definitionsos was defined as the duration from surgery to anydeath or last followup diagnosis of recurrence was calculated based on recist according to the chemotherapy criteria rfs was defined as the durationfrom surgery to any recurrence includes local recurrenceor distant metastasissolid meal low residue and full diet with symptomliquid or enteral nutrient intakeno oral intakerequiring continuous decompressionstatistical analysisstudents ttest and wilcoxon test for continuous variables and the Ï2 and fishers exact tests for categoricalvariables were conducted survival curves were generated using the kaplanmeier method and compared 0cohta bmc surgery page of table baseline characteristics and outcomes of endoscopicsems placementtable comparison of baseline characteristics in patientsundergoing emergency surgery and bridge to surgerybts n es n pmalefemalemedian range genderagelocationmalefemalemedian rangececumascendingtransversedescendingsigmoidrectumlength of obstructionmedian range cmtechnical successprocedurestentingmorbiditymortalityclinical successthrough the scopethrough the wirewall flex cmnitis cmoverall cda iiicda vcrossbts n a claviendindo classificationusing a logrank test univariate and multivariate survival analyses were performed using the cox proportional hazards regression model all statistical analysesused jmp version sas institute cary nc or statistical scripting language r httpwwwrprojectpvalues of ¤ twosided were considered statistically significant this prognostic study complied withthe reporting recommendationsfor tumor markerprognostic studies resultsa total of patients underwent endoscopic semsplacement which was technically safe for malignantcolorectal obstruction with the technical success rate of the clinical success rate was and the patientssymptoms and oral intake dramatically improved afterthe sems placement shown in table a total of patients were reviewed and patients underwentes and bts respectively as shown in table baselineclinical characteristics were balanced between the twogroups moreover cases of patients underwent es on the same day as in open surgery the median waiting period for surgery was days for bts thegenderagelocationtype of operationcecumascendingtransversedescendingsigmoidrectumstandbyemergency duration tooperationmedian range dayssurgical procedurelaparotomy laparoscopy timemedian range minblood lossmedian range ml before afterstoma creation morbidity30day complication cda iii anastomosticleakagehospital stayaclaviendindo classificationmedian range days primary anastomosis ratios were in es and in bts p postoperative complication rateswere in es and in bts p postoperative hospital stay was shorter in bts days comp patients withpared to esobstructive crc showed significantinpostoperative complication rate and hospital stay withsems placement operative procedures were dramatically changed and the primary anastomosis rate improved after the sems placementimprovement daysthe pathological tissue type accounted for of differentiated types shown in table tumordepth was similar between the two groups lymphatic vesselinvasion ratios were in es and in bts p and venous invasion ratioswere in es and in bts p recurrence rates were cases in es and cases in bts nodenegative patients stage iimorewhereas nodepositive patients stage iii more frequently had liver metastasis in the kaplanmeier survival analysis in fig 1a the 3year rfs waslung metastasisfrequentlyhad 0cohta bmc surgery page of table comparison of pathological characteristics of emergency surgery and bridge to surgerypt factortotal lymph nodespn factorhistologicallymphatic invasionvenous invasionsurgical clearancet4b t4a t3median rangen0 n1 n2 n3tub1 tub2 othersly v cur a b csignificantly different between the two groups es bts which was significantly low inbts than that in es logrank test p the3year os rate was also significantly different between thetwo groups es bts p shown in fig 1b the relationship between lymph node metastasis and sems placementwas also evaluated the pathological nodenegativestage ii 3year rfs rate was not different betweenthe two groups es bts as shown infig 2athe pathological nodepositivestage iii 3year rfs rate was different between thetwo groups es bts as shown in fig 2bthe stage ii 3year os rate was not different between thetwo groups es bts as shown in fig 2cwhereas stage iii 3year os rate was different between thetwo groups es bts as shown in fig 2dthese results suggestthat vascular invasiveness andpathological nodepositive status were correlated withtumor progression after sems placementthein contrastthuses n bts n p survival rate was affected by poor prognosis in the btsgroupresults of adjusted multiple cox proportional hazard regression for rfs and os in all stages and stage iii diseaseare presented in table after adjusting for possible confounders venous invasion and bts independently predicted poor rfs in all stages and venous invasionindependently predicted poor rfs in stage iii disease venous invasion and bts were also significantly associatedwith os in stage iii diseasediscussionacute colonic obstruction requires emergent surgicalintervention a mandatory conventional treatment skillemergent surgicalis associated with highmorbidity mortality and stoma creation rates affectingthe quality of life of patients malignant colorectal obstruction is not only an intestinal obstruction but alsoan advanced stage crc their prognosis was poorerthan that in patients with nonocclusive disease becausetreatmentfig kaplanmeier survival curves in patients undergoing emergency surgery vs bridge to surgery a recurrencefree survival b overall survival 0cohta bmc surgery page of fig kaplanmeier survival curves in patients undergoing emergency surgery vs bridge to surgery a rfs nodenegative patients b rfs nodepositive patients c os nodenegative patients d os nodepositive patientsof highly invasiveness and distant metastasis [ ]chen revealed that the prognosis in patients withperforation associated with obstruction was poor early intervention in the clinical setting before the colonic perforation has been established endoscopic placement of colonic stents improves the high decompressioneffect and reduces clinical symptoms high postoperative complication rates were correlatedwith poor prognosis in patients with cancer in severalans [] reducing complication rates can improve the prognosis our results showed high clinicalsuccess rate after sems placement and high primarysurgicalinterventions howeveranastomosis rate stentrelated complications requiredemergentthe stentplacement is safe and feasible in this study moreoverthe laparoscopic rate was high and postoperative complication rate was clinical results including shortterm outcomesin bts after sems were verifiedthrough a metaanalysis [ ]the prognosis was poor in patients with stent perforation and increased local recurrence rate after the colonic stent placementthe longtermprognosis in patients with colorectal obstruction afterbts was not different compared with that in patients however 0cohta bmc surgery page of table multivariate analysis of recurrencefree survival at all stages and stage iiipvaluevariablesrecurrence free suvivalall stages hazard ratio ± sd cistage iii hazard ratio ± sd cipvaluees vs semspt factor t3t4pn factor n0n1n2verous invasion v01v2lymphatic invasion ly01ly2hr ± hr ± hr ± hr ± hr ± overall suvivales vs semspt factor t3t4pn factor n0n1n2verous invasion v01v2lymphatic invasion ly01ly2hr ± hr ± hr ± hr ± hr ± without obstruction [] according to the europeansociety of gastrointestinal endoscopy clinical guidelinethat considers the risk of perforation due to colorectalstents only limited uses are allowed therefore colorectalstent placement is not a standard treatment []the prognostic outcomes of bts in this study were significantly worse than those of es particularly in lymphnodepositive patientslymphatic and venous invasionseemed to be a significant prognostic factor althoughreduced postoperative complication rate would improve the prognosis our results were contradictoryafter the stent replacement these results suggestedthat stent placement leads to poor prognosis a concern that colonic stents may be associated with adverse effects of mechanical expansion also exists mechanical expansion may be associated with thegrowth of solid tumors particularly lymphatic andvenous invasion [ ]we found that recurrence and os were associatedwith high vascular invasion after a colonic stent placement venous invasion was an independent factor for recurrence and prognosis the ck20 mrna level anepithelial marker is significantly increased in peripheralblood serum suggesting stent deployment into the vasculature alliteratively ki67 level associated withcellular proliferation and p27 gene assisting cell cycleprogression were measured using specimens obtainedbefore and after sems insertion next the ki67 leveldecreased in the specimen after an sems placementcompared with that before and cell proliferation wassuppressed the prognostic nutritionalindex andserum albumin levels were significantly decreased afterstenting suggesting its disadvantage as bts theduration from stent placement to surgery was daysoncological and nutritional factors might change in theblood and contribute to poor prognosis during the waiting period mechanical expansion of the replacement hr ± hr ± hr ± hr ± hr ± hr ± hr ± hr ± hr ± hr ± should be minimized to prevent perforation and molecular cytological factors to improve the materials expansion and establishment of new mechanism are necessaryin colorectal obstruction [ ]thisisafirstretrospectivethese findings should be considered in light of severallimitationsnonrandomized small sample sized study from a single institution thereby the heterogeneity of the surgical strategy may have affected the prognostic factors secondalthough validated endoscopic procedures were validated stent devices used in this study had differentlengths types and thickness and obtained from differentvendors lastly we performed stent placement in the patients with cross and who are not indicated forstent insertion until to investigate the oncological longterm prognosis ofcolonic sems placement as a bridge to elective surgerylarge sample size and prospective randomized controlledstudies are warranted to develop a treatment strategy forcrc with obstructionvascular invasiveness was correlated with tumor progression after a sems placement and os and rfs rateswere lower in bts sems placement potentially worsensprognostic outcomes in stage iiiii malignant colorectalobstructionabbreviationsbts bridge to surgery crc colorectal cancer cross colorectal obstructionscoring system es emergency surgery esge european society ofgastrointestinal endoscopy jsccr japanese society for cancer of the colonand rectum recist response evaluation criteria in solid tumors version os overall survival rfs recurrencefree survival sems selfexpandablemetallic stent wses world society of emergency surgeryacknowledgementsthe authors thank for contribution as endoscopic technical adviser kenkonishi md phd from department of surgery hyogo prefecturenishinomiya hospital 0cohta bmc surgery page of authors contributionsall authors have read and approved the manuscript ko protocolproject development data collection and management andmanuscript writingediting mi protocolproject developmentmanagement and manuscript writingediting mu protocolprojectdevelopment and data collection and management jm se jm and ikdata collection and management yt and sn data collection ki andtn data analysis and manuscript writingediting mt and ty dataanalysis and managementfundingauthors have no grant support and no financial relationship for this studyavailability of data and materialsthe datasets used and analyzed during this study are available from thecorresponding author upon reasonable requestethics approval and consent to participateall procedures performed in studies involving human participants were inaccordance with the ethical standards of the institutional researchcommittee and with the helsinki declaration and its later amendmentsor with comparable ethical standards all participants or their guardians haveprovided their written informed consent and that the study protocol wasapproved by higashiosaka city medical center ethical committee on humanresearch assignment number consent for publicationno applicablecompeting intereststhe authors declare that they have no conflict of interest to discloseauthor details1gastroenterological surgery higashiosaka city medical center osaka japan2department of gastroenterological surgery kindai university nara hospital otodacho ikomacity nara japan 3thoracic surgeryhigashiosaka city medical center osaka japan 4digestive surgery kawasakimedical school okayama japan 5gastroenterology higashiosaka citymedical center osaka japanreceived june accepted august referencesjemal a bray f center mm ferlay j ward e forman d global cancerstatistics ca cancer j clin pubmed pmid epub eng winner m mooney sj hershman dl feingold dl allendorf jd wright jd incidence and predictors of bowel obstruction in elderly patients withstage iv colon cancer a populationbased cohort study jama surg pubmed pmid pubmed central pmcid pmc45 epub engjullumstro e wibe a lydersen s edna th colon cancer incidencepresentation treatment and outcomes over years color dis pubmed pmid epub engcheynel n cortet m lepage c benoit l faivre j bouvier am trends infrequency and management of obstructing colorectal cancers in a welldefined population dis colon rectum pubmed pmid epub engcuffy m abir f audisio ra longo we colorectal cancer presenting assurgical emergencies surg oncol pubmed pmid epub eng mcardle cs hole dj emergency presentation of colorectal cancer isassociated with poor 5year survival br j surg pubmedpmid epub eng mainar a tejero e maynar m ferral h castanedazuniga w colorectalobstruction treatment with metallic stents radiology pubmed pmid epub engzhang y shi j shi b song cy xie wf chen yx comparison of efficacybetween uncovered and covered selfexpanding metallic stents inmalignant large bowel obstruction a systematic review and metaanalysiscolor dis 2012147e367 pubmed pmid epub engtilney hs lovegrove re purkayastha s sains ps westonpetrides gk darziaw comparison of colonic stenting and open surgery for malignantlarge bowel obstruction surg endosc pubmed pmid epub engsaito s yoshida s isayama h matsuzawa t kuwai t maetani i aprospective multicenter study on selfexpandable metallic stents as a bridgeto surgery for malignant colorectal obstruction in japan efficacy and safetyin patients surg endosc pubmed pmid epub engsaida y sumiyama y nagao j uramatsu m longterm prognosis ofpreoperative bridge to surgery expandable metallic stent insertion forobstructive colorectal cancer comparison with emergency operation discolon rectum suppls44 pubmed pmid epub engtomita m saito s makimoto s yoshida s isayama h yamada t selfexpandable metallic stenting as a bridge to surgery for malignant colorectalobstruction pooled analysis of patients from two prospectivemulticenter series surg endosc pubmed pmid pubmed central pmcid pmc6342866 epub eng chen hs sheenchen sm obstruction and perforation in colorectaladenocarcinoma an analysis of prognosis and current trends surgery pubmed pmid epub eng huang x lv b zhang s meng l preoperative colonic stents versusemergency surgery for acute leftsided malignant colonic obstruction ametaanalysis j gastrointest surg pubmed pmid epub engkim hj huh jw kang ws kim ch lim sw joo ye oncologic safetyof stent as bridge to surgery compared to emergency radical surgery forleftsided colorectal cancer obstruction surg endosc pubmed pmid epub engshigeta k baba h yamafuji k kaneda h katsura h kubochi k outcomesfor patients with obstructing colorectal cancers treated with onestagesurgery using transanal drainage tubes j gastrointest surg pubmed pmid epub engvan hooft je bemelman wa oldenburg b marinelli aw lutke holzik mfgrubben mj colonic stenting versus emergency surgery for acute leftsided malignant colonic obstruction a multicentre randomised trial lancetoncol pubmed pmid epub engvan hooft je fockens p marinelli aw timmer r van berkel am bossuyt pm early closure of a multicenter randomized clinical trial of endoscopicstenting versus surgery for stage iv leftsided colorectal cancer endoscopy pubmed pmid epub engsloothaak da van den berg mw dijkgraaf mg fockens p tanis pj vanhooft je oncological outcome of malignant colonic obstruction inthe dutch stentin trial br j surg pubmed pmid epub eng gorissen kj tuynman jb fryer e wang l uberoi r jones om localrecurrence after stenting for obstructing leftsided colonic cancer br j surg pubmed pmid epub eng ormando vm palma r fugazza a repici a colonic stents for malignantbowel obstruction current status and future prospects expert rev meddevices pubmed pmid epub englauro a binetti m vaccari s cervellera m tonini v obstructing leftsidedcolonic cancer is endoscopic stenting a bridge to surgery or a bridge tonowhere digestive diseases and sciences pubmed pmid epub engschwartz lh litière s de vries e ford r gwyther s mandrekar s recist 11update and clarification from the recist committee europeanjournal of cancer oxford england p pubmed pmid pubmed central pmcid pmc5737828 epub eng mcshane lm altman dg sauerbrei w taube se gion m clark gm reportingrecommendations for tumour marker prognostic studies remark eur jcancer pubmed pmid epub eng hashiguchi y muro k saito y ito y ajioka y hamaguchi t japanesesociety for cancer of the colon and rectum jsccr guidelines for thetreatment of colorectal cancer int j clin oncol pubmed pmid epub eng cortet m grimault a cheynel n lepage c bouvier am faivre j patterns ofrecurrence of obstructing colon cancers after surgery for cure a population 0cohta bmc surgery page of based study color dis pubmed pmid epub eng nojiri t maeda h takeuchi y funakoshi y kimura t maekura r predictive value of btype natriuretic peptide for postoperative atrialfibrillation following pulmonary resection for lung cancer eur jcardiothorac surg pubmed pmid epub engpubmed pmid pubmed central pmcid pmc4128744 epub engkaragiannis gs poutahidis t erdman se kirsch r riddell rh diamandis epcancerassociated fibroblasts drive the progression of metastasis throughboth paracrine and mechanical pressure on cancer tissue mol cancer res pubmed pmid pubmed central pmcidpmc4399759 epub eng nojiri t inoue m yamamoto k maeda h takeuchi y funakoshi y b maruthachalam k lash ge shenton bk han af tumour celldissemination following endoscopic stent insertion br j surg pubmed pmid epub eng matsuda a miyashita m matsumoto s sakurazawa n kawano y yamahatsuk colonic stentinduced mechanical compression may suppresscancer cell proliferation in malignant large bowel obstruction surg endosc pubmed pmid epub eng haraguchi n ikeda m miyake m yamada t sakakibara y mita e colonic stenting as a bridge to surgery for obstructive colorectal canceradvantages and disadvantages surg today pubmedpmid epub eng zhu z li b liao w lv n chen y shu x novel predictive nomogram foridentifying difficult guidewire insertion in patients with malignant colorectalobstruction and sphincterotomeassisted guidewire insertion for improvingthe success rate of selfexpandable metal stent insertion front oncol pubmed pmid pubmed central pmcid pmc7237730epub eng miyasako y kuwai t ishaq s tao k konishi h miura r newlydeveloped selfexpandable nitis md colonic metal stent for malignantcolonic obstruction world j gastrointest surg pubmedpmid pubmed central pmcid pmc7215972 epub engpublishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationstype natriuretic peptide as a predictor of postoperative cardiopulmonarycomplications in elderly patients undergoing pulmonary resection for lungcancer ann thorac surg pubmed pmid epub eng cowie mr struthers ad wood da coats aj thompson sg poolewilsonpa value of natriuretic peptides in assessment of patients withpossible new heart failure in primary care lancet pubmed pmid epub eng cuthbertson bh card g croal bl mcneilly j hillis gs the utility of btypenatriuretic peptide in predicting postoperative cardiac events and mortalityin patients undergoing major emergency noncardiac surgery anaesthesia pubmed pmid epub engsabbagh c browet f diouf m cosse c brehant o bartoli e isstenting as a bridge to surgery an oncologically safe strategy for themanagement of acute leftsided malignant colonic obstruction acomparative study with a propensity score analysis ann surg pubmed pmid epub engtung kl cheung hy ng lw chung cc li mk endolaparoscopic approachversus conventional open surgery in the treatment of obstructing leftsidedcolon cancer longterm followup of a randomized trial asian j endoscsurg pubmed pmid epub eng gianotti l tamini n nespoli l rota m bolzonaro e frego r aprospective evaluation of shortterm and longterm results from colonicstenting for palliation or as a bridge to elective operation versus immediatesurgery for largebowel obstruction surg endosc pubmed pmid epub eng yang sy park yy han yd cho ms hur h min bs oncologicoutcomes of selfexpandable metallic stent as a bridge to surgery andsafety and feasibility of minimally invasive surgery for acute malignantcolonic obstruction ann surg oncol pubmed pmid epub engvan hooft je van halsema ee vanbiervliet g beetstan rg dewitt jmdonnellan f selfexpandable metal stents for obstructing colonic andextracolonic cancer european society of gastrointestinal endoscopy esgeclinical guideline endoscopy pubmed pmid epub eng ansaloni l andersson re bazzoli f catena f cennamo v di saverio s guidelenines in the management of obstructing cancer of the leftcolon consensus conference of the world society of emergency surgerywses and peritoneum and surgery pns society world j emerg surg pubmed pmid pubmed central pmcid pmc3022691epub eng arezzo a balague c targarona e bhi f giraudo g ghezzo l colonic stenting as a bridge to surgery versus emergency surgery formalignant colonic obstruction results of a multicentre randomisedcontrolled trial esco trial surg endosc pubmedpmid epub eng amelung fj burghgraef ta tanis pj van hooft je ter b f siersema pd critical appraisal of oncological safety of stent as bridge to surgery inleftsided obstructing colon cancer a systematic review and metaanalysiscrit rev oncol hematol pubmed pmid epub eng domingo s puertolas s graciavilla l puertolas ja mechanical comparativeanalysis of stents for colorectal obstruction minim invasive ther alliedtechnol pubmed pmid epub engsuzuki n saunders bp thomasgibson s akle c marshall m halligan scolorectal stenting for malignant and benign disease outcomes incolorectal stenting dis colon rectum pubmed pmid epub eng voutouri c mpekris f papageis p odysseos ad stylianopoulos t roleof constitutive behavior and tumorhost mechanical interactions in thestate of stress and growth of solid tumors plos one 201498e104717 0c" | 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 |
" ovarian cancer is the second most common gynecologic cancer with high mortality rate andgenerally diagnosed in advanced stages the 5year diseasefree survival is below micrornas subset of thenoncoding rna molecules regulate the translation in post transcriptional level by binding to specific mrnas topromote or degrade the target oncogenes or tumor suppressor genes abnormal expression of mirnas were foundin numerous human cancer including ovarian cancer investigating the mirnas derived from the peripheral bloodsamples can be used as a marker in the diagnose treatment and prognosis of ovarian cancer we aimed to findbiological markers for early diagnosis of ovarian cancer by investigating brca1 gene mutation carrier monozygoticdiscordant twins and their high risk healthy family individuals mirnasmethods the study was conducted on monozygotic twins discordant for ovarian cancer and the liquid biopsyexploration of mirnas was performed on mononuclear cells that were isolated from the peripheral blood samplesthe mirna expression profile changes in the study were found by using microarray analysis mirna isolationprocedure performed from the lymphocyte in accordance with the kit protocol the presence and quality of theisolated mirnas screened by electrophoresis raw data logarithmic analysis was studied by identifying thethreshold normalization correlation mean and median values target proteins were detected for each mirna byusing different algorithmsresults after the comparison of monozygotic discordant twins for epithelial ovarian carcinoma upregulation of the mirnas mir6131 mir1305 mir1973p mir3651 and downregulation of mirnas mir3135b mir4430 mir664b5p mir7663p were found statically significantcontinued on next page correspondence hy2188istanbuledutrdepartment of cancer genetics istanbul faculty of medicine oncologyinstitute istanbul university istanbul turkey the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctuncer of ovarian research page of continued from previous pages the detected mirnas out of mirnas might be used in the clinic as new biological indicatorsin the diagnosis and follow up of epithelial ovarian cancer with complementary studies the mirna expressionprofiles were identified to be statistically significant in the evaluation of ovarian cancer etiology brca1 mutationstatus and ovarian cancer risk in accordance with the obtained datathere is a need for validation of the mirnas which were particularly detected between monozygotic twins and itsassociation with ovarian cancer was emphasized in our study in wider cohorts including ovarian cancer patientsand healthy individualskeywords monozygotic twins mirna expression profiles brca1 and brca2 biomarkers ovarian cancer is a significant cause of mortality in gynecologic cancers and one ofthe leading cause ofcancerassociated mortality in turkey ovarian cancer is the 7th most common type of cancer in women inaccordance with worldwide globocan datas showthat each year more than women are diagnosedwith ovarian cancer oc worldwide and approximately women die from it the data of globocan for turkey shows that annually women are diagnosed with ovarian cancer and women die fromthis malignancy the 5year survival rate was given as these data revealed that ovarian cancer is an important reason of gynecologic cancer associated mortality rate the epithelial ovarian cancerseocoriginating from the ovarian surface epithelium constitutes approximately of ovarian malignancy themajority of eoc patients are diagnosed in advanced stages stage iii and iv and year freesurvivalrate is below the standard treatment for newlydiagnosed ovarian cancer is the combination of cytoreductive surgery and platinbased chemotherapy significant advances in radical surgery and chemotherapystrategies have improved clinical outcomes but unfortunately no progress has been made with relapse andtreatment resistance ninety percent of ovarian cancer occurs sporadically in the population whereas hereditary type appears of ovarian cancer patientsbrca1 and brca2 genes are the most common breastovarian cancer syndrome associated genes both brca1and brca2 have roles in the control of the genomic stability cell cycle and apoptosis the mutations occurringin these genes result with the inability of dna repairand therefore results in the accumulation of the mutations in the cell the rate of the breast cancer susceptibility of women with brca1 gene mutation until theage of years was and the rate of ovarian cancersusceptibility rate is breast cancer susceptibilitywomen with brca2 gene mutation until the age of years is and ovarian cancer development risk is twin studies became important on genetics by theendandcentury geneticnineteenthofthethe differentiated genesepidemiologic studies with monozygotic twins were accepted as highly useful investigation models in the pastdecades and have been used recently when a similarity for a disease or a quantitative feature betweenmonozygotic and dizygotic twins is compared variationsare excluded according to studies conducted in thepopulation and thereforeit is easier to identify andmake etiological differences visible via twin studies because the affected siblings and dizygotic twins share theapproximately ofthephenotypic differences between twins are known to beassociated with the genetic variation in addition diversity may be revealed with a very limited patient population therefore the results of the twin studies can beapplied to the population and can make valuable contributions to the genetic studies monozygotic twins aregenetically similar and generally expected to be compatible for congenital malformations chromosomal abnormalities and mendelian disorders there are numerousstudies conducted via discordant monozygotic twins revealing the genetic contribution therefore investigating the genetic variability in monozygotic twins ishighly important and the majority of the human geneticsassociated research focus on finding genetic variability indiscordant monozygotic twins phenotypically discordantmonozygotic twins are used as the model systems inidentification of the variable in understanding the pathogenesis of a disease the most striking study is the oneconducted with monozygotic twins in canada and evidencing that multiple sclerosis ms was a genetic disease micrornas are one of the subset of the noncoding rnas generally consisting of single strand in nucleotide length not transformed to protein havingroles in post transcriptional regulation or suppression oftranslation of the target mrnas [ ] the regulatoryroles of mirnas were demonstrated to occur in tumorigenesis cell differentiation proliferation and apoptosis[] mirna genes are known to locate in thechromosomal breaks this dna breaks cause chromosomal abnormalities frequently associated with cancersusceptibility and tumor development [] the noninvasive biologicalindicators have been used for the 0ctuncer of ovarian research page of treatment resistance of ovarian cancer the most common of this indicators are the cancer antigen125 ca and cancer antigen153 ca153 these biological indicators can be used in the followup of thetreatment response in the diagnosed patients but cannotbe used in the early diagnosis and in differentiation ofthe malignant disease therefore there is a need forspecial therapeutic agents customized for patients thatmay be used target specific therapies and in the earlydiagnosis of the ovarian cancer in identification of theefficacy of therapy and in the follow up period thusstudies investigating the target molecules and biologicalindicators are required that will enable the early diagnosis and in the development of the better therapy optionsdifferentially synthesize mirnas such as mir mir141 mir125b mir2223p or let7 family has beenshown in studies with ovarian cancer patients however the use of these mirnas as a biomarker in ovariancancer is not yet available in order to clearly define therole of mirnas in the pathogenesis of ovarian cancerwe planned to investigate the brca mutant monozygotic twins with the same genetic profile but with discordant for ovarian malignant transformation in thisstudy mirnas which are thought to have the potential biological indicator role were studied from bloodsamples of both discordant monozygotic twins andbrca wild type healthy siblingsmethodspatients recruitmentthe peripheral blood lymphocytes of monozygotic twinsdiscordant for ovarian cancer and healthy individuals inthe same family were used in the study the patient diagnosed with ovarian cancer and all family members applied to the cancer genetics clinic of oncologyinstitute of istanbul university for brca breast cancersusceptibility gene testing were examined for brcagene mutation all family members in the study consisted of highrisk individuals with hereditary breast andovarian cancer hboc syndrome and the people included in the study were given as br codes according topatient file number the monozygotic ovarian cancer patient healthy monozygotic twin healthy sisters and niece were found to have brca1 gene mutation c5266dupc pgln1756profs74 rs397507247 on exon thepatients brother and daughter were found negative forbrca1brca2 gene mutations in this study lymphocyte cells separated from peripheral blood belonging tototal of cases including younger age ovarian cancer patient and healthy monozygotic twin a patients daughter elder sisters a younger sister a nephew and a brotherwere examined by mirna microarray method thepedigree of the family included in the study and theirhierarchical cluster analaysis via euclidean method isshown on fig the study was approved by the ethics committee ofthe istanbul faculty of medicine following institutionalethics committee approval informed consents were obtained from all participants before enrollment into thestudy ethics committee approval number atstoredthey werelymphocyte and mirna isolationficoll sigmaaldrich darmstadt germany density gradient was used to separate white blood cells mononuclear cells from other blood components mirnaisolation procedure was performed from the lymphocytein accordance with the kit protocol using the mirneasymini kit qiagen cat noid the proceduresteps in accordance with the protocol are as follows μl qiazol solution was included on the cells storedin nitrogen tank cell fractionation was enabled by mixturing using the vortex for complete nucleoproteinfractionation °c roomtemperature for min by adding μl chloroformthey were shacked and mixed on hand the tubes wereincubated for min at °c room temperature thenwere centrifuged for min at °c and g thesupernatant formed after centrifugation was transferredto collection tube using a pipette and was mixed usingvortex by inclusion of μl ethanol the supernatant formed after the ethanol centrifuging was transferred to collection tube was removed with a pipettewas mixed with vortex by including μl ethanol seven hundred microliters was taken from the obtained mixture and was transferred to the rneasyminielute spin colon placed on ml collection tubethe tubes were centrifuged for s at g at °croom temperature seven hundred microliters rwt buffer was added to spin colons and the colons werewashed by centrifuging at g for sthe centrifuging procedure was repeated by including μl rpe buffer twice consecutively to colons the colons placed into clean tubes with ml were dried bycentrifuging min in maximum speed the colonsplaced in ml sterile tubes were included μl distilled water by centrifuging at g in min and themirnas were collectthe quality control of the mirnasthe presence and quality of the isolated mirnas werescreened by electrophoresis at v on agarose gelthen the purity and concentrations of the mirnaswere measured on thermo scientific nanodrop spectrophotometer nanodrop technologies wilmington de usa device the mirna purity for each persondevicemeasurement result were obtained with the comparisonaccordance withthe nanodropin 0ctuncer of ovarian research page of fig the pedigree of the family included in the study and their hierarchical cluster analysis legend the pedigree of the family and using thecorrelations between samples plotted a dendrogram for sample grouped by hierarchical clustering euclidean distance complete linkage br healthy brother brca1 negative nonbrca1 mutation carrier br healthy niece brca1 positive brca1 mutation carrier br healthy daughter brca1 negative br healthy monozygotic twin brca1 positive brca1 mutation carrier br monozygotic twindiagnosed with ovarian cancer brca1 positive brca1 mutation carrier br healthy sister brca1 positive brca1 mutation carrier br healthy sister brca1 positive brca1 mutation carrier br healthy sister brca1 positive brca1 mutation carrierof the measurements at spectrophotometrically at nm and nm wave lengths the measurement rates at nm wave lengths is a sign of quality of the purity of the samples therefore the samples in the idealvalue interval of and for rna measurementswere included in the study the purity of mirnas wereevaluated using a bioanalyser device bioanalyseragilent technologies santa clara ca usa agilentrna nano kit agilent technologies santa claraca usa for confirming whether the mirnas were appropriate and in adequate level for microarray analysisthe evaluated sample concentrations and results wereanalyzed the samples with rna concentrations between ngμl and rrna rate over and rna integrity number values between and were evaluated asthe appropriate samples for array study 0ctuncer of ovarian research page of microarray trial protocolmicroarray protocol was performed by preparing thespikein solution sample marking hybridization sampledephosphorylation sample denaturation sample ligationhybridization of the samples slide loading preparationof the hybridization unit and elution and scanning ofslides the slide scanning procedure was performedusing the agilent microarray scanner agilent microarray scanner with surescan high resolution technology agilent technologies santa clara ca usadevice the scanning procedure of the slides were performed on sureprint g3 human mirna microarrayrelease 8x60k agilent inc santa clara ca platform and using the agilent technologies g2600d scanning protocol the analysis of the tiff tagged imagefile format extensioned files obtained after scanningprocedure was performed using the agilent feature extraction v11011 programthe success levels of stages developed in all experiment process with this analysis program the quality ofthe levels the process were monitored and evaluatedthen bioinformatic analysis procedure was performeddata analysisraw data logarithmic analysis was studied by identifyingthe threshold normalization correlation mean and median values then the mirnas demonstrating differentexpression profile among the samples were filteredusing the fold change rates and independent twosample t test the possible difference between the compared groups were evaluated all evaluations were performed to enable the cutoff values as the foldchangerates fc ¥ and pvalue hierarchical clusteranalysis was performed using the euclidean method fig and complete linkage cluster method the control ofthe experimental errors and the detection of the erroneous finding rate were identified using the hochbergmethodbioinformatic analysistarget proteins were detected for each mirna by usingtwo algorithms targetscan71 httpswwwtargetscanvert_71 and mirdbv5 httpsmirdbmirdbthe targeted genes thought for each mirna wereconfirmed by also both algorithms and the mirnatarget relations were also experimentally confirmed mirtarbase70httpsmirtarbasembcnctuedutwphpindexphp databasecomparison groupsin the study mirna analysis was performed at the genome level withwithout mutation in cases withwithoutovarian cancer the mirna data was evaluated by comparing different groups in order to investigate the effectof brca mutation in ovarian malignancy developmentand determine the mirnas that can be important in theovarian cancer pathogenesis in group the monozygotic twins discordant for ovarian cancer were comparedin order to find the effects of mirnas in the formationof ovarian cancer in group the family members withbrca1 mutation were compared with family memberswithout brca1 mutation to identify the changes ofmirnas expression levels according to brca positivityin group the monozygotic ovarian cancer patient withbrca1 mutation carrier and the other healthy familymembers with mutation carrier were compared for investigate the effects of both ovarian cancer developmentand brca positivity on mirnas expression level ingroup all family members were compared with ovarian cancer monozygotic twin in order to find the mirnas that might be important in the predisposition ofovarian cancer the comparison groups also showed intable resultswe identified differentially expressed comparisonof mirnas between the groups the raw data obtainedafter experimental studies were filtered before the comparisons between the groups the upregulated or downregulated mirnas expression levels more than foldfc and smaller than the p value p wereconsidered in evaluation and the comparisons betweenthe groups were performed based on these values allthese comparisons were evaluated for ovarian cancer etiology brca1 mutation carriage and the ovarian cancerrisk hierarchical cluster analysis of the expression of mirnas represents sharp separations of upregulatedyellow from downregulated blue in fig mirnas total of mirnas were found statistically different after the comparison of phenotypicallydiscordant monozygotic twin siblings the mirnasmir1273 g3p mir1305 mir1973p mir3651 mir and mir92a3p expressions were found to haveupregulated and the other mirnas let7i 5p mir125a5p mir15b5p mir22 3p mir3135b mirtable comparison groups and cases in the groupscasegroup br1639controlbr1447group br1639br1447br1547br2030br1546br1861br1850br2028group br1639group br1639br1447br1447br1547br2030br1546br1861br1547br2030br1546br1861br1850br2028 0ctuncer of ovarian research page of fig hierarchical cluster analysis of the expression of mirnas legend br healthy brother brca1 negative nonbrca1 mutationcarrier br healthy niece brca1 positive brca1 mutation carrier br healthy daughter brca1 negative br healthymonozygotic twin brca1 positive br monozygotic twin diagnosed with ovarian cancer brca1 positive br healthy sister brca1positive br healthy sister brca1 positive br healthy sister brca1 positive the mirnas that may be effective in the etiology ofovarian cancer were identified after the comparison of monozygotic twins who were phenotypically discordant for ovarian cancer diagnosis ingroup 320d mir3423p mir4430 mir451a mir664b5pand mir7663p expressions were found to have downregulated after the bioinformatic analysis a total of upregulated and downregulated statistically significantmirnas and their target molecules are given in table and fig different mirnas level were compared between group in order to determine the effect of brca1 gene mutation group was consisted after the comparison of thebrca1 gene mutation carrier family members and individuals not carrying brca12 gene mutation accordingto mirnas expression profiles after the comparisonsdownregulated and upregulated mirnasrelated tobrca1 gene mutation carrier were determined the expression of a total of mirnas including mir4449mir46533p mir4865p mir5739 mir6165 andmir 8743p associated with the brca1 gene mutationcarrying were upregulated and the expression of a totalof mirnas including mir1263p mir320a mir320b mir320c mir320d mir320e mir3243p mir mir mir4428 mir4516 mir4741 mir mir564 mir6089 mir68695p mir68915pmir71075p and mir78473p were found downregulated after the bioinformatic analysis a total of upregulated and downregulated statistically significantmirnas and their target molecules are shown in table and fig aftercomparisondifferent mirna levels were compared between group in order to determine the relation with ovarian cancerdevelopment and brca positivity group consists ofcomparison of mirnas of brca1 positive ovarian cancer patient with all other brca1 positive healthy individualsanddownregulated mirnas related to mutation carriage inbrca1 gene and epithelial ovarian cancer etiology weredetermined the expression of mirnas includingmir1260a mir1260b mir165p mir175p mir181b5p mir 26b5p mir4281 mir4286 mir5100mir68403p mir71145p mir7975 and mir7977were found to have upregulated and the expression of mirnas including mir12255p mir1423p mir 26a5p mir mir29a3p mir30d5p mir3196upregulated 0ctuncer of ovarian research page of table ovarian cancer etiology related upregulated and downregulated mirnas and target proteins in monozygotic twinsmirnassequence of mirnamirnastatustarget genesfold change fcvaluesaccacugcacuccagccugagupregulatedznf138 tmem239 bmp3uuuucaacucuaaugggagagaupregulatedptpn4 prkaa1 papd7frat2 depdc1 fbxo41uucaccaccuucuccacccagcupregulatedcd82 pmaip1 mthfd1 check1 ago1 casp10cauagcccggucgcugguacauga upregulatedggcuggucagaugggagugupregulatedracgap1 ola1 tex261ptgs1 nfic znf200pagr1 igf2bp1 cacng8uauugcacuugucccggccuguugagguaguaguuugugcuguuucccugagacccuuuaaccuguga downregulated erbb3 cdkn1a tp53 erbb2 egfr stat3mycstat3 pten atm notch2 cdh1 nfkb1upregulateddownregulated tlr4 bmp4 eif2c1 neurog1 socs1 igf1vegfamir1273 g3pmir1305mir1973pmir3651mir6131mir92a3plet7i5pmir125a5pmir15b5puagcagcacaucaugguuuacamir223paagcugccaguugaagaacugudownregulated bcl2 vegfa ccnd1 ccne1 cdk1 cdk4 cdk6 e2f3mapk1downregulated cdkn1a wnt1 erbb3 mycbp hmgb1 e2f2 ptenpoted sod2mir3135bmir320dmir3423pmir4430mir451amir664b5pggcuggagcgagugcaguggugaaaagcuggguugagaggaucucacacagaaaucgcacccguaggcuggagugagcggagaaaccguuaccauuacugaguuugggcuaagggagaugauuggguadownregulated birc5 abl2 mapk1 mycndownregulated dctn5 syncrip fbxo28downregulated gemin4 dnmt1 id4 srebf1srebf2 bmp7downregulated znf485abl2 mapk1 msh5 ptendownregulated cpne3 rab5a il6r akt1 mmp2downregulated cd55msn rhobtb3 plag1mir7663pacuccagccccacagccucagcdownregulated cox1 mapk1 nf2 rad51 stk4 stk24 vegfcfig the upregulated and downregulated mirnas and foldchanges associated with ovarian cancer etiology in monozygotic twins 0ctuncer of ovarian research page of table brca1 gene mutation positivity related upregulated and downregulated mirnas and target molecules an additional tablefile shows this in more detail [see additional file ]mirnassequence of mirnatarget genesmirnastatusfold changefc valuesmir4449mir46533pmir4865pmir5739mir6165mir8743pmir1263pmir320amir320bmir320cmir320dmir320emir3243pmir3656mir4284mir4428mir mir4741mir484mir564mir mir6869 5pmir68915pmir71075pmir7847 3pcgucccggggcugcgcgaggcaupregulatedzfhx3uggaguuaaggguugcuuggagaupregulatedatg2a crebl2 mat2a frs2 tmed4 ubn2uccuguacugagcugccccgaggcggagagagaauggggagccagcaggaggugaggggagcugcccuggcccgagggaccgaupregulatedupregulatedupregulatedupregulatedolfm4cd40arhgap5 igf1r dock3 cadm1dlx6cd207chic1ppl2a plxdc1per1tfap2afads1 amer1luzp1 cox6b1hdac1 aqp3 stat3 cdk9ucguaccgugaguaauaaugcgdownregulatedtom1 crk vegfa sox2 twf1 pitpnc1 igfbp2 krasaaaagcuggguugagagggcgadownregulatedmcl1 banp itgb3 bmi1 nrp1 nfatc3 trpc5aaaagcuggguugagagggcaadownregulatedcdk6dctn5syncriparf1 bcl9l znf600aaaagcuggguugagaggguaaaagcuggguugagaggaaaagcuggguugagaaggacugccccaggugcugcuggggcgggugcggggguggdownregulateddownregulatedsyncripfbxo28smarcc npm3dctn5 syncrip fbxo28dctn5 npm3 znf275 ddx19a ncapd2 txnl1downregulateddownregulated wnt9b crebbp dvl2 wnt2bdownregulatedmrpl12 lsp1 mnt prdm2znf770 cecr1gggcucacaucaccccaudownregulatedbcl2l11rbbp5hnrnpa1 znf264 trib3 crtapcaaggagacgggaacauggagcdownregulatedmsl1mapre3myh14casp2 ccnd2 cdk14tp63gggagaagggucggggcdownregulatedstat3m6prgpr137cccnd2 ccnt1 cdkn1a scoc tp53cgggcuguccggaggggucggcudownregulatedddx39bmapk1 zbtb39 hmga1ucaggcucaguccccucccgaudownregulatedfis1 pagr1 zeb1 slc11a2smad2 anapc7 tbrg1aggcacggugucagcaggcdownregulatedgid4 cnbp e2f3 rcan3 akt2 appl1 slc1a2 gpr155ggaggccgggguggggcggggcggdownregulatednkx2 tpt1 kctd5 bbx sgcd cdh7 ccnb1gugaguaguggcgcgcggcggcdownregulatedtubb2amapk1nrbf2 wee1hmga2 mapk1 stag2uaaggagggggaugaggggdownregulatedchd4 cd207 ddx6 chrdl1ccnd2 tp63ucggccuggggaggaggaagggdownregulatedvav3 casp16 ccnd1 casp16 mapk14cguggaggacgaggaggaggcdownregulatedhavcr1 poted dnajc10 sod2 m6pr cdk19mir3423p mir3665 mir3960 mir4466 mir4530mir46873p mir47875p mir4943p mir50015pmir50065p mir5787 mir6068 mir6087 mir mir6090 mir6124 mir6125 mir638 mir65105p mir68005p mir7704 mir8063 and mir were found to have downregulated after bioinformatic analysis a total of upregulated and downregulated statistically significant mirnas and the targetmolecules are given in table and fig for identifying the ovarian cancer predisposition allfamily members were compared with ovarian cancermonozygotic twins in group the upregulated ordownregulated mirnas in association with the epithelialovarian cancer risk were identified after the comparisonthe expression of the mirnas consisting of let7a5plet7b5p mir181a5p mir1973p mir215pmir2233p mir23a3p mir27a3p mir36533pmir4255p mir572 mir5745p mir6127 and mir were detected to be upregulated the expression ofthe mirnas consisting of let7i5p mir 125a5pmir15b5p mir1505p mir22 3p mir3283pmir4430 mir451a mir46975p mir664b5p andmir7663p were detected to have downregulated atotal of upregulated and downregulated statisticallysignificant mirnas and the target molecules are givenin table and fig discussionwomen are diagnosed with ovarian cancer at an advanced stage due to limited number of biologicalmarkers for ovarian cancer patients although existingovarian cancer biomarkers cancer antigen125 and cancer antigen153 ca125 ca15 are sensitive in thefollowup of diagnosed gynecological cancers they have 0ctuncer of ovarian research page of fig the upregulated and downregulated mirnas and foldchanges associated with brca1 gene mutation positivityofearlysensitivityin the diagnosislessstagegynecological cancers and separation of malignant tumorformations from benign formations therefore tounderstand the underlying mechanisms of ovarian cancer and to explore targeting drugs and to improve newtreatment protocols for ovarian malignancy revealingsignificant genetic changes is necessary the genetic andepidemiologic studies conducted on monozygotic twinsare known to provide accurate and direct informationabout the gene and environment interaction with thedisease occurrence mechanism the changes in genesthat result in the occurrence of tumors such as mirnaexpression level among the monozygotic twins providesinformation on the etiology of disease and may have arole as a biological indicator in identifying the early stagedisease and in the follow up of the prognosis we aimedto identify the noninvasive biological markers that maybe used in the early diagnosis of ovarian cancer throughinvestigating the mirnas in the peripheral blood ofmonozygotic twin siblings discordant for ovarian cancerwith the mirna molecules of the other healthy members in the family thus that may cause less bias thanthe controls to be selected from the population ninetynine different mirna molecules presented in the studywere detected after the comparison of monozygotic twinsiblings who were discordant for ovarian cancer andwith the other healthy individuals seventeen differentmirnas were found that could be used for detectingearly diagnosis and prognosis of ovarian cancer betweenthe monozygotic twin siblings who were discordant forovarian cancer in our study the association between out of mirnas and ovarian carcinoma is beingreported for the first time in this study due to the highnumber of newly detected mirnas in our study the discussion and comparison were only made between thecandidate mirnas although mir1973p mir1305mir6131 mir3651 mir3135b mir4430 mir664b5p and mir7663p have not been shown to be associated with ovarian cancer in literature but limited number of studies have suggested the association with othercancerswang found the elevated level of mir1973pinthe same way as we do the upregulated mir1973p expression level was shown to promote the cellular invasion and metastasis in bladder cancer in that studyresearchers reported that linc00312 gene was responsible for invasion and metastasis mechanisms and thisgene inhibited the cellular migration and invasion bysuppressing the mir1973p expression similar resultswere detected in thyroid cancer in the study of liu [ ] jin reported that increased expressionlevel of mir1305 caused pluripotent stem cells to accelerate the cell cycle g1s transfer in addition to causingthe cellular differentiation with the increased mir1305expression the expression levels ofreducedmirna125a5p and let7i5p found in the scope of ourstudy have been shown to parallel with other studies inthe literature langhe suggested thatlet7i5pmight be described as a diagnostic indicator in ovariancancer the mirna125a5p expression was upregulated to inhibit the cancer proliferation and migrationin the in vitro study of qin in human cervical carcinomas and mir125a5p upregulated expressionlevel was demonstrated to inhibit the cervical cancer 0ctuncer of ovarian research page of table brca1 mutation carriage and epithelial ovarian cancer etiology related upregulated and downregulated mirnas targetmolecules an additional table file shows this in more detail [see additional file ]mirnassequence of mirnamirnastatustarget genesaucccaccucugccaccaaucccaccacugccaccauuagcagcacguaaauauuggcgupregulatedupregulatedupregulatedcaaagugcuuacagugcagguagupregulatedpsat1unc13a rps27 brd7sfrp1 dkk2 smad4 psat1unc13a rps27ccne1 arl2 bcl2 hmga1 cdk6 ccnd1vegfa reck prdm4tgfbr2 pten cdkn1a bcl2l11e2f1tp53stat3aacauucauugcugucgguggguupregulatedtcl1a timp3 plag1 bcl2rnf2vsnl1 atmfold changefc valuesmir1260amir1260bmir16 5pmir175pmir181b5pmir26b5pmir4281mir4286mir5100uucaaguaauucaggauagguupregulatedgggucccggggaggggggaccccacuccugguaccupregulatedupregulateduucagaucccagcggugccucuupregulatedptgs2 epha2 chordc1 ezh2ccne1abca1 gata4ncdn cdkn1a bcl3ldlr znf354b nsd1 rabgap1taok1 mknk2cox10 dek kcnn3 rab11fip1dynlt1 notch2slfn12l ctc1 gxylt2gde1fads1per1 atg9amir68403pgcccaggacuuugugcggggugupregulatedmir71145pucuguggaguggggugccuguupregulatedm6pr hnrnpul1 shmt1 znf529acvr2b paics taf8mir7975mir7977auccuagucacggcaccauucccagccaacgcaccaupregulatedupregulatedmir12255pguggguacggcccaguggggggdownregulatedkbtbd8gulp1 casz1 rad51hspa1b znf703 tmem185bsf3b3cox6b1 ccdc9 cdh7orc4 odf2l mtrnr2l7psmg2 mtrnr2l3mir1423pmir26a5pmir2861mir29a3pmir30d5pmir3196mir 3423pmir3665mir3960mir4466mir4530mir46873pmir47875pmir4943pmir50015pmir50065pmir mir6068mir6087mir6088mir6090mir6124uguaguguuuccuacuuuauggadownregulatedarntltgfbr1 rac1 rock2 ccnt2 tab2 ptpn23uucaaguaauccaggauaggcudownregulatedezh2rb1adam17 hmga2ccnd2 cpeb3 dnmt3bggggccuggcggugggcgguagcaccaucugaaaucgguuauguaaacauccccgacuggaagcggggcggcaggggccucdownregulateddownregulateddownregulateddownregulatedly6eccdc64 ccnd1 dars2 apaf1mcl1cdk6sparc dnmt3adnmt3b col4a1gnai2 tp53 casp3 snai1 ezh2 bcl9 notch1 smad1pou3f3tulp1 h2afxpcgf3 casp16atg2a ccdc64ucucacacagaaaucgcacccgudownregulatedgemin4 dnmt1 id4 srebf1srebf2 bmp7 rmnd5aagcaggugcggggcggcgggcggcggcggaggcggggggggugcgggccggcggggcccagcaggacgggagcguggcuguuggagggggcaggcdownregulateddownregulateddownregulateddownregulateddownregulated | Colon_Cancer |
there is currently a major human pandemic caused by the novelsevere acute respiratory syndrome sars coronavirus2 sarscov2 that leads to coronavirusinduced disease covid191 itis primarily a viralinduced ammatory disease of the airwaysand lungs that causes severe respiratory issues sarscov2 usesthe angiotensin converting enzymeii receptor ace2 to bindand infect cells leading to internalization and proliferation23ammatoryinnate and adaptive immune responses areinduced to clear the virus but also cause host tissue damage45consequent hypoxia leads to systemic involvement particularlyofleads to vasoconstriction reducedperfusion and an failure6 much remains to be understoodof the ammatory and immune responses that are induced bythe infection and how they induce pathogenesis ventilation andoxygen therapy are primary treatments and it is emerging thatthose with severe disease who survive develop lung ï¬brosis7 themost effective pharmacological treatments remain illdeï¬nedwith varying results with hydroxychloroquine8 but more promising results with dexamethasone9the vasculature thatelucidating the mechanisms of pathogenesis will enable theidentiï¬cation of the most effective therapies animal models ofsarscov2 infection and covid19 that recapitulate the hallmarkfeatures of the human disease will undoubtedly be valuable inelucidating pathogenic mechanisms identifying new therapeutictargets and developing and testing new and effective treatmentshuman infection and diseasesarscov2 is a betacoronavirus closely related to sarscovthat caused a relatively small outbreak in the early 2000s210similar to sarscov sarscov2 binds the ace2 receptor andrequires proteases such as serine tmprss2 to cleave the viralspike s protein required for sarscov and sarscov21112 cellentry2 this step may be facilitated by endosomal proteases suchas cathepsinl and enhanced by the protein furin13 the virusthen enters the host cell by endocytosisa critical element of sarscov2 tropism in humans is theabundance of ace2 in the upper respiratory tract urt especiallythe nasopharynx14 the molecular conï¬guration of the sarscov21centre for ammation centenary institute and university of technology sydney faculty of science sydney australia 2zhejiang universityuniversity of edinburgh institutezhejiang university school of medicine zju international campus haining china 3second afï¬liated hospital zhejiang university school of medicine hangzhou china4department of genomes and genetics institut pasteur paris france 5centre for innate immunity and infectious diseases hudson institute of medical research clayton vicaustralia 6department of molecular and translational sciences monash university clayton vic australia 7ritchie centre hudson institute of medical research clayton vic australia 8department of paediatrics monash university clayton vic australia 9monash newborn monash childrens hospital clayton vic australia 10priorityresearch centre for healthy lungs hunter medical research institute and university of newcastle newcastle nsw australia 11school of chemistry and molecular biosciencesand australian infectious diseases research centre the university of queensland brisbane australia 12centenary institute and dermatology the university of sydney andcancer services royal prince alfred hospital sydney nsw australia 13centenary institute and faculty of medicine and health university of sydney sydney australia 14dr johnand anne chong lab for functional genomics charles perkins centre centenary institute and school of life and environmental sciences university of sydney sydney nswaustralia 15charles perkins centre and school of life and environmental sciences university of sydney and faculty of medicine and health concord clinical school anzacresearch institute and centre for education and research on ageing sydney australia 16discipline of infectious diseases and immunology central clinical school faculty ofmedicine and health and the charles perkins centre the university of sydney camperdown sydney australia 17woolcock institute of medical research sydney australia and18centenary institute the university of sydney and department of clinical immunology royal prince alfred hospital sydney nsw australiacorrespondence p m hansbro philiphansbroutseduaureceived july revised july accepted july society for mucosal immunology 0canimal and translational models of sarscov2 infection and covid19md johansen membrane binding component of the s protein binds with greaterafï¬nity to ace2 than does sarscov which likely contributes to thehigher infectivity of the former15 the clinical course commenceswith an incubation period with a median of days with illnesstypically developing by days16 this phase is characterized bymild symptoms with most people remaining asymptomatic andinfection thought to be conï¬ned to the urt although they arecapable of transmitting infection symptoms when they do occur aretypically acute viral respiratory illness with fever cough dyspnoeafatigue anosmia myalgia and confusion17 in of people thecourse remains mild and disease does not extend to the lowerrespiratory tractlrt however develop more severesymptoms with diffuse widespread pneumonia with havingsevere gas exchange problems acute lung injury and progress ontosyndrome ards1819 the clearestacute respiratory distresspredictor of mortality is age with the case fatality rate risingdramatically over years of age20 other predisposing factors forheightened mortality are male sex social deprivation and chronicdisease particularly chronic obstructive pulmonary disease copdcardiovascular disease cvd obesity and diabetes21a key issue is why some individuals progress to more severe lowerrespiratory disease but others do not one factor is the ability of theammatory and immune responses to conï¬ne the infection to theurt ace2 is expressed in the lrt but at lower levels than in thenasopharynx22 also while ciliated airway epithelial cells are readilyinfected and transmit to surrounding cells the reduction in ace2may be a barrier to lrt infectionin those that progress severesystemic ammatory response or cytokine storm develop thepneumonia associated with severe infection bears all the pathological features of ards with diffuse alveolar damageinterstitialpneumonitis and lymphocytic ltrates2324 unique features ofcritical disease are extravascular ï¬brin deposition neutrophiltrapping microvascular thrombosis and large vessel pulmonaryemboli24 widespread thrombosis and microangiopathy in criticalcovid19 occurs at higher rates than in ards associated withuenza and dysregulated coagulation and angiogenesis are alsofeatures25increased and dysregulated th1 and th17 responses werepresent in ards in middle eastern respiratory syndrome merscov and uenza2627 the occurrence of severe lung disease at days postinfection dpi reï¬ects the dual features of spreadof infection to the lrt and coincident development of adaptiveimmune responses with heightened activation of virusspeciï¬c teffector cells this coincides with lymphopenia associated withsevere disease and is a predictive marker earlier in disease withworse outcome28 there is also evidence that tcells aredysfunctional with increased expression of exhaustion moleculesrelated to heightened systemic ammation29 the role ofelevated systemic immune dysregulation is supported by analysisof the transcriptomic responses in people with sarscov2infection demonstrating that impaired interferon ifn responseswere related to persistent viremia and increased systemicil6 and il1030 elevatedammation with elevated tnfαsystemic levels of il17 are also present in critical illness with ardsand heightened ammation and it is plausible that increasedth17 responses drive ongoing ammation31 while interestingthese are observations often performed with limited patientnumbers where ards and disease severity are associated withheightened ammatory and immune activation and a causativerole cannot be established2732 interrogation of representativeanimal models is needed to deï¬ne cause and effect elucidatemechanisms of pathogenesis and test treatmentssmall animal modelsa range of animal models have been used to study covid19 withvarying susceptibility likely dependent on species speciï¬c makeup for ace2 fig fig ace2 protein phylogenetic divergence and in vivo modelseverity left panel fast minimum evolution distance tree for ace2protein sequences using griphin for evolutionary distance unsortedspecies included are gallus gallus chicken anas platyrhynchos duckcavia porcellus guinea pig mustela putoris furo ferret canis lupusfamiliaris dog felis catus cat sus scrofa pig rousettus aegyptiacusfruitbat mesocricetus auratus golden hamster mus musculus mousecallithrixjacchus common marmoset macaca mulatta rhesus macaque macaca fascicularis cynomolgus macaque chlorocebus aethiopsafrican green monkey and homo sapiens macaques are representedas one image due to close divergence severity of disease is colorcoded from refractory to infection blue no virus detected to severered shedding common marmoset and guinea pig have only beenassessed for sarscov all others with sarscov2 scale indicates amino acid divergencemouse modelssmall animal models are widely used to study emerging virusesbut often they need to be genetically modiï¬ed or the virus needsto be adapted for different species to be susceptible and this is thecase for sarscov2 in most studies to date the mouse strainshave been incompletely or incorrectly described and should beincluded in future publicationsgenetically modiï¬ed miceinbred mouse strains were used to model sarscovseveralinfection including balbc33 c57bl634 and 129svev incorrectlyreported name35 as well as factordeï¬cient mice such ascd1 rag1 and stat1 the identiï¬cation of ace2 asthe host receptor for sarscov38 initiated considerable globalinterest in developing murine models that are representative ofhuman disease this led to the generation of transgenic mice thatexpress human hace2 k18hace2 in the epithelia of tissues andans including lungs liver kidney spleen heart and gut k18hace2 mice still express the mouse mace2 however this isprimarily localised in the ileum39 they are highly susceptible tosarscov infection and experience high virallung titressigniï¬cant weight loss and morbidity from dpi39 viral dissemination to the brain was the main cause of death40 similar resultswere obtained in another transgenic strain expressing hace2under the control of the mace2 promoter41mucosal immunology __ 0cwith the mers outbreak in and identiï¬cation of that virusentry receptor dipeptidyl peptidase4 dpp4 cd26 it was foundthat neither wildtype wt nor immunodeï¬cient mice weresusceptible to merscov infection42 transgenic mice expressinghuman hdpp4 in epithelial and endothelial cells in the lungbrain heart liver kidney spleen and intestine were generated43they were highly susceptible to merscov infection withsigniï¬cant weight loss high virallung titres and ammatorycytokine production from dpi and mortality from day inother studies exons of the mdpp4 locus were modiï¬ed toresemble hdpp4 expression44 which led to merscov lungreplication but not disease development4748with the discovery of sarscov2ace2 interactions21249 globalinterest in hace2 mouse models reemerged however due tofalling interest with the resolution of the sars outbreak most labsceased maintaining hace2 mice to satisfy the global demand forhace2 mice the jackson laboratory reanimated k18hace2 micewhich are becoming available the lagtime has slowed theunderstanding of disease mechanisms in covid19 and theidentiï¬cation of effective drug and vaccine candidates to progressto clinical trialsthe ï¬rst live sarscov2 infection model used transgenic miceexpressing hace2 under the control of the mace2 promoter4150mice had signiï¬cant weight loss over a 14day infection periodand high viral lung titres dpi histological lung examinationrevealed moderate interstitial pneumonia ltration of lymphocytes mucus accumulation and desquamation of bronchoepithelial cells from day there were no detectable viral titres orpathology in other tissues or ans except on day in theintestine suggesting that infection is localized almost exclusivelyto the lungssimilarly transgenic mice overexpressing hace2 under thecontrol of hfh4foxj1 lung ciliated epithelial cellspeciï¬cpromoters are also susceptible to sarscov2 infection5152 mostinfected hfh4hace2 mice had minimal weight loss over 7days ofinfection however mice that later became moribund showedsigniï¬cant weight loss from day and signiï¬cant lymphopeniaand neutrophilia in peripheral blood at day which recapitulatessevere human disease2831 lung histology showed initial macrophage and lymphocyte ltration and ï¬brin exudation from dpiwhich steadily progressed to severe pneumonia blockage ofterminal bronchioles extensive ï¬broplasia and alveolar necrosisby day in contrastlung tissuespeciï¬city50 hfh4hace2 infected mice had detectable viral titresin the lung eyes brain and heart suggesting that the virus mayhave additional tissue tropisms following initial lung infection5051reinfection following recovery from initial sarscov2 infectionresulted in reduced weight loss and viral titres and improvedsurvivalindicating the development of protective immunityfollowing initial challengeto previous ï¬ndings ofrecentlythe ï¬rst k18hace2 sarscov2 infection wasexamined53 mice exhibited no clinical symptoms or weight lossuntil dpi by day mice had weight loss with variableclinical presentation ranging from reduced activity to increasedrespiration and lethargy infected mice also had moderate virallung titres suggesting productive infection bronchoalveolarlavage revealed increased ltrating granulocytes monocytesand eosinophils accompanied by alveolar debris and septalthickening on histopathology analysis while these results areencouraging the study only examined ï¬ve mice up until dpi inagreement with these initial ï¬nding53 a recent preprint papersimilarly shows that sarscov2 infected k18hace2 mice lost weight by dpi which steadily decreased to bodyweight by dpi54 they had high virallung titres from dpiaccompanied by modest viral titres in the heart brain kidney andspleen declines in pulmonary function were notable at dpievidenced by reduced inspiratory capacity and increased tissueresistance and elastance rnasequencing analysis of infectedmucosal immunology __animal and translational models of sarscov2 infection and covid19md johansen type i and iiinnate immunelung tissues identiï¬ed the upregulation ofsignatures particularly nfkbdependentifnsignalling and leucocyte activation pathways another preprintpaper shows that sarscov2 infected k18hace2 mice produce arobust th1217 cytokine storm in the lungs and spleens from dpi55 highlighting the relevance of this mouse model thatrecapitulates critical human clinical features of covid19 importantly multiple reports have shown that sarscov2 infection offrom dpi55k18hace2 mice is dosedependently fatalsuggesting that it is similarly lethal as sarscov39 in thesetransgenic miceadenoviral systemsadenovirus vectorbased systems can be used to insert humanreceptors into mouse genomes and are valuable for use withalready factordeï¬cient or transgenic mice replicationdefectiveadenovirus vectors have been used to insert hddp4 into wt micerendering them susceptible to merscov infection58 infectedmice developed pneumonia with extensive pulmonary immunecell ltration and viral clearance by dpi58 but were less affectedthan fully hdpp4 transgenic micesimilarly transduction of balbc mice with adenovirus containing hace2 advhace2 led to stable hace2 expression in thelungs from h posttransfection59 sarscov2 infected advhace2 mice had weight loss over days high viral lungtitres and modest titres in the heart brainliver and spleenextensive neutrophil accumulation in perivascular and alveolarlocations and vascular congestion upon histological examinationadministration of antiifnar1 monoclonal antibodies to transiently inhibit typei ifn signalling resulted in up to weight lossand more severe lung ammation compared to infection alonein this system neutralizing antibodies against sarscov2 sprotein 1b07 were protective against severe disease mice lostless body weight and had lower viral titres in the lung heart andspleen at dpi and reduced expression of proammatorycytokines and chemokines ifnb il6 cxcl10 cxcl1 ccl2 ccl5 andimmune cell ltrates in the lungs at dpicrispr systemsusing crisprcas9 an alternative humanised hace2 mousemodel was developed where the mace2 was disrupted byinserting hace2 linked to the red ï¬uorescent protein tdtomato60hace2 expression is under the control of the mace2 promoter inthe native locus with expression predominantly in the lungs andkidneys similar to mace2 in wt mice sarscov2 infectedhumanized hace2 mice had high viral titres in the brain tracheaand lungs with no differences between young and aged micehowever infected aged mice had lung neutrophilia and extensivealveolarinjury and focal hemorrhagingcompared to young mice intragastric infection resulted in highviral titres in the trachea and lungs suggesting that the oraladministration route can lead to productive pulmonary infectionthickening vascularmouseadapted virusesviruses can be adapted to infect wt mice through serial passagingor targeted mutation their use is advantageous as they reducebiological risks to researchers and may more closely resemblenatural hostpathogen interactions in mice however they arelimited due to mouse adaptation that may result in infection thatdoes not recapitulate all aspects of human diseasethis approach was applied to sarscov which was seriallypassaged in the respiratory tracts of young balbc mice this ledto the generation of a mouseadapted sarscov strain ma15which was lethal to mice from dpi61 infection resulted in highviral titres in the lungs from dpifollowed by viremia anddiffusion to extrapulmonary sites including the brain liver andspleen signiï¬cant lymphopenia and neutrophilia mild and focalpneumonitis and necrotic cellular debris in the airways and alveoli 0canimal and translational models of sarscov2 infection and covid19md johansen another mouseadapted sarscov strain v2163 was producedby serial passage in 6weekold balbc mice62 infection resultedin more severe symptoms and higher mortality rate than withma15 with greaterimmune responses and lung pathologymouseadapted sarscov strains lacking the critical viral envelopei protein induce varying degrees of protection against reinfectionwith virulent strains highlighting the potential for liveattenuatedvaccines6364serial passage of merscov through the lungs of mice withhuman modiï¬ed exons of dpp4 resulted in a virus thatinduced signiï¬cant weight loss and mice became moribund from dpi mice had high stable viral titres in the lungs and bloodammatory cellltrates and oedema necrotic debris andvascular permeabilization in lungs47mouseadapted sarscov2 has been reported in a preprint withmutations in the receptor binding domain rbd of the spike proteinfollowing serial passageinducing productive infection of bothyoung and aged wt balbc mice65 infected mice had high virallung loads up to dpi and displayed mild pneumonia withammatory cell ltration alveolar damage focal exudation andhaemorrhage and endothelial cell denaturation the efï¬cacy of arbdfc based vaccine was examined in this model which inducedthe production of neutralizing antibodies which potently inhibitedthe infection a similar preprint describes the modiï¬cation of therbd of the sarscov2 s protein which facilitated the efï¬cientbinding of the s protein to mace2 for host cell entry66 infectionwith this virus resulted in viral replication in the upper and lowerairways of young and aged balbc mice aged mice had greaterweight loss and pulmonary function decline compared to youngmice reproducing important aspects of human diseasea summary of the different mouse models that are permissiveto sarscov2 infection table and a comparison of featuresbetween mouse models and human covid19 fig are showncovid191875 animal models that combine these risk factorswith infection will be invaluable in elucidating mechanisms ofincreased susceptibility and severity8081chronic respiratory diseasesboth cigarette smoking cs and copd are strong independentrisk factors for severe covid19 with extensive lung damage andincreased mortality82 cs upregulates ace2 expression in theairways which may increase infection risk83 the use of shorttermmurine models thatreplicate csinduced copd has greatlyincreased our understanding of disease and identiï¬ed and testednovel therapeutic interventions808184early data from china showed asthma prevalence in covid19patients were lower than the general population suggesting thatasthma may be protective89 however emerging reports showthat asthma is one ofthe most prevalent comorbidities inhospitalized covid19 patients90 and is associated with higher riskof death especially in severe asthma based on oral corticosteroiduse21 elucidating how severe asthma predisposes to severecovid19 is needed and can be achieved using preclinical animalmodels of severe asthma that recapitulate the human disease91k18hace2 mice and sarscov2 infectionlung ï¬brosis is the most severe sequelae of covid19 in up to of the patients after months94 the mechanisms areunclear but ammation and cytokine storm likely contribute7sarscov2 infection of human alveolar epithelial cells results inaltered proï¬brotic gene expression similar to pulmonary ï¬brosisincluding ace2 tgfβ connective tissue growth factor tissueinhibitor of metalloproteinase3 and ï¬bronectin98 fibrotic sequalae can be assessed in mouse models with long rest periods afterinfection8699 bleomycininduced experimental pulmonary ï¬brosiscould be combined with sarscov2 infection to investigatesequalae8699diversiï¬ed modelsthe immediate aim of producing infectionpermissive mice ormouseadapted viruses is to generate models with high respiratory viral titres and lung lesions resembling those observed inhumans host factors are associated with increased risk of severecomplications including age and obesity67 and comorbidities likecopd cvd and diabetes68 human genetic variantslikelymodulate susceptibility to covid1969in mice the impact of host genetic variations were investigatedin sarscov using the collaborative cross cc a collection ofmouse inbred strains produced by crossing eight founder inbredstrainsincluding ï¬ve classic laboratory strains aj c57bl6j129s1svimj nodshiltj and nzohiltj and three wildderivedcasteij pwkphj and wsbeij strains70 cc strains segregate million polymorphisms and have more genetic diversity thanthe human population71 this resource is ideally suited forinvestigating the role of host genetic variants in the pathophysiology of infectious diseases72 ma15 infected cc mice had abroad range of phenotypes including weight loss and increasedlung viral titres and lung pathology73 susceptibility varied fromabsence of symptoms to mortality with normallungparenchyma and lung viral titres at dpi that varied over logunits genetic analysis of cc strains identiï¬ed trim55 and ticam2as host susceptibility genes7374 this shows that the geneticbackground of mice is important in replicating distinct humanclinical presentations investigating multiple genetic backgroundsis possible using adenovirustransduced hace2 or mouseadaptedvirusespredisposing risk factors and mouse modelschronic diseases like copd asthma lung ï¬brosis cvd diabetesmellitus as well as obesity male sex and old age are associatedincreased susceptibility to sarscov2 infection and severeobesity diabetes and cvdmouse models of genetic or dietary modiï¬cations to inducefeatures of human disease are widely available8687100101 inbredmouse strains such as c57blb6 have genetic susceptibility toobesity and diabetes while male mice and rats are more pronecompared to females101 ace2 is highly expressed in hearttissues and so it is expected that cvd models willincreasedsusceptibility to sarscov2 infection104 the link between type andor diabetes and ace2 expression is unknown withconï¬icting reports on the levels of expression in diabetesprogression and severity105106 there are numerous mousemodels of type and diabetes that are chemically or geneticallyinduced and for type diabetes typically incorporate obesity107sarscov and sarscov2 infection has not yet been explored inthese models but these mice will likely be highly susceptible toinfection with pulmonary decline and increased mortality asobserved in humansagerelated susceptibilityold age is the main risk factor for severe covid19108 which isattributed to comorbidityimmunosenescence malnutritionresidential care and biological aging changes109 ace2 expressionin human lungs may either be unchanged83 or decreased104 withold age while expression is increased in the olfactory epitheliumwhich may contribute to susceptibility to sarscov2 infection110the signiï¬cance of age is exempliï¬ed in murine models whereinfection of young balbc and c57blb6 mice with sarscovresulted in high viral titres in the upper and lower airways butwith no disease or mortality33 however aged balbc mice hadsigniï¬cant weight loss and severe disease with extensive alveolardamage and bronchiolitis111 ace2 levels are decreased in agedcompared to younger mice which may explain differences indisease severity112 itis unknown whether aged mice aresusceptible to sarscov2 infection however evidence frommucosal immunology __ 0cybsaytilatrommrofinuhtiwipdybssolithgewllewsaipdmorfgnulehtnisertitlarivhghiipdinarbdnasetanbrutilasanehtnisertitlarivetaredomdnasgnulnidevresbomrotsienkotycipdmorfipdmorfneepslsgnulehtnisertitlarivhghiipdybssolithgewmorfyticapacyrotaripserdecuderipdmorfneepsldnayendkiinarbtraehehtnisertitlarivtsedomdnasyawhtapesnopserenummietanniroflnoitaugerpuhghiswohseussitgnuldetcefnifoqesanripdlygoohtapotsihufpÃufpÃecnereferemoctuoesaesidsesodsuoitcefninoitasilacoldnanoisserpxeecahretomorpenilesuomlarivhghiderevocerllatubithgewydobtsoliytidbromonsngislacinilcisuovboongnulehtniecimsertitdctiÃotpuyendkidnaenitsetnininoisserpxehghitraehninoisserpxeetaredomsgnulninoisserpxewolretomorpecambhecahsisenegohtapvocsrasienmaxeotdesusledomesuomeballiavafoyrammuselbatdnanoitammaï¬niinarbdnagnulevisnetxeipdybsertitgnullarivhghiipdybssolithgewufpÃotpuinarbninoisserpxeegamadgnulerevesdnaailihportuendahecimdnubiromhghiipdybesaesidcitamotpmysdnassolithgewlygoohtapotsihybdecnedveisgnulnistnuoclygoohtapotsihllecdnayrotammaï¬ninoitammaï¬nidnasertitlarivgnulevisnetxesyendkidnarevilninoisserpxewoljxofdctiÃwoltraehdnarevilyendkineepslenitsetnillamsninoisserpxeetaredomnoocldnasgnulninoisserpxehghiknitarekotycecahkdedidnaithgewydobtsolecimfonoitroporpufpÃinarbdnatugsgnulninoisserpxehghihfhrotcafnoitpircsnartdaehkrofecahhfhanimal and translational models of sarscov2 infection and covid19md johansen gnullarivhghisyadrevoithgewydobtsolytilatromdetroperondetroperecimsertituffninoisserpxesgnulninoisserpxedetropertonseussithghirehtolsurivoagemotycfolortnoctnetepmocnirednuhfhniretomorpecahvdadegagnuldnaaehcartinarbnisertitlarivhghdahisngislacinilcisuovboonithgewydobtsolecimecimufpÃninoisserpxewolyendkidnaneepsltraehdnayravoinarbenitsetnillamssgnulninoisserpxehghiretomorpecamevitanecadesinamuhrotcevlarivonedamucosal immunology __ 0canimal and translational models of sarscov2 infection and covid19md johansen fig comparison of disease features shared between humans with covid19 and mouse models of sarscov2 both humans and micedisplay similar clinical signs such as weight loss and pneumonia severe infections are often associated with increased proammatorycytokine production accompanied by high viral lung titres which correlates with extensive lung damage and signiï¬cant pulmonary declinehumanised ace2 mice shows that aged mice display greaterdisease severity60 aged mice showed a reduced response toexperimental vaccination for sarscov with lowerlevels ofneutralizing antibodies than young mice113 similar to responsesto many vaccines in older people thus studying aged mice islikely to be pivotal for developing treatments and vaccines forolder peoplewas less efï¬cient with low levels of viral antigen detectable innasal washes from two of six ferrets and infection was mild withno changes in body temperature overall the ferret model ofsarscov2 is limited as the infection is mild with little lrtinvolvement and no reported characteristics of severe disease inhumans such as oedema or ards they may provide a model tostudy sarscov2 transmissionferretsferrets have been extensively used to study uenza a virus iavpathogenesis and transmission since their respiratory tract isanatomically comparable to humans114 ace2 is most abundantlyexpressed on typeii alveolar and glandular epithelial cells in thetrachea and bronchi of ferrets115 ferrets are susceptible to sarscov infection with replication in the urt and lrt increased bodytemperature sneezing and alveolar damage115recently ferret ace2 was shown to contain the critical residuesrequired for binding by sarscov2 rbd118 the animals weresusceptible to sarscov2 infection with viral replication in theurt however only low levels of virus were detected inlungs119120 viral loads in the lung and nasal turbinates peakedat dpi with clearance by and dpi respectively whileinfectious virus was not detected outside the respiratory tract viralrna was found in the intestine saliva urine rectal swabs andfaeces up to 8dpi this suggests that sarscov2 preferentiallyreplicates in the urt of ferrets in line with this disease is mildwith reduced activity and occasional cough from days in moststudies elevated body temperatures are observed from dpireturning to baseline by day with no change in bodyweight119121 shi 120 reported only out of ferretsdeveloped fever and loss of appetite following intranasalinoculation with different sarscov2 isolates suggesting isolatevariability and dose may alter disease outcomes limited studieshave examined immune responses to sarscov2 in ferretsintranasal infection induced serum neutralizing antibodies119121another study compared transcriptional responses in cells fromiav and sarscov2infected ferrets122 thenasal washes ofmagnitude of antiviral and ifn responses were higher with iavcompared to sarscov2 infection with unique enrichment forcell death and leucocyte activation in the latter kim 119 usedthe ferret model to study sarscov2 transmission viral rna wasdetectable in nasal washes from ferrets h after direct contactwith intranasally inoculated animals suggesting transmission israpid and can be transmitted prior to the peak of disease dpi allsix ferrets that had direct contact developed elevated bodytemperatures in contrast to direct contact airborne transmissiongolden hamstersyrian golden hamsters have been used to model a broad rangeof viral infections123 and genetically modiï¬ed animals have beengenerated124 hamsters are susceptible to sarscov infectionwith comparable viral replication in the urt and lrt but noclinical signs of disease other than reduce activity111127128in silico structural analysis predicts that sarscov2 s proteinrbd effectively binds hamster ace2129130 infection of hamsterswith sarscov2 resulted in clinical signs such as rufï¬ed fur rapidbreathing and weight loss from day with recovery by dpi129 high levels of infectious virus were detected in the lungand nasal turbinates on days and and signiï¬cant histologicalchanges were observed including proteinrich lung ï¬uid exudatemononuclear cell ltration severe cell death and haemorrhageand alveolar damage transmission studies revealed efï¬cient virusspread via direct contact between cohoused infected hamsters129 infectious virus was detectable in the lungs and nasalturbinates accompanied by lung histology changes cohousedhamsters did not lose signiï¬cant body weight suggesting theinfection was less severe than with intranasally inoculation likelydue to differences in inoculum dose this suggests that sarscov infection of hamsters is not dissimilar to humans and may be amodel to study pathogenesis and transmission and test potentialtherapeuticsnonhuman primates nhpsnhp experiments account for only of all animal researchnevertheless clinical translation of nhp studies is much greaterthan for other animal models as they are closest to humanp | Colon_Cancer |
objective this study aimed to test the hypothesis that levobupivacaine has antitumour effects on breast cancer cellsresults colony formation and transwell assay were used to determine breast cancer cells proliferation flow cytometry annexin v and pi staining was used to investigate breast cancer cells apoptosis the effects of levobupivacaine on cellular signalling and molecular response were studied with quantitative polymerase chain reaction and western blot induction of apoptosis was confirmed by cell viability morphological changes showed cell shrinkage rounding and detachments from plates the results of the western blot and quantitative polymerase chain reaction indicated activation of active caspase and inhibition of foxo1 the results of the flow cytometry confirmed that levobupivacaine inhibited breast cancer cell proliferation and enhanced apoptosis of breast cancer cells quantitative polymerase chain reaction and western blot analysis showed increased p21 and decreased cyclin d quantitative polymerase chain reaction and western blot analysis showed that levobupivacaine significantly increased bax expression accompanied by a significant decreased bcl expression and inhibition of pi3kaktmtor signalling pathway these findings suggested that levobupivacaine inhibits proliferation and promotes breast cancer cells apoptosis in vitrokeywords levobupivacaine proliferation invasion apoptosis breast cancerintroductionbreast cancer is one of the most recorded cancer illness among women in the united states it is estimated that more than women die every year from breast cancerrelated illness despite the advance in chemotherapy and targeted treatments correspondence yanqiu63126com wqp89163com department of anaesthesiology dalian medical university dalian china department of biochemistry and molecular biology dalian medical university dalian chinafull list of author information is available at the end of the molecular signalling pathways that are involved in breast cancer transformation have become targets for treatment the mechanisms of the pi3kaktmtor signalling pathway have present some promising targets for cancer treatments this signalling pathway hinders the functions of several tumour suppressor genes such as bad gsk3 foxo transcription factors and tuberinhamartin complex which control cell survival proliferation and growth [] suppressing this signalling pathway may inhibit cancer cells proliferation and also stimulate them toward cell deaththe growing evidence of local anaesthetics inhibiting cancer cell growth seems promising though limited the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0ckwakye a0et a0al bmc res notes page of at the tissue level administration of a certain amount of local anaesthetics topical or local has shown to have a direct inhibitory effect on the action of epidermal growth factor receptor egfr which is a potential target for antiproliferation in cancer cells evidence also shows that ropivacaine and lidocaine hinder cancer cells growth invasion migration and enhance apoptosis of lung cancer cells [] to the best of our knowledge the effect of levobupivacaine on breast cancer cells is yet to be determined the present study therefore aimed to investigate the antitumour effects of levobupivacaine on breast cancer cellsmain textmaterials and a0methodsethics statementthe ethical committee of the dalian medical university first affiliated hospital approved for this study to be carried outcell culturewe purchased mcf7 and mdamb231 breast cancer cells from the atcc beijing zhongyuan limited china we maintained the mcf7 and mdamb231 cells with highglucose dmem or dmemf12 gibco usa medium the medium was supplemented with fetal bovine serum fbs gibco usa penicillin a0unitsml and streptomycin a0µgml transgen biotech china to maintain the cells the mcf7 and mdamb231 cells were then maintained in an incubator at a0ºc humidified air with co2 atmospheric condition the cells were routinely subcultured subsequentlyantibodies and a0reagentsepr17671 akt monoclonal antibody abcam china y391 mtor polyclonal antibody abcam china a2845 bcl2 polyclonal antibody abclonal technology a11550 bax polyclonal antibody abclonal technology a0265 pik3ca polyclonal antibody abclonal technology a2934 foxo1 polyclonal antibody abclonal technology epr21032 active caspase monoclonal antibody abcam china afo931 cyclin d1 polyclonal antibody affbiotech china af6290 p21 polyclonal antibody affbiotech china antimtor phospho s2448 antibody abcam china pa517387 phosphopi3k p85p55 tyr458 tyr199 polyclonal antibody themofisher scientific posphopanakt123 ser473 antibody affbiotech china peroxidaseconjugated goat antirabbit igg proteintech china prap antibodies proteintech china and gapdh antibodies proteintech chinacell viability assay and a0ic50we determined the mcf7 and mdamb cells viability using cck8 assay levobupivacaine at a concentration of or a0mm was used to treat mcf7 and mdamb cells plated in 96well plates a0cellswell and then incubated for or a0h respectively in an incubator at the atmospheric condition of a0 °c with co2 the rest of the procedures used for the cck8 assay were the same as described elsewhere flow cytometryannexin v and propidium iodide pi staining assay were used to investigate the apoptosis of mcf7 and mdamb cells following levobupivacaine treatment after treating the cells for a0h trypsin was used to harvest the treated cells and centrifugation at rcf for a0min the mcf7 and mdamb treated cells were again suspended with binding buffer and then a0 µl of fluorochromeconjugated annexin v sigmaaldrich saint louis usa was added into a0µl of the cell suspension to stain intracellular phosphatidylserine ps the cells were then incubation in a dark under room temperature the cells were again suspended and a0 µl propidium iodide staining solution sigmaaldrich saint louis usa added into a0µl of the cell suspension we detected the percentage of the apoptotic cells via flowjo software treestar ashland usa and flow cytometry facs calibur becton dickinson and sunnyvale ca usaquantitative polymerase chain reaction qpcrthe procedures used for the qpcr were the same as previously described the primers sequences were bax 5tgg cag ctg aca tgt ttt ctg3 f 5tcc cgg agg aag tcc aat g3 bcl2 5acg gtg gtg gag gag ctc tt3 f 5gcc ggt tca ggt act cag tcat3 r p21 5gcg act gtg atg cgc taa tg3 f 5gaa ggt aga gct tgg gca gg3 r gapdh ²cat gtt cgt cat ggg tgt gaa² f ²ggc atg gac tgt ggt cat gag3² rr western blotat the log phase of treated mcf7 and mdamb cells growth we harvested the cells and then washed twice with icecold pbs the rest of the procedures used for the western blot were the same as described elsewhere colony formation assaythe procedures used for the colony formation assay were the same as previously described 0ckwakye a0et a0al bmc res notes page of transwell assaythe mcf7 and mdamba231 cells that were pretreated with different dose of levobupivacaine a0mm for a0h and resuspended in culture medium with the same concentrations of levobupivacaine were seeded onto the coated membrane in the upper chamber of the transwell 24well millicell cell culture insert a0mm diameter a0μm pores merck kgaa p18p01250 china the procedures used for the transwell assay were the same as previously describe data analysisvalues were expressed as the mean ± sd statistical analysis was performed with graphpad prism version 501graphpad software la jolla ca us oneway anova was used to measure significance p dunnetts post hoc tests were used to test the difference between groupsresultslevobupivacaine decreases breast cancer cell invasiontranswell assay analysis showed significantly decreased in the invasion ability of mcf7 and mdamb231 cells in a dosedependent manner compared with the untreated cells additional file a0 fig s1a b levobupivacaine inhibits proliferation in a0breast cancer cellsthe mcf7 and mdamba231 cell viability decreased as the concentrations of levobupivacaine or a0mm increased the mcf7 cells showed a cytotoxic effect while the mdamb231 cells showed a similar cytotoxic effect of fig a01a under a fluorescence microscope cells treated with levobupivacaine showed morphological changes including cell rounding cell shrinkage and cells detachment from the plates additional file a0 fig s2a b the viability of breast cancer cells decreased in a dosedependent manner the results showed significantly decreased in the number of clones of the treated cells compared with the untreated cells fig a01b c the data showed that the mrna level of p21 significantly increased following levobupivacaine treatment fig a0 1d e western blot analysis showed a similar increased in p21 and decreased in foxo1 and cyclin d1 expressions in a dosedependent manner compared with the untreated cells fig a01f g additional file a03f glevobupivacaine promote apoptosis in a0breast cancer cellslevobupivacaine significantly reduced the number of cells showing nuclear staining when compared with the untreated cells fig a0 2a b the qpcr data showed a decreased in bcl2 and increased in bax expressions in mcf7 and mdamb231 cells compared with the untreated cells fig a0 2c d western blot analysis also showed a similar decreased in bcl2 and increased expressions of active caspase and bax compared with the untreated cells fig a02e f additional file a03e flevobupivacaine inhibits proliferation and a0promotes apoptosis in a0breast cancer through a0pi3kaktmtor signalling pathwaywestern blot analysis showed a significant decreased in the expression of the nuclear localization of ppi3k pakt and pmtor compared with the untreated cells fig a03a b additional file a03a bdiscussionbreast cancer remains a common cause of mortality among women worldwide though current orthodox drugs have demonstrated promise in breast cancer therapy its treatment options remain limited these therefore supports the concept that effective therapeutic approaches for breast cancer are critically needed several retrospective studies have demonstrated that regional anaesthesia is associated with a decreased risk of recurrence or metastasis of multiple carcinomas including breast prostate and cervical cancers [] recent growing evidence demonstrates that local anaesthetics have an antitumour effect and may suppress the motility of cellular function and invasiveness more likely via voltagegated sodium channel inhibition a study report indicates that lidocaine inhibits the growth of human hepatocellular carcinoma cells hcc by increasing the caspase activity whereas ropivacaine inhibits the growth of hcc cells by stopping the cell cycle in g2 phase lee et a0al demonstrated that local anaesthetics potentiate tnfα mediated apoptosis in hk2 cells the cellular modification of treated cells is likely dependent on the duration of exposure and the dose of the local see figure on next pagefig levobupivacaine inhibits proliferation in breast cancer cells mcf and mdamb cells were treated with different concentrations of levobupivacaine a cell viability was measured by cck assay ic50 results of levobupivacaine on mcf and mdamb cells b c colony formation of mcf and mdamb cells treated with various concentrations of levobupivacaine and stained with crystal violet d e the mrna expression levels of p21 and gapdh were analysed by qpcr f g protein expression assessment of mcf and mdamb cells by western blot against antibodies foxo1 p21 cyclin d1 and gapdh used as control the data was statistically significant at indicates p indicates p indicates p compared with untreated cells this data corresponds to the mean ± sem of three independent experiments 0ckwakye a0et a0al bmc res notes page of 0ckwakye a0et a0al bmc res notes page of fig effects of levobupivacaine on apoptosis of breast cancer cells a b mcf and mdamb cells were treated with different concentrations of levobupivacaine for h the cells were then stained with fluoresceinconjugated annexin v and pi and analysed by flow cytometry error bars represent standard error of the mean p versus the control c d relative gene expression of bax and bcl following the treatment of breast cancer cells with different concentrations of levobupivacaine for h and analysed by qpcr e f mcf and mdamb cells were treated with different concentrations of levobupivacaine for h and the activities of bax bcl and active caspase were examined by western blot analysis using specific antibodies gapdh was used as internal controls the data was statistically significant at indicates p indicates p compared with control the data correspond to the mean ± sem of three independent experiments 0ckwakye a0et a0al bmc res notes page of fig mcf and mdamb cells were treated with different concentrations of levobupivacaine for h a b the cells were lysed and subjected to sdspage and analysed by western blotting and probed with specific antibodies ppi3k pakt and pmtor the results showed a decrease in the expressions of ppi3k pakt and pmtor proteins gapdh was used as internal controls the data represent the mean ± sd of three independent experimentsanaesthetic [] in this study we employed mcf and mdamb231 cells as models and found that different concentrations of levobupivacaine could effectively inhibit breast cancer cell proliferation and promote apoptosis in a0vitro the antiproliferation and apoptosis effects observed in this study suggest that levobupivacaine may have therapeutic effects on breast cancerpi3kaktmtor signalling pathway plays a vital role in cell proliferation survival development metabolism motility and regulation of the immune response breast cancer cell resistance to therapies can result from the activation of pi3kaktmtor signalling pathway [] this has made the pi3kaktmtor signalling pathway an important object of study for understanding the development and progression of breast cancer in patients with breast cancer pi3kaktmtor signalling pathway can be a target for diagnostic prognostic and treatment purposes [] akt plays a role in the activation and inactivation of many transcription factors activation of akt correlated with the activation of mtor phosphorylation of the foxo proteins by akt may results in cytoplasmic retention by interacting with other proteins thereby isolating them from their targeted genes cyclin d1 classified as a pro oncogene is often overexpressed in several human malignancies including breast colon lung and prostate cancers reports show that overexpression of cyclin d1 and underexpression of tumour suppressor p21 is required for cancer initiation as it is confirmed that downregulation of cyclin d1 and overexpression of p21 in xenograft model discontinues the formation of cancer in the early stages datta et a0al reported that akt can phosphorylate the proapoptotic bcl2 family member bad causing its isolation from the mitochondrial membrane by other proteins local anaesthetics modify the protein levels of key members of the bcl2 family in a manner that presents an increase in the ratio of baxbcl2 which may contribute to the response of cancer cells to apoptosis in the present study the role of levobupivacaine on the expression of pi3k akt and mtor was investigated to illustrate the potential molecular mechanism we observed a significantly decreased expression of pakt ppi3k pmtor and subsequent decreased expression of foxo cyclin d1 and bcl2 following levobupivacaine treatment which correlated with decreased breast cancer cells proliferation and increased apoptosis these emerging pieces of evidence suggest that levobupivacaine may inhibit proliferation and promote apoptosis by suppressing pi3kaktmtor signalling pathway which demonstrated an antitumour effect on breast cancer cells in this studyconclusionlevobupivacaine has the potency of reducing breast cancer cell viability proliferation and also causes cell death by suppressing the pi3kaktmtor signalling pathway these findings could lead to clinical studies which will seek to examine the anticancer effects of levobupivacaine and may also increase the benefits in cancer patient as well as improve patient care 0ckwakye a0et a0al bmc res notes page of limitationsnumerous studies have reported on the antitumour effects of local anaesthetics on various cancer cells [] however our work is not without limitations in a0vivo and clinical studies on the antitumour effects of levobupivacaine are neededbiology dalian medical university dalian china department of anaesthesia and critical care school of medicine university of health and allied sciences ho ghana department of biochemistry and molecular medicine school of medicine and health sciences university for development studies tamale ghana departments of anaesthesia and critical care ridge hospital accra ghana department of medicine princefied university ho ghana received june accepted july supplementary informationsupplementary information accompanies this paper at https doi101186s1310 additional file a0 figure s1 levobupivacaine decreases breast cancer cell invasionadditional file a0 figure s2 effect of levobupivacaine on the morphology of mcf and mdamb cellsadditional file a0 original gelsblots scan used in fig 1f g fig 2e f and fig 3a b for mcf and mdamb cellsabbreviationsegfr epidermal growth factor receptor hcc hepatocellular carcinoma cells nc nitrocellulose pi propidium iodide ps phosphatidylserine qpcr quantitativepolymerase chain reactionacknowledgementswe thank the first affiliated hospital and the department of biochemistry of dalian medical university for making available all the necessary materials needed for this work we also thank the key laboratory of liaoning provincial education department grant no lz2016002 and liaoning natural science foundation grant no of china for supporting this work our thanks also go to the china scholarship council and the government of the republic of ghana for giving financial aid to some of the authors to study at dalian medical universityauthors contributionsakk sk qy and qpw conceived and designed this study qpw and qy were responsible for the supervision and coordination of this study akk sk jl mnr qy and qpw conducted the data collections sk led the data analysis with inputs from akk qy and qpw akk and sk wrote the first draft of the manuscript and jl mnr sar aaf ja and ean contributed to revising and reviewing the manuscript all authors read and approved the final manuscriptfundingthis study was supported by the key laboratory of liaoning provincial education department grant no lz2016002 and liaoning natural science foundation grant no availability of data and materialsthe data used andor analysed in this study are available from the corresponding author upon reasonable requestethics approval and consent to participatethe ethical committee of the first affiliated hospital of dalian medical university approved the study protocol and because this study used breast cancer cells consent to participate was not applicable for the studyconsent for publicationsnot applicablecompeting interestsauthors declare that they have no competing interestsauthor details department of anaesthesiology dalian medical university dalian china department of anaesthesiology first affiliated hospital of dalian medical university dalian china department of biochemistry and molecular references american cancer society breast cancer facts and figures atlanta american cancer society american cancer society cancer facts and figures atlanta ameri can cancer society siegel r naishadham d jemal a cancer statistics ca cancer j clin chang yc hsu yc liu cl huang sy hu mc cheng sp local anaesthetics induce apoptosis in human thyroid cancer cells through the mitogenactivated protein kinase pathway plos one 20149e89563 gomezgutierrez jg souza v hao hy de montes ocaluna r dong yb zhou hs mcmasters km adenovirusmediated gene transfer of fkhrl1 triple mutant efficiently induces apoptosis in melanoma cells cancer biol ther sunters a de fern¡ndez mattos s stahl m brosens jj zoumpoulidou g saunders ca coffer pj medema rh coombes rc lam ew foxo3a transcriptional regulation of bim controls apoptosis in paclitaxeltreated breast cancer cell lines j biol chem fu z tindall dj foxos cancer and regulation of apoptosis oncogene barnes dm gillett ce cyclin d1 in breast cancer breast cancer res treat sherr cj roberts jm cdk inhibitors positive and negative regulators of g1phase progression genes dev pelengaris s khan m evan g cmyc more than just a matter of life and death nat rev cancer di padova m barbieri r fanciulli m arcuri e florida a effect of local anaesthetic ropivacaine on the energy metabolism of ehrlich ascites tumour cells oncol res 199810491e8 xing w chen dt pan jh chen yh yan y li q xue rf yuan yf zeng wa lidocaine induces apoptosis and suppresses tumour growth in human hepatocellular carcinoma cells in vitro and in a xenograft model in vivo anesthesiology drasner k lidocaine spinal anaesthesia a vanishing therapeutic index anesthesiology wang hw wang ly jiang l tian sm zhong td fang xm amidelinked local anaesthetics induce apoptosis in human nonsmallcell lung cancer j thorac dis https doi1021037 jtd20160966 piegeler t vottavelis eg liu g place at schwartz de beckschimmer b minshall rd beat a antimetastatic potential of amidelinked local anaesthetics inhibition of lung adenocarcinoma cell migration and inflammatory src signalling independent of sodium channel blockade anesthesiology lirk p berger r hollmann mw feigl h lidocaine time and dosedependently demethylates deoxyribonucleic acid in breast cancer cell lines in vitro br j anaesth villargarea a fraga mf espada j esteller m procaine is a dnademethylating agent with growthinhibitory effects in human cancer cells cancer res 4984e634984e9 kampo s ahmmed b zhou t owusu l anabah tw doudou nr kuugbee ed cui y lu z yan q wen qp scorpion venom analgesic peptide bmk agap inhibits stemness and epithelialmesenchymal transition by downregulating ptx3 in breast cancer front oncol hirata m sakaguchi m mochida c sotozono c kageyama k yoshihiro k munetaka h lidocaine inhibits the tyrosine kinase activity of the epidermal growth factor receptor and suppresses proliferation of corneal epithelial cells anesthesiology 0ckwakye a0et a0al bmc res notes page of grouselle m tueux o dabadie p geescaud d mazat jp effect of local anaesthetics on mitochondrial membrane potential in living cells biochem j fraser sp diss jkj chioni am mycielska me pan h yamaci rf pani f siwy z krasowska m grzywna z brackenbury wj theodorou d koyuturk m kaya h battaloglu e de tamburo bella m slade mj tolhurst r palmieri c jiang j latchman ds coombes rc djamgoz mba voltagegated sodium channel expression and potentiation of human breast cancer metastasis clin cancer res le gac g angenard g clement b laviolle b coulouarn c beloeil h local anaesthetics inhibit the growth of human hepatocellular carcinoma cells anesth analg lee ht xu h siegel cd krichevsky ie local anaesthetics induce human renal cell apoptosis am j nephrol unami a shinohara y ichikawa t baba y biochemical and microarray analyses of bupivacaineinduced apoptosis j toxicol sci villargarea a fraga mf espada j esteller m procaine is a dnademethylating agent with growthinhibitory effects in human cancer cells cancer res sakaguchi m kuroda y hirose m the antiproliferative effect of lidocaine on human tongue cancer cells with inhibition of the activity of epidermal growth factor receptor anesth analg chang yc liu cl chen mj hsu yw chen sn lin ch chen cm yang fm hu mc local anaesthetics induced apoptosis in human breast tumour cells anesth analg kawasaki c kawasaki t ogata m sata t chaudry ih lidocaine enhances apoptosis and suppresses mitochondrial functions of human neutrophil in vitro j trauma hodgkin al huxley afa quantitative description of membrane current and its application to conduction and excitation in nerve j physiol besson p driffort v bon © gradek f chevalier s roger s how do voltagegated sodium channels enhance migration and invasiveness in cancer cells biochim biophys acta roger s gillet l le guennec jy besson p voltagegated sodium channels and cancer is excitability their primary role front pharmacol roger s potier m vandier c besson p le guennec jy voltagegated sodium channels new targets in cancer therapy curr pharm des driffort v gillet l bon e marionneaulambot s oullier t joulin v collin c pag¨s jc jourdan ml chevalier s bougnoux p le guennec jy besson p roger s ranolazine inhibits nav15mediated breast cancer cell invasiveness and lung colonization mol cancer nelson m yang m dowle aa thomas jr brackenbury wj the sodium channelblocking antiepileptic drug phenytoin inhibits breast tumour growth and metastasis mol cancer yuan t li z li x yu g wang n yang x lidocaine attenuates lipopolysaccharideinduced inflammatory responses in microglia j surg res brocco mc paulo dns almeida ced carraretto ar cabral sa silveira ac gomez rs baptista jf a study of interleukin il and tumour necrosis factoralpha tnfα serum levels in rats subjected to faecal peritonitis and treated with intraperitoneal ropivacaine acta cirurgica brasileira piegeler t schl¤pfer m dull ro schwartz de beat a minshall rd beckschimmer b clinically relevant concentrations of lidocaine and ropivacaine inhibit tnfαinduced invasion of lung adenocarcinoma cells in vitro by blocking the activation of akt and focal adhesion kinase br j anaesth shankar s chen q srivastava rk inhibition of pi3kakt and mekerk pathways act synergistically to enhance antiangiogenic effects of egcg through activation of foxo transcription factor j mol signal qian j zou y rahman jsm lu b massion pp synergy between phosphatidylinositol kinaseakt pathway and bclxl in the control of apoptosis in adenocarcinoma cells of the lung mol ortega ma frailemartınez o asunsolo a bujan j garcıahonduvilla n coca s signal transduction pathways in breast cancer the important role of pi3kaktmtor j oncol royds j khan ah buggy dj update on existing ongoing prospective trials evaluating the effect of anaesthetic and analgesic techniques during primary cancer surgery on cancer recurrence or metastasis int anesthesiol clin 2016544e76 burgering bmt medema rh decision on life and death foxo forkhead transcription factors are in command when pkbakt is off duty j leukoc biol chen q ganapathy s singh kp shankar s srivastava rk resveratrol induces growth arrest and apoptosis through activation of foxo transcription factors in prostate cancer cells plos one 20105e15288 arnold a papanikolaou a cyclin d1 in breast cancer pathogenesis j clin oncol santarius t shipley j brewer d stratton mr cooper cs a census of amplified and overexpressed human cancer genes nat rev cancer datta sr brunet a greenberg me cellular survival a play in three akts genes daev lirk p hollmann mw fleischer m weber nc feigl h lidocaine and ropivacaine but not bupivacaine demethylate deoxyribonucleic acid in breast cancer cells in vitro br j anaesth 2014113suppl 1i32i3838 li k yang j han x lidocaine sensitizes the cytotoxicity of cisplatin in breast cancer cells via the upregulation of rarβ2 and rassf1a demethylation int j mol sci publishers notespringer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your ï¬eld¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold open access which fosters wider collaboration and increased citations maximum visibility for your research over 100m website views per year ¢ at bmc research is always in progresslearn more biomedcentralcomsubmissionsready to submit your research choose bmc and benefit from 0c' | Colon_Cancer |
objectives paired box protein8 pax8 immunohistochemical expression can be used as a diagnostic marker for epithelial cells tumors this study aimed at investigating the immunohistochemical expression of pax8 among sudanese females diagnosed with cervical endometrial and ovarian cancers between december and may by studying their formalinfixed paraffin embedded blocksresults sixty patients diagnosed with female reproductive tract cancers were included who aged ± years range cervix was the most common cancer site in patients regarding cancer stage there was and of the study population had stage 3b and 2b respectively the histopathological diagnosis included and poorly moderately and well differentiated cervical squamous cell carcinoma scc as well as and endometrial adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma respectively pax8 was positively expressed in endometrial adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma poorly and moderately differentiated scc all patients diagnosed with well differentiated scc and metastatic adenocarcinoma showed no expression of pax8 a statistically significant was seen for pax8 expression and the different histopathological diagnosis p value keywords female reproductive cancer paired box protein8 immunohistochemical expressionintroductionpaired box protein8 pax8 is a member of the family paired box proteins paxs [ ] pax8 consists of amino acids with a molecular weight of approximately kilo dalton and its molecular properties are located on chromosome 2q13 [] pax8 is a transcription factor that regulates ans development during the embryonic period as well as to maintain normal cellular functions in some cells after birth [ ] during the embryonic period pax8 also plays a significant role correspondence nouh_saadoutlookcom alfarrabi college for science and technology khartoum sudanfull list of author information is available at the end of the in the development of genital ans derived from the mesonephric and the m¼llerian ducts [] in a previous experiment the deletion of the pax8 gene resulted in dysfunctional uterus absence of the endometrium and the vaginal opening also resulted in poor development of the myometrial tissue several studies have described the immunohistochemical utility of pax8 as a diagnostic marker for epithelial cells neoplasms of many glands and ans such as thyroid thymus and kidney as well as some female reproductive tract tumors [ ]in a healthy female reproductive tract pax8 shown to be overexpressed in the epithelial cells of the endocervix and the endometrium [] pax8 was found to be expressed among endometrioid carcinomas transitional the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cali a0et a0al bmc res notes page of undifferentiated cell carcinomas and the metastatic carcinomas at a range of and [ ] whereas for the ovarian carcinomas pax8 was under expressed considering that few studies have investigated the immunohistochemical expression of pax8 in carcinomas of the endometrium and uterine cervix in the different parts of the world but none from sudan yet [ ] this study aimed at investigating the immunohistochemical expression of pax8 among sudanese females diagnosed with cervical endometrial and ovarian carcinomasmain textmaterials and a0methodsstudy design and a0population characteristicsthis is a descriptive retrospective hospital based study conducted at different histopathology laboratories during the period from december till may in khartoum state sudan we retrieved archived formalin fixed paraffin embedded blocks previously collected from female patients with cervical endometrial or ovarian carcinomas the retrieved formalin fixed paraffin blocks represent all the female population admitted at the hospitals for reproductive malignancies diagnosis the participants demographic data was collected including age place of residence the clinical data including site of cancer cancer grade and the histopathological diagnosis were also collectedsections preparation for a0immunohistochemistry stainingtwo sections were cut using rotary microtome leica germany from each histopathological block then one slide was stained by hematoxylin and eosin staining technique the other slide was mounted onto 3aminopropyltriethoxysilane coated slides for immunohistochemistry to retrieve pax8 tissues antigen we treated the sections with citrate buffer at ° a0c for a0min in a waterbath then the tissue sections were rinsed first in distilled water and later with tris buffer saline tbs this was followed by treatment with peroxidase block hydrogen peroxide in methyl alcohol for a0min to quench endogenous peroxidase activity the slides were then placed in a humid chamber then the slides were drained and rinsed in two successive changes of tris buffer wash buffer for a0 min each nonspecific proteinprotein interactions were blocked by incubating and treating the tissue sections in a humid chamber with the power block casein in phosphate buffered saline for a0 min then the remaining solution was drained from the slides the sections were then incubated in the primary antibody pax8 antipax8 rabbit antihuman monoclonal antibody ab189249 abcam united kingdom at room temperature in the humid chamber according to the manufacture instructionsobserving the yellowishbrown or brown appearance of the nucleus was considered a positive result for the pax8 for the negative control we omitted the incubation with the primary antibody step instead we incubated the section in the phosphate buffer saline pbsresults interpretationsfor the interpretation of the results we depended on the intensity as well as the number of the cells that expressed the marker and the expression was graded into categories negative no staining less than of the cells were expressing the marker of the cells were expressing the marker more than of the cells were expressing the marker more than of the cells were expressing the marker the slides were interpreted and validated by two expert pathologists blindly of each other results photomicrographs were taken using olympus sp350 camera olympus imaging america inc usastatistical analysisthe statistical analysis of the results was done using ibm spss statistics vs the chisquared test was performed to compare the frequencies of categorical variables statistical significance level was defined as p value at confidence intervalresultscharacteristics of a0the a0study participantsthe study included patients diagnosed with female genital tract cancer patients aged ± a0years range a0years patients were grouped into age groups those aged a0 years constituted half of the study participants the remaining were and patients distributed across the remaining age groups of a0 years a0 years and a0 years respectively according to patients place of residence patients were originating from the four regions of sudan most of the patients were from western part of sudan followed by from the central part of sudanregarding the site of cancer the cervix was the most commonly involved patients there were and endometrial and ovarian cancer respectively based on the international federation of gynecology and obstetrics figo cancer grading the majority of the study population was diagnosed with stage 3b and 2b cancer and of the patients respectively the were and stage 4b 3a 2a 1b and 4a respectively 0cali a0et a0al bmc res notes page of no statistically significant association between figo staging and age group was found p value histologically there were squamous cell carcinoma scc all of which were cervical cancers and adenocarcinoma scc and adenocarcinoma were further classified into poorly differentiated scc moderately differentiated scc and well differentiated scc endometrium adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinomabased on age groups age group showed no statistically significant relationship with either patients place of residence cancer site cancer histological type figo staging and cancer histopathological type table a0immunohistochemical expression of a0paxthe immunohistochemical expression of pax8 was shown as a yellowishbrown or brown staining of the nucleus fig a0 based on site of cancer all endometrium carcinoma showed positive expression of pax8 with p value there were only patients who had positive expression of pax8 including adenocarcinoma and scc a statistically significant difference was noted for the pax8 staining and cancer type with p value the analysis of pax8 staining results based on the histopathological diagnosis showed that all patients who were diagnosed with well differentiated scc and metastatic adenocarcinoma had negative results for the pax8 expression while of the endometrium adenocarcinoma were found positive for the pax8 expression a statistically significan was t seen for pax8 expression and the different histopathological diagnosis p value table a0table classification of a0participants demographic and a0clinical diagnosis based on a0age groupage group no total no p value a0years a0years a0years a0yearsresidence of patient central sudan east sudan west sudan north sudansite of cancer cervix endometrium ovarycancer histological type scc adenocarcinomafigo staging stage stage 2a stage 2b stage 3a stage 3b stage 4a stage 4bhistopathological cancer grades well differentiated scc poorly differentiated scc moderately differentiated scc endometrium adenocarcinoma endocervical adenocarcinoma metastatic adenocarcinoma ovarian mucinous cyst adenocarcinomascc squamous cell carcinoma 0cali a0et a0al bmc res notes page of fig immunohistochemical expression of pax8 among the different histopathological cancer types and grades the immunohistochemical expression of pax8 is shown as a yellowishbrown or brown staining of the nucleus a well differentiated scc negative b metastatic adenocarcinoma negative c poorly differentiated scc positive d moderately differentiated scc positive e endometrium adenocarcinoma positive f ovarian mucinous cystadenocarcinoma positive g endocervical adenocarcinoma positive and h endometroid adenocarcinoma positive 0cali a0et a0al bmc res notes page of table association of a0clinical diagnosis and a0the a0immunohistochemical expression of a0pax8pax results no total no p valuepositivenegativecancer histological type scc adenocarcinomacancer site cervix endometrium ovaryfigo staging stage stage 2a stage 2b stage 3a stage 3b stage 4a stage 4bcancer histopathological grading well differentiated scc poorly differentiated scc moderately differentiated scc endometrium adenocarcinoma endocervical adenocarcinoma metastatic adenocarcinoma ovarian mucinous cyst adenocarcinomascc squamous cell carcinoma discussionprevious studies on the immunohistochemical expression of pax8 in the normal female reproductive tract showed that pax8 was expressed in the endometrial endocervical and ovarian epithelial cells as well as in nonciliated epithelial cells of the fallopian tubes [ ] this study investigated the immunohistochemical expression of pax8 in sudanese patients who were diagnosed with female reproductive tract cancers patients on the 5th decade of life were constituting half of the study participants with no statistically significant association between age group and the type of cancer however previous studies had suggested other risk factors which could contribute in the development of certain gynecological cancer [ ]regarding the place of residence the majority of patients coming from western sudan this result is in contrary with a previous study in sudan conducted by saeed et a0al in which they showed that the percentage of patients suffering from different types of cancers residing in central and northern sudan were higher compared to the other regions in sudan nevertheless these findings could suggest the involvement of environmental risk factors however the limited study samples size is insufficient to support this suggestion therefore further research with a larger samples size investigating the potential environmental risk factors is essential for strategic prevention and protection measuresthe reported number of female patients with cervical cancer was high compared to ovarian and endometrium cancer similar results were seen previously among sudanese females also the high frequency of stages 3b and 2b compared to the other stages were comparable to previous study conducted in sudan this similarity underscores a delayed response among sudanese females in seeking healthcare and urge the need for health promotion and education to encourage young sudanese females for the early signs detection and seeking healthcare as early as possible for a better treatmentregarding the classification based on the histopathological diagnosis most of the female diagnosed with scc this result was also similar to previous study investigated the prevalence of the different gynecologic cancer in sudan however the expression of pax8 among the studied samples was relatively low compared to previous studies [ ] this could be attributed 0cali a0et a0al bmc res notes page of to the site of cancer development while agrees with another study where pax8 was expressed only in patient interestingly a high frequency of pax8 expression was noted among females diagnosed with endometrium cancer compared to scc this finding is in contrary with a previous report where pax8 was expressed among only of the studied samples also the result was strongly in accordance with other studies [ ] besides that the lack of pax8 expression among those who were diagnosed with well differentiated scc and metastatic adenocarcinoma could play a significant role in either gynecologic cancer differentiation or in detection of endometrium adenocarcinoma progression to metastatic adenocarcinoma [ ]conclusionalthough pax8 showed a significant expression among adenocarcinomas lesions and negative expression was noted among those with well differentiated scc and metastatic adenocarcinoma pax8 might not be beneficial when used alone as a diagnostic marker for the tumors that occur in the female reproductive tractlimitations¢ the small sample size investigated in this study reduced the ability of using the expression of pax8 as a diagnostic marker therefore a largescale study is needed and it should include other types of malignant tumors encountered in the female reproductive systemacknowledgementsthe authors would like to acknowledge the medical staff for their interest and cooperation during the study and thanks to all who participated in completing this studyauthors contributionseta nsm and ees provided conceptual framework for the study guidance for interpretation of the data and performed data analysis eta ees irs lah and amm performed laboratory work nsm ees msm aay and aa performed the statistical analysis nsm msm ees and aa participated in the manuscript preparation revision and coordination all authors read and approved the final manuscriptfundingnot applicableavailability of data and materialsthe datasets used andor analyzed during the current study are available from the corresponding author on reasonable requestethics approval and consent to participateethical approval was obtained from the research ethics committee of the faculty of medical laboratory sciences university of khartoum sudan ethical approval no fmlsrec002042 all participant approved to participate by signing an informed consentconsent for publicationnot applicablecompeting interestsno competing interests to discloseauthor details department of histopathology and cytology faculty of medical laboratory sciences university of khartoum khartoum sudan department of histopathology and cytology faculty of medical laboratory sciences national university khartoum sudan alfarrabi college for science and technology khartoum sudan faculty of medicine sinnar university sennar sudan molecular biology department faculty of medical laboratory sciences nile university khartoum sudan faculty of dentistry ibn sina university khartoum sudan department of neurology mayo clinic jacksonville fl usa department of radiology mayo clinic jacksonville fl usa institute of endemic diseases university of khartoum khartoum sudan mycetoma research center university of khartoum khartoum sudan faculty of medicine nile university khartoum sudan received july accepted august references gruss p walther c pax in development cell mansouri a hallonet m gruss p pax genes and their roles in cell differentiation and development curr opin cell biol macchia pe lapi p krude h pirro mt missero c chiovato l souabni a baserga m tassi v pinchera a pax8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis nat genet vilain c rydlewski c duprez l heinrichs c abramowicz m malvaux p renneboog bt parma j costagliola s vassart g autosomal dominant transmission of congenital thyroid hypoplasia due to lossoffunction mutation of pax8 j clin endocrinol metab park s vk c genetics of congenital hypothyroidism j med genet dahl e koseki h balling r pax genes and anogenesis bioessays lang d powell sk plummer rs young kp ruggeri ba pax genes roles in development pathophysiology and cancer biochem pharmacol stoykova a gruss p roles of paxgenes in developing and adult brain as suggested by expression patterns j neurosci mittag j winterhager e bauer k grummer r congenital hypothyroid female pax8deficient mice are infertile despite thyroid hormone replacement therapy endocrinology bouchard m de caprona d busslinger m xu p fritzsch b pax2 and pax8 cooperate in mouse inner ear morphogenesis and innervation bmc dev biol mittag j winterhager e bauer k grummer rje congenital hypothyroid female pax8deficient mice are infertile despite thyroid hormone replacement therapy endocrinolog laury ar perets r piao h krane jf barletta ja french c chirieac lr lis r loda m hornick jl a comprehensive analysis of pax8 expression in human epithelial tumors am j surg pathol wong s hong w hui p buza n comprehensive analysis of pax8 expression in epithelial malignancies of the uterine cervix int j gynecol pathol ozcan a shen ss hamilton c anjana k coffey d krishnan b truong ld pax expression in nonneoplastic tissues primary tumors and metastatic tumors a comprehensive immunohistochemical study mod pathol bowen nj logani s dickerson eb kapa lb akhtar m benigno bb mcdonald jf emerging roles for pax8 in ovarian cancer and endosalpingeal development gynecol oncol 0cali a0et a0al bmc res notes page of ozcan a liles n coffey d shen ss truong ld pax2 and pax8 expression in primary and metastatic m¼llerian epithelial tumors a comprehensive comparison am j surg pathol distinguishing ovarian mucinous neoplasms from colonic and appendiceal mucinous neoplasm bmc res notes nesrin r kilic d risk factors for cervical cancer results from a hospital ozcan a liles n coffey d shen ss truong ldjtajosp pax2 and pax8 based casecontrol study int j hematol oncol expression in primary and metastatic m¼llerian epithelial tumors a comprehensive comparison am j surg pathol nonaka d tang y chiriboga l rivera m ghossein r diagnostic utility of thyroid transcription factors pax8 and ttf2 foxe1 in thyroid epithelial neoplasms mod pathol tacha d zhou d cheng l expression of pax8 in normal and neoplastic tissues a comprehensive immunohistochemical study appl immunohistochem mol morphol bowen nj logani s dickerson eb kapa lb akhtar m benigno bb mcdonald jfjgo emerging roles for pax8 in ovarian cancer and endosalpingeal development gynecol oncol k¶bel m kalloger se boyd n mckinney s mehl e palmer c leung s bowen nj ionescu dn rajput a ovarian carcinoma subtypes are different diseases implications for biomarker studies plos medicine 2008512e232 nonaka d chiriboga l soslow ra expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas am j surg pathol tong gx devaraj k hamelebena d yu wm turk a chen x wright jd greenebaum e pax8 a marker for carcinoma of m¼llerian origin in serous effusions diagn cytopathol laury ar perets r piao h krane jf barletta ja french c chirieac lr lis r loda m hornick jljtajosp a comprehensive analysis of pax8 expression in human epithelial tumors am j surg pathol tong gx devaraj k hamelebena d yu wm turk a chen x wright jd greenebaum ejdc pax8 a marker for carcinoma of m¼llerian origin in serous effusions diagn cytopathol chu pg chung l weiss lm lau sk determining the site of origin of mucinous adenocarcinoma an immunohistochemical study of cases am j surg pathol brunner ah riss p heinze g meltzow e brustmann h immunoexpression of pax in endometrial cancer relation to highgrade carcinoma and p53 int j gynecol pathol ozcan a shen ss hamilton c anjana k coffey d krishnan b truong ldjmp pax expression in nonneoplastic tissues primary tumors and metastatic tumors a comprehensive immunohistochemical study mod pathol aldaoud n erashdi m alkhatib s abdo n almohtaseb a graboskibauer a the utility of pax8 and satb2 immunohistochemical stains in saeed me cao j fadul b kadioglu o khalid he yassin z mustafa sm saeed e efferth t a fiveyear survey of cancer prevalence in sudan anticancer res saeed me cao j fadul b kadioglu o khalid he yassin z mustafa sm saeed e efferth tjar a fiveyear survey of cancer prevalence in sudan anticancer res mohamed keh ashmeig aaa cervical cancer our experience in sudan philadelphia aacr elhasan lme bansal d osman of enan k abd farag eab prevalence of human papillomavirus type in sudanese women diagnosed with cervical carcinoma j cancer res ther tacha d zhou d cheng ljai morphology m expression of pax8 in normal and neoplastic tissues a comprehensive immunohistochemical study appl immunohistochem mol morphol ord³±ez ng value of pax immunostaining in tumor diagnosis a review and update adv anat pathol gailey mp bellizzi am immunohistochemistry for the novel markers glypican pax8 and p40 δnp63 in squamous cell and urothelial carcinoma am j clin pathol yemelyanova a gown am holmes bj ronnett bm vang r pax8 expression in uterine adenocarcinomas and mesonephric proliferations int j gynecol pathol liang l zheng w liu j liang sx assessment of the utility of pax8 immunohistochemical stain in diagnosing endocervical glandular lesions arch pathol lab med wong s hong w hui p buza njijogp comprehensive analysis of pax8 expression in epithelial malignancies of the uterine cervix int j gynecol pathol de andrade dap da silva vd de macedo mg de lima ma de andrade vm andrade cemc schmidt rl reis rm dos reis r squamous differentiation portends poor prognosis in low and intermediaterisk endometrioid endometrial cancer plos one 20191410e0220086publishers notespringer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations ¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your ï¬eld¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold open access which fosters wider collaboration and increased citations maximum visibility for your research over 100m website views per year ¢ at bmc research is always in progresslearn more biomedcentralcomsubmissionsready to submit your research choose bmc and benefit from 0c' | Colon_Cancer |
" 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 |
" curcumin is herbal compound that has been shown to have anticancer effects in preclinical andclinical studies the anticancer effects of curcumin include inhibiting the carcinogenesis inhibiting angiogenesisand inhibiting tumour growth this study aims to determine the clinical effects of curcumin in different types ofcancers using systematic review approachmethods a systematic review methodology is adopted for undertaking detailed analysis of the effects of curcuminin cancer therapy the results presented in this paper is an outcome of extracting the findings of the studiesselected from the s published in international databases including sid magiran iranmedex irandoc googlescholar sciencedirect scopus pubmed and web of science isi these databases were thoroughly searched andthe relevant publications were selected based on the plausible keywords in accordance with the study aims asfollows prevalence curcumin clinical features cancerresults the results are derived based on several clinical studies on curcumin consumption with chemotherapydrugs highlighting that curcumin increases the effectiveness of chemotherapy and radiotherapy which results inimproving patients survival time and increasing the expression of antimetastatic proteins along with reducingtheir side effects the comprehensive systematic review presented in this paper confirms that curcumin reduces the sideeffects of chemotherapy or radiotherapy resulting in improving patients quality of life a number of studiesreported that curcumin has increased patient survival time and decreased tumor markers levelkeywords prevalence curcumin clinical feature cancer systematic review research over the past years has significantly increased our understanding of the molecular genetic basisof cancer it is now well known that cancer is caused bya set of molecular genetic changes that lead to loss ofgrowth control and cellular differentiation resulting in correspondence nsalarikumsacir masoudmohammadi1989yahoocom5department of biostatistics school of health kermanshah university ofmedical sciences kermanshah iran7department of nursing school of nursing and midwifery kermanshahuniversity of medical sciences kermanshah iranfull list of author information is available at the end of the uncontrollable cell growth that eventually leads to tumorformation more than half of all cancers occur in developingcountries including those located in southern americaand asia nearly threequarters of people of these countries are classified into low or middleincome categoriesthe cancer survival rates in developing countries aregenerally onethird ofthe patients in the developedcountries there are million new cases of cancereach year with million new cancer cases in the developed countries and million in developing countries in the next decades cancer will be one of the leading the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cmansouri bmc cancer page of causes ofillness worldwide and the number of newcases of various types of cancer is expected to rise to million by furthermore cancer is predicted to bethe leading cause of death by given that cancerstatistics are on the rise and their treatments are costlyit is very crucial to find effective and economically viablemethods for patients in low and middle income countries therefore this study is motivated by using effectiveand relatively cheap treatments for cancer therapy asystematic review of the clinical studies on curcumin useand its effectiveness in inhabiting and treating varioustypes of cancer is carried out to obtain comprehensiveinformation aboutthe curcumin effects on cancertherefore a structured review of all published sand other relevant documents on the use of curcuminfor cancer therapy creates a more complete picture ofcurcumin effects on cancer patients from different angles in the process of this review only evidence from thestudies with highest quality are selected to gather information and derive on curcumin effects andeffectiveness at various stages of cancer therapyamong the medical herbs flavonoids are a large subgroup of the family of natural polyphenolic compoundsthat are the result of secondary metabolism in plants in recent years research has shown that flavonoids havebeen very effective in the prevention and control of common diseases complex such as cancer cardiovasculardiseases alzheimers stroke diabetes osteoporosisand rheumatoid arthritis furthermore there are robustevidence of antiviral antiinflammatory and antiallergic effects of flavonoidsin the recent years the use and effectiveness of medicinal herbs in treatment of various diseases has been received enormous attention huge research efforts weremade on extraction and examination of the properties ofthe herbal compounds in the treatment of different typesof diseases eg cancers and providing detailed mechanisms of drug performance of these compounds amongst the wide range of the medical herbs curcuminis an effective ingredient of turmeric plant with the scientific name of longa curcuma chemical name ofdiferuloylmethane and the chemical formula of c21h20 o6 as illustrated in fig curcumin makes up between to of turmericcompounds and is considered as the main cause of yellowgolden colour of turmeric and it has also been identified as responsible for many ofthe properties ofturmeric [ ] however curcumin has low inherenttoxicity and various properties with great impact and applications on a wide range of pharmacological developmentsantiinflammatoryantimicrobial and anticancer drugs []antioxidantincludingcurcumin has been shown to have preventive and therapeutic effects on various types of cancers the findingsfrom several studies suggest that curcumin compound canprevent the formation and spread of tumors or reduce theirsize it was shown that curcumin can inhibit the formationof cancer and spread the cancerous cells by exerting antiangiogenic effects inducing apoptosis and interfering withthe cell proliferation cycle [ ] curcumin exerts itsanticancer effects through a variety of mechanisms curcumin inhibits and suppresses the proliferation of a widerange of cancer cells which exerts its effects by reducingthe modulation of antiapoptotic gene products activatingcaspase and upregulating cancersuppressive genes such asp53 [] recent studies confirm the preventive andtherapeutic effects of curcumin on various types of cancersindicating that it can prevent or reduce the formation orspread of tumors curcumin inhibits tumor invasion by reducing the modification of matrix metalloproteasesmmps the cell surface adhesive molecules nfκ ap1tnfα lox and cox2 chemokines growth factorsher2 and egfr inhibits nterminal activity and tyrosine kinase protein [ ] curcumin inhibits angiogenesisin some tumors by suppressing angiogeniccytokines such as il6 il23 and il1 [] due tothe strong relationship between inflammation and cancerthe antiinflammatory effects of curcumin would well resultin its antitumor effects it was reported that curcumin hasprevented the development of several types of cancer by reducing the production of mediators of the inflammatoryprocess such as cox2 lipoxygenase inos and relatedcytokines one of the possible mechanisms for suppressing tumor proliferation is the chemical inhibitor effectof curcumin as a result topical use of curcumin considerably inhibits inflammation due to tetradecanoylphorbol13fig the chemical expansion of curcumin by coreldraw graphics suite 0cmansouri bmc cancer page of acetate 12o tpa hyperplasia cell proliferation odcactivity production of active oxygen species oxidativedna changes and papillomavirus formation [] multiple human gastrointestinal cell interactions with curcumininhibit lipid peroxidation inhibit cox2 expression inhibitpge2 production and increase glutathionestransferaseenzyme levels [ ] the other mechanism of the anticancer effects of curcumin is due to its interference in thecell cycle and reduction in cdk expression cdks are actually serine threonine kinases that control cell cycle progression furthermore curcumin inhibits the stat3phosphorylation which is responsible for signalling carcinogenic pathways given that cancer statistics are on the rise and theirtreatments are quite costly it is very crucial to find someeffective methods and economically viable for low andmiddleincome patients therefore this paper providesupto date evidence and findings of clinical studies onthe effects of curcumin contributions in tumor cells survival and metastasis using a systematic review approachmethodssystematic review approach is adopted for undertakingthis study by extracting the findings of the relevant studies selected from the s published in national andinternational databases including sid magiran iranmedex irandoc google scholar sciencedirect scopuspubmed and web of science isi these databases werethoroughly searched and the relevant publication records were selected based on the plausible keywords inaccordance with the aim of this study as follows prevalence curcumin clinical features cancerthe selection of relevant studies for the systematic review and the output quality control process involvedseveral steps first all related s were collectedbased on the search keywords mentioned in the nextstep the specifications including the name of thejournal and authors were hidden and the full text of thes were made available to the reviewers each was investigated independently by two reviewersmm shr and if an was excluded in the studyfig the flowchart on the stages of including the studies in the systematic review prisma 0cmansouri bmc cancer page of detailed rationale were give accordingly in the case ofdisagreement between the two reviewers the wasjudged by a third reviewer in this paper all studies related to clinical investigations of curcumin use and impacts at varioustreatment weresystematically examined without any time constraintsand according to prisma guidelines fig stages of cancer selection criterias with the following characteristics were selectedfor metaanalysis original research s clinical trialstudies s that their full text and data are availableand studies that examined the clinical effects of curcumin in various types of cancers we prepared a list of s specifications based on prisma includingthe researchers name the title the year and placeof the study sample size and number of patients duration of study dosage of the drug and the result of theintervention table including review papers exclusion criteriastudiessystematic reviewmetaanalysis cohort casecontrol crosssectional descriptive and those which didnt present samples fromcancer patients and those which conducted with secondary data were excluded from the review duplicate publication and multiple publicationssamepopulation will be removed using citation managementsoftware endnote version x7 for windows thomsonreutersfrom thequality assessmentthe quality of the selected s was evaluated basedon criteria outlined by the consort checklist the lastconsort statement published in included items the consort statement has been shown to improve the scientific quality of rct reporting [ ]each was blindly assessed by two independentevaluators mm shr the result of each item wasassessed by yes point or no point and some itemswere assessed as not applicable due to the features ofstudies accordingly the maximum quality score of was considered and papers with a score of less than were considered to have low quality and thus they wereexcluded from the studycurcumin role in the prevention of cancersfree radicals and toxic products resulted from oxidativestress play an important role in the early stages of cancerformation therefore compounds that have antioxidanteffects can be helpful in preventing cancer formationcurcumin has the property of trapping free radicals andthus can play a crucial role in inhibiting the onset ofcancer several cellular and preclinical studies havereported that curcumin inhibits dna damage caused byoxidative factors such as ionizing radiation by inhibitingfree radicals and active oxygen species the nfkappab plays an important role in the formation of nitricoxide synthase and oxidative stress and as a resultcauses cancer curcumin suppresses the onset ofcancer by inhibiting nfkappab from formation [ ]curcumin was reported to be effective on liver enzymescytochrome p450 which has an imminent role in theoxidation and detoxification of toxins it also inhibits thephase i enzymes that is involved in the production oftoxic metabolites and carcinogens furthermore curcumin activates the phase ii enzymes which plays a crucialrole in detoxification of toxic metabolites curcumin prevent tumor formation and growth by inhibitingand activating these two enzymes phase i ii effect of curcumin on metastasis angiogenesis andinflammation in cancer cellsangiogenesis is the process of new blood vessel formation from preexisting vessels that is dependent on aprice equilibrium between antiangiogenic and angiogenicfactors however under pathological conditions for example tumor growth this tight regulation becomes lostwhich can result in tumor metastasis many gene products that are produced by different cells have a role inangiogenesis process hypoxia usually occurs in tumorsites in order to overcome to hypoxia tumor cells regulate and control the expression of genes related to angiogenesis cell cycle metastasis and drug resistance usinghypoxiainducible factor hif1 hif1 was first recognized as a transcription factor involved in hypoxiainduced erythropoietin expression this factor has beenpresented as a main transcription regulator for thesemolecules [ ] several studies have shown thathif1 activation of genes including vascular endothelialvegf angiopoietin1 ang1 andgrowth factorangiopoietin2 ang2 nfkb etc induced angiogenesis in the tumor cells furthermore the activation ofgenes such as insulinlike growth factor igf2 transforming growth factor a tgfa and mapk and pi3ksignalling pathway will also enable the survival proliferation and metastasis of tumor cells hif1 by activating genes involved in angiogenesis and also activatessignalling pathways associated with cell survival and proliferation plays an important role in the stability andgrowth of tumors as above mentioned hif1α is apotent activator of angiogenesis and curcumin inhibitsits expression ap1 is a transcription factor that is activated in response to hypoxia which is the principlephysiological stimulus that induces angiogenesis it isalso involved in the conversion of epithelial cells to mesenchymal cells which is the primary stage of metastasisand causes the expression of mmp and upa urokinase 0cmansouri bmc cancer page of table examines the characteristics extracted in the studiesauthors name yearcountry duration ofdosage of the drughejazi2013 belcaro2014 iranitalystudy years g per daynumber ofpatientsresultscurcumin reduces the severity of urinary symptoms mg with soy lecithin curcumin reduced side effectsbayetrobert2010 france months to mgryam2013 kanai2011 hemati2011 garcea2005 sharma2001 sharma2004 usajapaniranukukukcruzcorrea m2006 usa years months months months months months g g g to mg per day g per day g per day g per dayyu he2010 china days g per daydurgaprasad2005 india weeks g per daydhillon2008 usa g per dayide2010 japan months g per daygolombick2009 australia months g per daypolasa1992 hastak1997 indiaindia days g per day months g per daycheng2001 taiwan months g per dayrai2010 uk days g per daymarcia cruz correa2018 richard greil2018 newyorkusa month weeks mg orally twice adaydoses between and mg per minutelynne m howells2019 unitedkingdom days g per dayplasminogen activator genes that are involved in tumorangiogenesis and its invasion curcumin inhibits the expression of this transcription factor curcumin mayinhibit angiogenesis directly by regulating angiogenicgrowth factors growth factors as well as the genes including angiopoietin1 hif1 ho1 and transcription factors such as nfkappab fig [] it isknown that hypoxic stress and activation of betagrowthfactor tgf stimulate vegf expression by activatingap1 and the hypoxiainducible factors hif1 curcumin is an important inhibitor in ap1 activationand it has recently been shown that curcumin is a directinhibitor of hif1 transcription factor activity whichcauses the transcription of many genes associated withcurcumin lowers the concentration of the cea markertumorcurcumin reduces some skin complicationscurcumin increased patient survivalcurcumin reduces some skin problemscurcumin increased the effectiveness of the coloncurcumin reduced glutathione stransferase activitycurcumin reduces prostaglandin e2 productionreduces the number and size of polyps without anysignificant toxicitycurcumin has been shown to improve the overallhealth of patients with colorectal cancercurcumin reduced lipid peroxidation and increasedglutathione content in patientswelltolerated limited absorption and showedactivity in some patientsreduced the serum prostatespecific antigen contentin combination with isoflavonesdecreased para protein load and urinary n telopeptideof type i collagenreduced the urinary excretion of mutagens in smokersreduced the number of micronuclei in mucosal cellsand in circulating lymphocytesimproved the precancerous lesionsincreased vitamins c and e levels decrease dmalondialdehyde and 8hydroxy deoxy guanosine contents inthe serum and salivano difference in polyp size and number betweenplacebo and curcuminno variation in tumor size according to recist criteriacurcumin was a safe and tolerable adjunct to folfoxchemotherapy in patients with metastatic colorectal cancerangiogenesis in tumors [ ] it is also shown thatcurcumin will reduce the expression of membrane surface moleculesadhesionmolecule1 vascular cell adhesion molecule1 and eselectin which play a role in cellular adhesion fig intracellularincludingcurcumin affects a number of adhesive cellular molecules involved in tumor growth and metastasis processes curcumin caused reduction in the expression ofadhesive molecules inside the cells of icam1 vcamvcam or vascular cell adhesion molecule and mmpswhich play an important role in cellular adhesion andmetastasis furthermore curcumin results in increase ofthe expression of various antimetastatic 0cmansouri bmc cancer page of fig the effect of curcumin on angiogenesis and metastasis in cancer cells by coreldraw graphics suite including tissue inhibitor metalloproteinaseproteinstimp the nonmetastatic gene nm23 and ecadherin lack of ecadherin would increase the possibility of metastasis because ecadherin are essential tomaintain cellular adhesion angiogenesis is alsolinked with neoplasia angiogenesis means the formationof new blood vessels which is generally a major step intumor survival and growth curcumin inhibits cancer invarious ans [ ]easyto assess whetherthe antiinflammatory effects of curcumin have beenproven in many studies since oxidative stress leads tochronic inflammatory diseases antioxidant compoundscan be useful in the prevention and treatment of inflammatory disorders [ ] on the other handsince curcumin has a high antioxidant activity it willnot beantiinflammatory activity is also dependent on its antioxidant activity [ ] since many of the antioxidantsthat have been already identified do not have antiinflammatory properties it seems unlikely that the antiinflammatory effects of curcumin are due solely to itsantioxidant properties curcumin as a potent antiinflammatory factor expresses its own effects throughseveral mechanisms first curcumin inhibits the activation of the nfκ factor [ ]curcuminsthe lab based studies have revealed that curcuminneutralizes oxidative stress caused by tumor and restorestnfαnfkappabcurcuminactivityinhibitsproduction thus tcell apoptosis caused by tumor willbe minimised resultsin the initial screening of databases s wereidentified after deleting duplicate s studieswere obtained after deleting unrelated s studies were obtained17 s were also deleted due tolack of access to their fulltext or falling into the lowquality category at the end studies entered the finalphase and analysis as illustrated in fig the specifications and details of the studies considered in this systematic review are summarized in table according to the studies presented in table curcumin has reduced side effects including skin complications depending on the different doses of curcuminprescribed for the patients suffering from cancer theirsurvival rate was increased and their symptoms ofchemotherapy were reduced in studies examining theeffect of curcumin on colorectal cancer curcumin hasincreased efficacy in the large intestine reduced glutathione stransferase activity and reduced prostaglandin e2production curcumin also reduces the number and sizeof polyps without any significant toxicity curcumin inpancreatic cancer reduces lipids peroxidation and increases glutathione content in the patients with this typeof cancer in prostate cancer curcumin reduces theserum levels of prostatespecific antigen in combination 0cmansouri bmc cancer page of with isoflavones and also reduces the severity of urinarysymptoms according to the published studies the useof curcumin during radiation therapy for breast cancerpatients improved treatment outcomes for these patients such as preventing skin symptoms reducing painand suffering of patients improving their quality of lifeduring treatment and reducing delays or unwantedstops during the course of radiation therapy curcumincan regulate multiple signalling pathways and affect different moleculartargets low cost pharmacologicalsafety efficiency and multiple molecular targets makecurcumin a promising product for the prevention andtreatment of various human diseases table after collecting various s from reputable databases and deleting duplicate s and removing unrelated s to the main aim of this paper we finallyconsidered s for further investigation and analysis the main aim of this paper is to review the clinicalstudies about curcumin and its various purposeseffectson cancer the results reported from numerous clinicalstudies that have examined the effects of curcumin onthe patients who are suffering from cancer and undergoing radiotherapy and chemotherapy were very promising here we briefly describe some of these studieswhich are summarised in table garcea evaluated patients in the ukin this study each patient received to mg ofcurcumin per day at the same time that these patientsreceived curcumin they were treated with radiotherapyand chemotherapy the results of this study revealedthat curcumin increased the effectiveness of the treatment plan for colorectal cancer in the patients receivedwith curcumin importantin bayetrobert conducted a study consists offourteen patients with advanced breast cancer who werebeing treated with docetaxel chemotherapy and simultaneously received curcumin at different doses up to amaximum dose of g per day for days per each treatment cycle finally patients participated in this studywere able to complete this treatment plan nutropeniaand leukopenia were the mosttoxicitiescaused by docetaxel administration after days two patients refused to continue treatment because they received curcumintreatment continued by reducing the dosage to a maximum of g per day nine patients were screened fortumor response six weeks after completing the courseof treatment patients partially responded well butthree patients still suffered from the disease in thisstudy the ca tumor marker did not decrease butthe cea tumor marker decreased compared to the initial value prior to the treatment in patients the vegfvascular endothelial growth factor as a tomur markerwhichandcurcumin capsules howeverindicatestumrowth metastasismalignancy was reduced by compared to the baseline before treatment in the effect of curcumin on reducing the sideeffects of radiotherapy and chemotherapy in patientswith ovarian lung colon liver kidney and stomach cancers was investigated eighty patients received mg ofcurcumin simultaneously with radiotherapy the duration of this study was days the incidence of side effects such as nausea diarrhea constipation and weightloss decreased in patients who treated with both radiotherapy and curcumin in the patients who are simultaneously under radiotherapy and received curcumin theprevalence of side effects such as skin lesions mouthand throat ulcers swallowing problems nausea vomitingfatigue weakness and common medications required for treating side effects were statistically lowerthan the control group in a study conducted by hemati in iran patientsreceiving radiation therapy to the breast area due tobreast cancer from days before the start to the end ofradiotherapy mgcapsules containing curcuminwere taken orally times a day yu he evaluated patients in their study andstated that a dose of g of curcumin per day for days improved the general health of patients withcolorectal cancer through increasing the expression ofp53 molecules in tumor cells in a study conductedby cruzcorrea it was stated that a dose of g of curcumin per day will reduce the number and size of polypswithout any significant toxicity discussionin the recent years several studies have been conductedon the biological effects of curcumin in more than studies have been recently published curcumin hasshown to have various effects in cancer treatments curcumin has antioxidant antibacterial antifungal antiviralantiinflammatory antiproliferative proapoptotic effects etc curcumin has tremendous potential for treatment of neurodegenerative diseases arthritis diabetespsoriasis allergies intestinal inflammation kidney poisoning alzheimers depression aids multiple sclerosis cardiovascular disease and especially cancer [] the numerous and multifaceted effects of curcuminin determining the cellular targets and molecular mechanisms involved in curcumin pathways have attractedmuch attention from researchers curcumin is a multifaceted molecule and has many therapeutic effects themultifaceted effects of curcumin are due to its capacityto interact with different molecules and to regulate multiple molecular pathways and their targets one of the compelling properties of curcumin whichmakes it appropriate for therapeutic use is its low toxicity so that even its consumption up to a dose of g 0cmansouri bmc cancer page of per day does not cause any side effects consumption of curcumin in highdose prevents cancer cells frommultiplying although it does not damage healthy cells[ ]minimaltoxicity alongside with possessing manytherapeutic effects have led to the widespread use of natural plantderived compounds in the treatment of cancer the compounds found in nature target various cellular and molecular aspects of cancer cells the researchers have demonstrated that curcumin regulatessignalling pathways in cancer cells reduces the expression of proteins related to drug resistance and increasesthe performance of antitumor drugs at various levelscurcumin reverses drug resistance mechanisms and results in increasing the sensitivity of chemotherapyresistant cells in the research conducted by keyvanighamsari they demonstrated that curcumin is aneffective chemical in cancer treatment in laboratory studies which have been performed onthe cellular categories of colorectal cancer the derivedresults show that curcumin inhibits cell growth and alsostimulates apoptosis by interacting with several molecular targets furthermore curcumin has been used as partof dietary formulations to prevent colon cancer in vitroand in vivo these compounds have been shown to haveanticancer properties for colon cancer and its inflammation the results of this study show that curcuminwould be effective in preventing colorectal cancer in animals this property offers promising expectations inhumans due to the limited number of the human clinical studiesthe corresponding results are somehowcontradictory on the other hand there exist several unanswered questions about dosage bioavailability optimalsigns and potential toxicity which should be investigatedin future studies using sufficiently large samples inaddition curcumin can induce autophagy apoptosisand cell cycle arrest in order to reduce the survival andproliferation oflung cancer cells curcumin has thispromising capability to increase the effectiveness ofradiotherapy in the treatment of lung cancer by targetingdifferent signalling pathways such as epidermal growthfactor receptor and nf κb curcumincontaining nanocarriers increase bioavailability cell uptake and curcumin antitumor activity [ ]in a study conducted by cruzcorrea oral curcumin was prescribed to the patients with adenomatouspolyposis this research was implemented to determinethe safety and efficacy of curcumin in patients with adenomatous polyposis in this study mg of oral curcumin was administered twice per day over monthsto patients with adenomatous polyposis the resultsshowed that there was no significant difference betweenthose who received oral curcumin and those receivingplacebo in another study conducted by grell patients were subjected to receive doses between and mg per minute the main aim of their studywas to evaluate the safety of curcumin locally in patientswith advanced or metastatic cancer the results obtainedfrom their study showed that no change in tumor sizewas observed based on the recist criteria in howells evaluated patients with the age over and with metastatic colorectal cancer using the histological diagnosis quality oflife and neurotoxicity ofthese patients were assessed using questionnaires thederived results showed that curcumin is a safe and tolerable adjunct for folfox chemotherapy in patients withmetastatic colorectal cancer overall the results suggest that curcumin can be usedas an effective combination in inhibiting and controllingcancersimproving clinical symptoms and preventingtumor spread and metastasis this compound wouldaffect various molecular pathways and inhibits vasodilation cell proliferation and metastasiscurcumin is a natural product found in turmeric thathas a unique chemical structure with particular biological and medicinal properties through various cellular and molecular mechanisms curcumin inhibits thecarcinogenesis and their growth due to the fact that nospecific toxic effects of this natural product have beenreported its use has been considered as a drug supplement in therapeutic diets of cancer patients in a number of studies considered in this systematic review haveshown that taking curcumin would increase the expression of antimetastatic proteins in several other studiesit was reported that curcumin has also increased patientsurvival and decreased tumor marker concentrationabbreviationsmmps matrix metalloproteases vcam vascular cell adhesion moleculewho world health anization sid scientific information database prisma preferred reporting items for systematic reviewsacknowledgementsthe authors thank the faculty members of the faculty of nursing andmidwifery kermanshah university of medical sciencesauthorscontributionsshr and ns and km contribute | Colon_Cancer |
" nlr plr and lmr have been associated with pancreatic ductal adenocarcinoma pdac survivalprognostic value and optimal cutpoints were evaluated to identify underlying significance in surgical pdac patientsmethods nlr plr and lmr preoperative values were available for pdac patients who underwent resectionbetween and os rfs and survival probability estimates were calculated by univariate multivariable andkaplanmeier analyses continuous and dichotomized ratio analysis determined bestfit cutpoints and assessed ratiocomponents to determine primary driversresults elevated nlr and plr and decreased lmr represented and of the cohort respectively osp and rfs p were significantly decreased in resected pdac patients with nlr ¥ compared to thosewith nlr optimal prognostic os and rfs cutpoints for nlr plr and lmr were and respectivelylymphocytes alone were the primary prognostic driver of nlr demonstrating identical survival to nlrs nlr is a significant predictor of os and rfs with lymphocytes alone as its primary driver weidentified optimal cutpoints that may direct future investigation of their prognostic value this study contributes tothe growing evidence of immune system influence on outcomes in earlystage pancreatic cancerkeywords neutrophil lymphocyte ratio platelet lymphocyte ratio lymphocyte monocyte ratio pancreatic cancerbiomarker correspondence mokengemalafamoffitt1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl 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 0cpointer bmc cancer page of pancreatic ductal adenocarcinoma pdac is the thirdleading cause of cancerrelated death in the us with anestimated deaths in and a 5year overall survival os rate of among newly diagnosed pdacpatients only to present with resectable diseasewith resection as the only chance for cure prognosis isgenerally poor with reported 5year os of afterresection [] ajcc tnm staging is the only widelyaccepted indicator of prognosis for resectable pancreaticcancer however its performance in earlystage diseasehas been questioned additionally controversy regarding initial treatment of earlystage pancreatic cancerpersists yielding no uniform treatment algorithm giventhe wide variation in the biological behavior of pdacand treatment algorithms for this disease there is an unmet need for enhanced prognostic biomarkers biomarkers derived from easily obtainable laboratory valueshave shown potential to meet this need and may help tostratify patients with earlystage pancreatic cancer andguide future treatment plansconventionally survival outcomes among cancer patients have been determined by the disease stage and receipt of treatment more recently howeverincreasedattention has been directed toward the role of inflammation and immune response in the tumor microenvironment and their effects on tumor behavior quantifyingthe systemic inflammatory response by creactive protein and various nutritional parameters has shown prognostic significance in gastrointestinal gynecological andthoracic cancers additionally inflammatory indicesand immunologic ratios including ratios comprised ofintratumoral or circulating neutrophils plateletslymphocytes and monocyte counts have been proposed tobe prognostic biomarkers for a wide range of malignancies []the neutrophil to lymphocyte ratio nlr platelet tolymphocyte ratio plr and lymphocyte to monocyte ratio lmr are among the many surrogate biomarkers forinflammation that have been associated with outcomesin gastrointestinal cancers although these ratios havebeen reported to have promising prognostic value fewstudies have examined the effect of these inflammatoryratios in us surgical cohorts [] moreover manysingleinstitution studies have reported inconsistentprognostic outcomes for these surrogate biomarkers wepreviously reported an inverse association between survival and nlr in patients with borderline resectable disease to expand the scope of our previous analysiswe evaluated the prognostic significance of the nlrplr and lmr in a cohort of patients with resectedpdac who were treated at a highvolume cancer centerfurthermore we aimed to establish optimal nlr plrand lmr cutpoints for determining os and recurrencefree survival rfs and define the primary factor drivingthe prognostic value of these ratios for survival outcomes we hypothesized that preoperatively increasednlr and plr and decreased lmr were associated withworse os in patients with resectable pdacmethodsa retrospective review was conducted using our institutional prospective pancreatic cancer database as part ofour ongoing outcomebased study the study was approved by our institutional review board mcc16446and patient consent was unable to be obtained as thisstudy was conducted retrospectively on deidentified dataposing less than minimal risk patients diagnosed withpdac who underwent curativeintent resection for thetreatment of their disease were identified resectable andborderline resectable pdac patients were defined and included on the basis of the nccn guidelines applied at thetime of diagnosis pancreatic resection included open orminimally invasive pancreaticoduodenectomy total pancreatectomy and distal pancreatectomy performed at ourinstitutionpatient characteristics were summarized using descriptive statistics including median and range for continuous measures and proportions and frequenciesforcategorical measures kaplanmeier plots were made todetermine os and rfs for the nlr plr and lmrsurvival probability estimates were calculated using thekaplanmeier method univariate and multivariable coxproportionalhazard models for os and rfs were runfor each ratio as continuous predictors and dichotomized forms the nlr plr and lmr were calculatedby dividing the absolute neutrophil count by thelymphocyte count the platelet count by the lymphocytecount and the lymphocyte count by the monocytecount respectively dichotomized analyses included neutrophil and lymphocyte counts and percentages whichwere defined as the proportion of neutrophils or lymphocytes to all white blood cells in the sample valuesused for these calculations were part of the last completeblood count and differential obtained after neoadjuvanttherapy and before operative intervention cutpoints of and were used for nlr plr and lmr respectively nlr cutpoints were determined on the basisof values used in previously published studies [ ]cutpoints for plr and lmr were not well establishedtherefore the medians of the observed data were usedoptimal nlr plr and lmr cutpoints for the prediction of os and rfs were determined using maximallyselected rank statistics based on the logrank method the resulting cutpoint for each ratio provided thebest separation of the responses into groups in whichthe standardized rank statistics take their maximumthe p value approximation was based on the improved 0cpointer bmc cancer page of bonferroni inequality variables were evaluated inrelation to os and rfs for predetermined cutpoints andnewly identified bestfit cutpoints all analyses were performed using r software version resultsa total of patients treated at our institution between and were eligible for this study two hundredseventyseven patients with complete data met the inclusion criteria and were included in the analysis the meanage was ± years of whom were maletwentyfive percent of patients had a charlson comorbidity index cci ¤ had a cci of to and had a cci ¥ medicare with a private supplement wasthe largest represented insurance provider among patients sixtyfour percent of our cohort was classified as resectable and treated with upfront resection and received neoadjuvant systemic therapy marginnegative r0 resection was achieved in of our patients with and demonstrating lymphovascularand perineural invasion respectively table mean preoperative nlr plr and lmr was ± ± and ± respectively additional file using the predetermined cutpoints described above and of patients demonstrated preoperative nlr ¥ plr ¥ and lmr ¤ respectivelyos was significantly shorter among patients with annlr ¥ than patients with an nlr in univariatehr [ ci ] p and multivariable hr [ ci ] p analysestable neither the plr nor lmr demonstrated a significant association with os table and fig patients with a high nlr also demonstrated significantlyworse rfs in univariate hr [ ci ]p and multivariable hr [ ci ] p analyses table and fig this wasnot observed with plr or lmr in multivariable analyses pathologic t stage presence of grade complications cci ¥ nlr node positivity and perineuralinvasion were found to be significant predictors of osand rfs tables and maximally selected rank analyses of nlr plr andlmr were performed to identify optimal cutpoints forpredicting os and rfs os optimal cutpoints for nlrplr and lmr were and respectively forrfs cutpoints were and respectively because neutrophil percentage is highly correlated with nlrwe found the corresponding cutpoint for determining ahigh neutrophil percentage to be resulting in patients being above the cutpoint similarly lymphocytepercentage was highly negatively correlated with nlrwith a corresponding cutpoint percentage of thecomponents of nlr was analyzed separately to evaluatetheir prognostic importance the lymphocyte percentagealone yielded a survival curve that was identical to that ofthe nlr whereas the neutrophil percentage km plot wasnot statistically significant additional file discussionwe demonstrated a statistically significant associationbetween preoperative nlr and both os and rfs inpdac patients who underwent curativeintent resectionat a highvolume cancer center plr and lmr failed todemonstrate any correlation with survival in additionwe identified optimal cutpoints for immunologic ratiosurvival analyses on the basis of our cohort data finallywe identified the lymphocyte component of nlr to bethe primary driver of survival prognosis to our knowledge this is the largest us cohort utilized to analyzeimmunologic ratio biomarkerassociated outcomes andperform dichotomized analyses for the purpose of identifying the prognostic driver of the nlr in surgical pdacpatientsinflammation and the inflammatory response have beendiscussed extensively in the literature in relation to tumorigenesis progression and metastasis furthermorelinkshave been established between the inflammatory responseand oncogenic signaling pathway interactionstumormicroenvironment analyses and use of immunetargetedtherapies surrogate biomarkers of inflammation haveproven useful in predicting disease progression recurrenceand overall prognosis across a wide range of malignancies[ ] in a metaanalysis evaluating the role of thesystemic immuneinflammation index zhong showedthat an elevated systemic immuneinflammation index isassociated with worse os in hepatocellular carcinomaurinary cancers gastrointestinal cancers and smallcell lungcancer in a review of patients with gastrointestinalmalignancies nora demonstrated nlr and plr to besignificant predictors of lymph node positivity metastaticdisease and recurrence especially when used in combination the use of the nlr plr and lmr have shownpromise in pancreatic adenocarcinoma demonstratingprognostic value in both resectable and palliative populations [ ]the nlr has shown substantial potential for prognostic utility in pancreatic adenocarcinoma patients in alarge retrospective analysis of surgical pdac patients alow nlr was associated with longer median survival vs months p and an nlr ¥ independently predicted poor prognosis hr [ ci] p giakoustidis further explored pretreatment nlr in surgical pdac patients andidentified decreased os rates to be associated with a highnlr in univariate analyses which maintained independent prognostic significance in multivariable analyses two recent metaanalyses including a total of patients have also suggested an association between 0cpointer bmc cancer page of table descriptive statistics of study cohortsnlr demographicsn overalln age median range ynlr ¥ n plr n pvalue plr ¥ n lmr ¤ n pvalue lmr n sex no femalemalerace no blackotherwhite bmi median range cci no ¥ tumor sizepathologic stage no t0t1 no t2 no t3preoperative resectabilityno neoadjuvant therapy nonoyesmargin no negativepositivelymphovascular invasionno pvalue borderlineresectable noyes perineural invasion no noyes complication 34a no noyes completion of adjuvanttherapy no 0cpointer bmc cancer page of table descriptive statistics of study cohorts continuednlr ¥ demographicsn nlr n overalln nopvalueplr n plr ¥ n pvaluelmr ¤ n lmr n pvalueyes aclaviendindo classification of surgical complicationsabbreviations bmi body mass index nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratio cci charlesoncomorbidity indexnlr and os in which elevated nlr carried poor prognoses zhou found elevated nlr to be associatedwith shorter rates of os hr [ ci ]p and diseasefree survival hr [ ci] p evaluating os alone mowbray also demonstrated that significantly shorter rates ofos were associated with elevated nlr hr [ci ] p we corroborated these results in our own resected pdac patients and similarlydemonstrated that decreased rates of os were associatedwith an nlr ¥ in multivariable analyses additionallywe showed a significant association between hightable univariate and multivariate cox proportional hazard models for overall survivalvariablep valueunivariate analysishr cimultivariable analysishr ciap valuegenderfemalemaleage¤ pathologic staget0t1t2t3ccinlr ¥ plr ¥ lmr ¥ perineural invasionnoyesnananananananananana reference reference reference nanananananananananananananacomplication grade 4bpositive nodesamodel includes age gender pathologic stage cci complication score nlr nodal and perineural invasion status b claviendindo classification ofsurgical complicationsabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte rationananana reference reference reference reference reference nananana reference nananana 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating overall survival in a nlr b plr and c lmrpreoperative nlr and a decrease in rfs our study further supports the nlr as a valid prognostic biomarkerfor earlystage pdacalthough a cutpoint of has been widely used to define highlow nlr variations in cutpoints exists withsome groups using values ranging from to [ ] with no clearly defined cutpoint we chose to perform a continuous analysis to identify an optimal cutpoint for the nlr in relation to survival based solely onthe data from our cohort optimal cutpoints of foros and for rfs were obtained our study supportsthe prognostic value of the commonly used nlr cutpoint of as the nlr was the only significant ratio inour cohort we elucidated its prognostic driver by analyzing the components of the ratio the denominatorthe lymphocyte count percentage alone yielded a survival curve identical to the nlr whereas the numeratorthe isolated neutrophil count percentage was not statistically significant suggesting that lymphocyte count percentages have equal prognostic value and perhaps offera simpler alternative to the nlr biomarker this findingis supported by those from previous studies that showedlow lymphocyte counts to be poor prognostic indicatorsin pancreatic and colorectal cancers [] the finding also has immunotherapeutic implications which corroborate basic science findings on a population level[]in contrast to our study other studies have found noprognostic significance of the nlr in some pdac patient populations recently chawla described a cohort of resectable pdac patients whose nlr atdiagnosis did not correspond to os jamieson similarly reported patients who underwent pdacresection and found no relationship between nlr and 0cpointer bmc cancer table univariate and multivariate cox proportionalhazard models for recurrencefree survivalvariablep valueunivariate analysishr cimultivariable analysishr ciapage of p value reference reference reference reference nananana reference nanagenderfemalemalepathologic staget0t1t2t3ccinlr ¥ plr ¥ lmr ¥ perineural invasionnoyesnananananananana reference reference reference nanananananananacomplication grade 4bpositive nodesabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratioa model includes age gender pathologic stage cci complication score nlr nodal and perineural invasion statusb claviendindo classification of surgical complicationsnanananasurvival similar findings have been reported byother groups [ ] the reasons for this variability include diverse patient populations differences in ratiocutpoints timing of blood collections and receipt ofneoadjuvant therapy in the current study of patients received neoadjuvant therapy before pancreatic resection which may havecellpopulationsinfluenced immuneincreased monocyte presence in the tumor microenvironment or in circulation has been implicated inangiogenesis tumor growth and poor prognosis in cancer patients circulating monocytes are commonlyquantified by the lmr which has demonstrated an inverse association with survival and prognosis in solidtumor malignancies few studies have investigatedthis parameter in surgical pdac patients in a large review and metaanalysis of patients li reported a favorable prognosis associated with elevatedlmr in pooled analyses hr [ ci ]p although this study included a range oflmr cutpoints and both resected and nonoperablepdac patients a prognostic value of the lmr was observed in surgical patients in subgroup analyses sierzega reported a series of resectable pdacpatients demonstrating prolonged median survival vs months p in the lmr ¥ group anlmr was an independent predictor of poor prognosis hr [ ci ] p in contrastaldemonstrated no association between lmr and os ordiseasefree survival in a large retrospective analysis ofthe prognostic effects of patientspecific nutritional andimmunologic factors in resected pdac patients we also did not show a prognostic value of lmr in ouranalyses of resected pdac patients differences in prognostic outcomes were likely due to the paucity of dataevaluating lmr and survival inconsistency in evaluatedpatient cohorts and variation of cutpoint delineationto studies previously discussed abeet 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating recurrencefree survival in a nlr b plr and c lmrwe used mean values for lmr cutpoints in our analysesbecause of the variation of cutpoints reported in the literature an optimal cutpoint analysis of lmr for osand rfs was performed to clarify the reporting of lmrassociated outcomessurvival outcomes have similarly been linked to elevated plr in solid tumor malignancies comparedto other commonly described ratios the application ofplr to pdac is less clear with mixed outcomes reported giakoustidis also investigated pretreatmentplr in surgical pdac patients and identified decreasedos with high plr in univariate analyses the plrdid not maintain independent prognostic significance inmultivariable analysis interestingly patients with concurrently high nlr and plr experienced significantlydecreased os when compared to those with normalnlr and plr or those with an elevation of either ratio respectively p in a subsequentanalysis of resected and inoperable pdac patients stotz found no association between os hr [ci ] p and plr hr [ ci] p in either cohort similarly nodemonstrable association between plr and os was observed in several separate resected pdac patient series[ ] consistent with the literature discussedabove our study did not find a significant correlation between survival os or rfs and plr in resected pdacpatientshowever some authors have demonstrated the plr tobe an important predictor of survival smith and 0cpointer bmc cancer page of watanabe reported elevated plrs as the most significant determinant of survival in their resected pdaccohorts of and patients respectively [ ]reasons for inconsistent results may have included differing plr cutpoint values small patient cohorts andvariations in multidisciplinary treatments of these patients with complex pdac furthermore the plr wassynthesized using surrogates that are fundamental tomany biologic functions ie coagulation cascade whichmay explain the variability of correlation in oncologicoutcomes in our study mean values were initially usedfor plr cutpoints because of the variation reported inthe literature again an optimal plr cutpoint analysiswas performed to provide clarity and consistency in thereporting of plrassociated factorsthereforsettingis potentialthe limitations of this study include those inherent inreviewing retrospective data although our data set wasrobust and associated with an electronic medical recordthe potential for selection bias exists additionally although all blood specimens were collected in the preoperativevariationregarding the date and time blood draws were done inrelation to the surgery date the present study did notstratify patients based on receipt of neoadjuvant therapythis stratification was previously investigated by ourgroup who reported significantly decreased rates of osamong patients with increased nlr after neoadjuvanttherapy when compared to those with stable nlr finally we did not analyze pretreatment immunologicratios in patients who received neoadjuvant chemotherapy therefore we were not able to determine whetherchemotherapy significantly altered preoperative valuesthere continues to be little doubt about the importanceof inflammation and immunity in cancer biology thenlr and other immunologic ratios are derived from easily obtainable standard laboratory values with littleadded expense when obtained in the preoperative setting the nlr is a biomarker with the potential to guidetreatment algorithms in earlystage pdac patients andprovide clarity on common unresolved management dilemmas routinely debated today given their demonstrable poor outcomes patients with high nlr maybenefitfrom neoadjuvant systemic therapy variationmore detailed preoperative staging or stratification inclinical trials additionally consistent with the findingsof developing research on the tumor microenvironmentand immunotherapy lymphocytes alone may be significant drivers of survival in the context of improving outcomes ourtargeting inflammatorypathways may be relevant in chemoprevention prospective trials would serve to elucidate the provided prognostic information and provide insightinto alternativesuggestresultstreatment algorithms that can improve outcomes amongpatients with pdacsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020071829additional file summary statistics of immunologic ratiosadditional file kaplanmeier plot demonstrating overall survival osin dichotomized nlr values a neutrophil and lymphocyte bpercentageabbreviationscci charlson comorbidity index lmr lymphocyte to monocyte rationlr neutrophil to lymphocyte ratio os overall survival pdac pancreaticductal adenocarcinoma plr platelet to lymphocyte ratio r0 marginnegative resection rfs recurrencefree survivalacknowledgmentseditorial assistance was provided by the moffitt cancer centers scientificediting department by dr paul fletcher daley drucker no compensationwas given beyond their regular salaries this work was presented as a posterat the ahpba meeting and the pancreas club meeting theabstract of this work was previously published in hpb journalauthors contributionsdp conception and design acquisition of data analysis and interpretation ofdata drafting of original critical revision gave final approval ofcompleted manuscript dr conception and design acquisition of dataanalysis and interpretation of data drafting of original critical revisiongave final approval of completed manuscript bp conception and designacquisition of data analysis and interpretation of data critical revision gavefinal approval of completed manuscript gm conception and designacquisition of data critical revision gave final approval of completedmanuscript se conception and design acquisition of data critical revisiongave final approval of completed manuscript zt statistical analysis andinterpretation of data critical revision gave final approval of completedmanuscript ms statistical analysis and interpretation of data critical revisiongave final approval of completed manuscript ph conception and designanalysis and interpretation of data critical revision gave final approval ofcompleted manuscript jp conception and design analysis andinterpretation of data critical revision gave final approval of completedmanuscript jf conception and design analysis and interpretation of datacritical revision gave final approval of completed manuscript mmconception and design primary investigator supervision analysis andinterpretation of data critical revision gave final approval of completedmanuscriptfundingthis work was supported by the h lee moffitt cancer center researchinstitute nci cancer center support grant p30ca076292 the funders hadno role in study design data collection and analysis decision to publish orpreparation of the manuscriptavailability of data and materialsthe data that support the findings of this study are available from thecorresponding author upon reasonable requestethics approval and consent to participatethis study was approved by the moffitt cancer center institutional reviewboard mcc because of the retrospective nature of this studypatient consent was not required no personally identifiable data for anypatients were included the study was performed in accordance with thedeclaration of helsinkiconsent for publicationthis study was approved by the moffitt cancer center institutional reviewboard mcc due to the retrospective nature of this study patientconsent was not required 0cpointer bmc cancer page of competing intereststhe authors have no conflicts of interest to declareauthor details1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usa2department of surgery university of texas southwestern dallas tx usa3department of biostatistics and bioinformatics h lee moffitt cancer centerand research institute tampa fl usareceived april accepted july referencessiegel rl miller kd jemal a cancer statistics ca cancer j clin ryan dp hong ts bardeesy n pancreatic adenocarcinoma n engl j medkatz mh wang h fleming jb longterm survival aftermultidisciplinary management of resected pancreatic adenocarcinoma annsurg oncol neoptolemos jp palmer dh ghaneh p comparison of adjuvantgemcitabine and capecitabine with gemcitabine monotherapy in patientswith resected pancreatic cancer espac4 a multicentre openlabelrandomised phase trial lancet oettle h neuhaus p hochhaus a adjuvant chemotherapy withgemcitabine and longterm outcomes among patients with resectedpancreatic cancer the conko001 randomized trial jama chen dt davisyadley ah huang py prognostic fifteengenesignature for early stage pancreatic ductal adenocarcinoma plos one2015108e0133562helm j centeno ba coppola d histologic characteristics enhancepredictive value of american joint committee on cancer staging inresectable pancreas cancer cancer proctor mj morrison ds talwar d a comparison of inflammationbased prognostic scores in patients with cancer a glasgow inflammationoutcome study eur j cancer bindea g mlecnik b tosolini m spatiotemporal dynamics ofintratumoral immune cells reveal the immune landscape in human cancerimmunity hong x cui b wang m yang z wang l xu q systemic immuneinflammation index based on platelet counts and neutrophillymphocyteratio is useful for predicting prognosis in small cell lung cancer tohoku jexp med zhong jh huang dh chen zy prognostic role of systemic immuneinflammation index in solid tumors a systematic review and metaanalysisoncotarget templeton aj mcnamara mg seruga b prognostic role of neutrophiltolymphocyte ratio in solid tumors a systematic review and metaanalysisj natl cancer inst 20141066dju124 giakoustidis a neofytou k costa neves m identifying the role ofneutrophiltolymphocyte ratio and plateletstolymphocyte ratio asprognostic markers in patients undergoing resection of pancreatic ductaladenocarcinoma ann hepatobiliary pancreatic surg glazer es rashid om pimiento jm hodul pj malafa mp increasedneutrophiltolymphocyte ratio after neoadjuvant therapy is associated withworse survival after resection of borderline resectable pancreatic ductaladenocarcinoma surgery sierzega m lenart m rutkowska m preoperative neutrophillymphocyte and lymphocytemonocyte ratios reflect immune cellpopulation rearrangement in resectable pancreatic cancer ann surg oncolli w tao l zhang l xiu d prognostic role of lymphocyte to monocyteratio for patients with pancreatic cancer a systematic review and metaanalysis oncotargets ther abe t nakata k kibe s prognostic value of preoperative nutritionaland immunological factors in patients with pancreatic ductaladenocarcinoma ann surg oncol quigley da dang hx zhao sg genomic hallmarks and structuralvariation in metastatic prostate cancer cell e759 halazun kj aldoori a malik hz elevated preoperative neutrophil tolymphocyte ratio predicts survival following hepatic resection for colorectalliver metastases eur j surg oncol lausen b schaumacher m maximally selected rank statistics biometricslausen b sauerbrei w schumacher v classification and regression treescart used for the exploration of prognostic factors measured on differentscales in university of essex research repository p mantovani a allavena p sica a balkwill f cancerrelated inflammationnature giakoustidis a neofytou k khan az mudan s neutrophil to lymphocyteratio predicts pattern of recurrence in patients undergoing liver resectionfor colorectal liver metastasis and thus the overall survival j surg oncolli c wen tf yan ln postoperative neutrophiltolymphocyte ratioplus platelettolymphocyte ratio predicts the outcomes of hepatocellularcarcinoma j surg res nora i shridhar r huston j meredith k the accuracy of neutrophil tolymphocyte ratio and platelet to lymphocyte ratio as a marker fastrointestinal malignancies j gastrointest oncol ye s bai l comparison and validation of the value of preoperativeinflammation markerbased prognostic scores in resectable pancreaticductal adenocarcinoma cancer manag res zhou y wei q fan j cheng s ding w hua z prognostic role of theneutrophiltolymphocyte ratio in pancreatic cancer a metaanalysiscontaining patients clin chim acta mowbray ng griffith d hammoda m shingler g kambal a alsarirehb a metaanalysis of the | Colon_Cancer |
" patients who have undergone radical cystectomy for urinary bladder cancer are not sufficientlyphysically active and therefore may suffer complications leading to readmissions a physical rehabilitationprogramme early postoperatively might prevent or at least alleviate these potential complications and improvephysical function the main aim of the canmore trial is to evaluate the impact of a standardised and individuallyadapted exercise intervention in primary health care to improve physical function primary outcome and habitualphysical activity healthrelated quality of life fatigue psychological wellbeing and readmissions due tocomplications in patients undergoing roboticassisted radical cystectomy for urinary bladder cancermethods in total patients will be included and assigned to either intervention or control arm of the study allpatients will receive preoperative information on the importance of early mobilisation and during the hospital staythey will follow a standard protocol for enhanced mobilisation the intervention group will be given a referral to aphysiotherapist in primary health care close to their home within the third week after discharge the interventiongroup will begin weeks of biweekly exercise the exercise programme includes aerobic and strengtheningexercises the control group will receive oral and written information about a homebased exercise programmephysical function will serve as the primary outcome and will be measured using the sixminute walk test secondaryoutcomes are gait speed handgrip strength leg strength habitual physical activity healthrelated quality of lifefatigue psychological wellbeing and readmissions due to complications the measurements will be conducted atdischarge ie baseline postintervention and year after surgery to evaluate the effects of the intervention mixedor linear regression models according to the intention to treat procedure will be usedcontinued on next page correspondence andreaporserudkise1department of neurobiology care sciences and society division ofphysiotherapy karolinska institutet stockholm sweden2allied health professionals function medical unit occupational therapy andphysiotherapy karolinska university hospital stockholm swedenfull 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 0cporserud bmc cancer page of continued from previous pagediscussion this proposed randomised controlled trial has the potential to provide new knowledge withinrehabilitation after radical cystectomy for urinary bladder cancer the programme should be easy to apply to otherpatient groups undergoing abdominal surgery for cancer and has the potential to change the health care chain forthese patientstrial registration clinicaltrialsgov clinical trial registration number nct03998579 first posted june keywords abdominal surgery behaviour bladder neoplasm complications exercise physical activity primaryhealth care process evaluation the most common treatment for solid cancer tumoursis surgery often in combination with chemotherapy orradiotherapy or both minimising postoperative complications is important in health care for the individual patient and in reducing health care costs for society earlymobilisation at the ward and physical activity at homeafter discharge are important activities to reduce complications common complications after abdominal surgery are postoperative pulmonary complications andvenous thrombosis [ ] which generally are thought tobe partially avoidable with early mobilisationafter radical cystectomy for urinary bladder cancerthere is a high risk for postoperative complications thecomplications could be directly related to the patientshigh age to a high degree of comorbidity or both the major risk factor for developing urinary bladdercancer is smoking most of the patients are men andthe median age of undergoing a radical cystectomy is years [ ] as much as of patients are at severe nutritional risk before a radical cystectomy afterroboticassisted radical cystectomy rarc for urinarybladder cancer ofthe patients need to bereadmitted to hospital after discharge because of complications [ ]there is strong evidence that aerobic physical activityhas a positive impact on health survival and quality oflife qol patients diagnosed with cancer shouldfollow the general recommendations on physical activityand exercise for health [ ] yet most patients areinsufficiently active consequently with an increasing number of cancer survivors the importance of supporting high physical function and qol increases research has shown that exercise has a positive effect onhealthrelated qol hrqol in patients who have completed active cancer treatment moreoverin patients living with or beyond a diagnosis of cancerbehaviouraleg goalsetting andgraded tasks are important components in the exerciseinterventions with high adherence and positive physicaloutcomes support methodsin a recent study we evaluated the activity board®phystec sweden as a method to enhance mobilisationand recovery after abdominal surgery for cancer theactivity board is a tool based on techniques to supportbehaviour change [ ] the evaluation showed thatthe activity board resulted in a higher level of mobilisation in the group with the activity board compared withthe group who received standard treatment although evidence for exercise after abdominal surgery isscarce a few studies have evaluated exercise programmes for patients postoperatively atthe hospitalward with promising results [ ]despite the lack of exercise interventions after surgeryit has been shown that functional performance after aradical cystectomy for urinary bladder cancer correlatesto overall survival a large proportion of patientswith urinary bladder cancer do not achieve the recommendations on physical activity and exercise it isalso common that patients who undergo radical cystectomy have not performed physical exercise for a longtime before surgery finally after surgery patientsreport a low level of physical exercise recently tworeviews have been published on physical and psychological interventions to improve healthrelated outcomesin this patient group [ ] both reviews include thesame two postoperative exercise studies [ ] onelarger rct showed that early physical exercise and enhanced mobilisation after radical cystectomy positivelyaffected some domains of hrqol in addition in apilot study we tested a model for physical rehabilitationafter radical cystectomy the model consisted of weeks of individually tailored exercise after dischargefrom the hospital the exercise programme conductedat the hospital showed both short and longterm effectson physical function and hrqolconsequently the few studies within the field raiseseveral research questions for future exercise interventions in patients with urinary bladder cancer undergoingradical cystectomy current recommendations proposethe following areas supervised exercise after dischargethe optimal type of exercise fidelity and adherence ofthe intervention if shortterm outcomes are sustainedclinical relevance longterm outcomes and readmissionsto hospital [ ] we also need to understand thekinds of support that are optimal use behaviour change 0cporserud bmc cancer page of screened for eligibility in medical records by the responsible researcher rr and given written information by a registered nurse at a preoperative meetingafter days the rr will phone the patient provideoral information and then ask about participation informed consent will be signed before surgery the rrkeeps a protocol for enrolment the patient flowchartis depicted in fig eligibility criteriainclusion criteria will be patients who are planned for ararc for urinary bladder cancer the patients shouldbe able to talk and understand swedish without an interpreter be mobile with or without a walking aid and livein the stockholm regionstrategies and implement the intervention as a part ofthe patients clinical pathway through the healthcare system [ ]in summary patients who have been treated forurinary bladder cancer are not sufficiently physicallyactive and suffer from readmissions to hospital due tocomplications therefore there is a need for developing a physical rehabilitation programme to supportpatients who have a radical cystectomy in the earlypostoperative period in this paper we present a studyprotocol for the canmore trial a physical rehabilitation programme after rarc for urinary bladdercancerimpact ofamethodsdesignmain objectiveis to evaluatethe main aim of the canmore trialthestandardised and individuallyadapted exercise intervention in primary health carephcfunction primary outcome and habitual physical activity hrqol fatiguepsychological wellbeing and readmissions due tocomplications in patients undergoing rarc for urinary bladder cancerto improve physicalhypothesiswe hypothesise that the canmore programme is morebeneficial than homebased exercises in increasing physical function primary outcometrial designthe canmore trialis a randomised controlled trialrct with a singleblinded design the interventiongroup will receive a 12week h twice a week standardised and individually adapted exercise intervention inphc and behavioural support for daily physical activitythe control group will receive a homebased exerciseprogramme as well as recommendations on daily physical activity based on general guidelines we will followthe spirit standard protocol items recommendationsfor interventional trials statement and guidelinesfor reporting the study protocol the clinical trial registration number for this trial is nct03998579study settingthe study will be conducted in two settings a universityhospital and a phc context in region stockholmrecruitment and screeningparticipants will be recruited through theme canceratthe karolinska university hospital solna andscreened for eligibility recruitment will be performedconsecutively based on power analysis patientswill be included potential participants will initially befig patient flowchart 0cporserud bmc cancer page of exclusion criteriapatients with planned palliative surgery or cognitive impairment identified by screening of medical records willnot be includedrandomisation assignment of interventionpatients who fulfil the criteria for inclusion will afterhaving given their oral and written consent be randomised in the alea system operated by the clinical trials office cto atthe centre for clinical cancerstudies theme cancer karolinska university hospitalsolna swedenrandomisation will be conducted in blocks of patients stratified by sex and age ¥ years a confirmation email will be sent to the entering investigatorand an enrolment log will be filed at the centre the patient will receive the next consecutive code number inthe trial and treatment arm according to the randomisation schemeislogic model for the canmore programmeit is recommended that programme design should bebased on a theory to improve evidence synthesis the theoretical framework underpinning the canmoreprogrammethe movement continuum theorymct and the evidencebased calore taxonomy forbehavioural change techniques the mct posits that anindividual has three stages of movement capability amaximum a current and a preferred the canmoreprogramme identifies the patients current physical function capability and intervenes regarding the patientsneed for function several models and theories are supporting a behaviour change which results in increasedphysical activity however research has shown thatit is most often not necesssary for a complete theorybut instead the different components in the theory thatsupport the behaviour to consider the patients need forsupport the calore taxonomy for behavioural changetechniques has been added the taxonomy is recommended to be used to improve the specification of interventions behavioural techniques proven effective tosupport behaviour change are goalsetting graded tasksselfmonitoring feedback and reward all of these areused in the canmore programmea conceptual framework visualising the inputs theoryintervention components and its intermediate and possible longterm outcomes is depicted in a logic modelfig interventionall patients receive preoperative information on the importance of early mobilisation and postoperative individual physiotherapy during the hospital stay the activityboard is used for enhanced mobilisation before discharge from the hospital the patients receive standardised information about avoiding the lifting of heavyobjects and the importance of physical activity the patients are then randomised to either the intervention orthe control groupintervention grouppatients in the intervention group get a referral to aphysiotherapist in phc close to where they live the patients can choose from phc settings spread throughout the stockholm region within the third week afterdischarge the patients begin weeks of biweekly exercise the patients pay for their primary care visits physiotherapists in the targeted primary care units receive afig logic model of the intervention 0cporserud bmc cancer page of leaflet and a short education before starting comprisinginformation about rarc restrictions potential adverseevents the trial process and the exercise programmethe physical exercise is individually targeted but basedon international recommendations for persons with cancer disease the exercise programme includes aerobicexercise aiming at moderate intensity minsessionand strengthening exercises comprising endurance training with à repetitions see additional file theprogramme is gradually increased based on the patientscapability the programme also includes exercises forabdominal muscles including pelvic floor exercises tominimise the risk of developing stoma hernia however to avoid heavy strain on the surgery wounds restrictions regarding abdominal muscles are followed weeks postoperatively the exercise programme hasbeen approved by the responsible medical surgeons inaddition to the structured exercise sessions the patientsare advised to take daily walks in their neighborhoodthe number of recommended steps per day is set together with the physiotherapist based on the patientscapability to support the patient individual goalsettingfeedback and selfmonitoring of daily steps are usedsimilarly to those of the activity board at the end ofthe exercise periodthe physiotherapist recommendscontinued physical activity according to their clinicalroutinesanasapplicationactivitydiarypedometeroraphoneoutcomesthe measurements will be conducted using validated instruments at discharge ie baseline postintervention months and year after surgery table with thepurpose to receive information on the patients healthand physical function before surgery eg to adjust forin the analysis measurements will also be conductedbefore surgery all measurements will be conducted byexperienced physiotherapist blinded to the interventiona protocol for the measurements has been developedand the physiotherapist will receive specialised trainingby the research staffprimary outcomephysical function the primary outcome will be measured using the validated sixminute walk test 6mwt[ ] the test reproduces activity of daily living at asubmaximal level which is particularly applicable to elderly patients the patients are asked to walk as faras possible for min the number of meters m oxygensaturation and heart rate measured with a pulse oximeter will be recorded at the end of the test according tostandard procedures the primary outcome variablewill be walking distance in meterscontrol groupthe control group will receive oral and written information of a gradually increased homebased exerciseprogramme that includes daily walks and sittostandexercises they will also receive information on supportive techniques to improve physical activity suchsecondary outcomesgait speed will be assessed with the 10m walk test the test is used to determine walking speed in metersper second ms over a short distance the test is performed as three 10m walks without assistance one testwalk one in preferred walking speed and one in thepreop testingxbaselinexposttestingx1year followupxxxxxxxxxxxxxxxxxxxxxxxxxxxxxtable outcome measures and test occasionsvariablephysical functionmeasure6min walk testgait speedleg strength10m walk testchair stand testhandgrip strengthjamar hand dynamometerhabitual physical activityprevious physical activity levelahealthrelated quality of lifefatiguepsychological wellbeingpainactivpal3 microstanford brief activity surveyeortc qlqc30eortc qlqblm30piper fatigue scalehadsnrslength of hospital stay dayscomplicationsmedical recordsmedical recordsxxxxxxxxreadmissionsaprevious physical activity level is only used for adjustment purposes in the analysismedical records 0cporserud bmc cancer page of fastest speed possible time is measured for the intermediate m to allow for acceleration and decelerationthe outcome variable is msgrip strength will be assessed with the validated jamarhydraulic hand dynamometer the patients will sitin a chair and hold the dynamometer the test is performed three times for each hand and a mean value foreach hand is calculated the outcome variable is the gripstrength reading in kgleg strength will be assessed with the 30s sec chairstand test the patient is asked to rise from a chairas many times as possible for s the outcome variableis the number of sittostand transitionshabitual physical activity will be measured usingthe activpal3 micro activity monitor pal technologies ltd glasgow uk [ ] the activpal is asmall device which when attached to the thigh provides information based on position and accelerationof the body the information is then transferred tobody posture the transition between postures stepping and stepping speed the activity monitor is attached to the anterior midline ofthe thigh withdressing and does not provide feedback to the patientthe monitor will be worn for seven consecutive daysafter discharge from hospital and after the intervention period outcome variables will be time spentsittinglying standing stepping numbers of stepcounts and sittostand transitionsselfreported previous physical activity level will bemeasured using the 2item stanford brief activitysurvey sbas the sbas assesses the usualamount and intensity of physical activity during thepast year that a person performs the first item describes five patterns of work activity ranging frommostly sedentary to hard physical labour the seconditem describes five patterns ofleisuretime physicalactivity ranging from sedentary to regular vigorousintensity aerobic activities for respondents who areretired and have no job or regular work they wouldselect the response not applicable for both itemsthe outcome variable was categorical and rated on a5point scalehrqol will be assessed using the eortc qlqc30 with addition of the eortc qlqblm30 questionnaire the eortc qlqblm30 is explicitlydeveloped for patients with muscleinvasive urinarybladder cancerfrom theworst to the best for functional health statusand from the best to the worst for symptoms outcome variables will range from to fordifferent domains scoring rangesfatigue will be assessed using the piper fatigue scale the questionnaire consists of items and scoringranges from noneto severe the score ispresented in four domains plus a total fatigue scoreoutcome variables will range from to psychological wellbeing will be assessed using the hospital anxiety and depression scale hads thescale consists of questions with each scored on ascale from to where represents more symptomsthe questions are equally divided into the domains anxiety or depression each domain can result in a maximum score of outcome variables will range from to pain will be assessed using the numeric rating scalenrs the nrs is an eleven point scale and scoring ranges from no pain to worst pain the nrsis verbally delivereddata on length of stay at the hospital and frequency ofreadmission to hospital due to complications will be extracted from patient medical records readmissions willbe extracted as and days after surgery and complications will be registered using the claviendindo classification [ ]ethicsthe project is approved by the regional board of ethicsin stockholm dnr and the swedishethical review authority dnr the new model for rehabilitation is compared withsimilar care the patients are given in todays care delivery yet the control group will receive less attentionthan the intervention group we find it unethical to askthe patients to come to phc pay their visit and only receive for example stretching exercises in addition itwill be challenging to motivate the physiotherapists inphc to offer such treatmentsample sizefunction evaluatedthe primary outcome is physicalwith the validated 6mwt based on data from our pilotstudy we calculate an increase in m in theintervention group m in the control group and astandard deviation of m to obtain a statistical powerof with a type error set at patients areneeded in each group however as the test is highlycorrelated with sex and age we will stratify the analysis and increase the sample sizefor the secondary outcome readmissions due to complications we will estimate readmissions in theintervention group compared with the proportion of patients being readmitted today which is in the control group to obtain a statistical power of with atype error of patients are needed in eachgroup taken all this into account and guard againstdropout patients in each group will be includedin the study 0cporserud bmc cancer page of data management and study databasedata will be entered using an electronic system pheedit which is based on the sas system provided and operated by the cto at the centre for clinical cancerstudies theme cancer karolinska university hospitalsolna sweden a data management plan is delivered bycto documenting the database and all procedures fordata management the investigator verifies that all dataentries in the case report forms crfs are accurate andcorrect if certain assessments according to the protocolare not performed for any reason or if certain information is not available not applicable or unknown this willbe indicated in the crf by the investigator the investigator is required to sign off all reported datasource datain this study physicaltests movement sensor datapatientreported outcome measures and medical recordswill be regarded as source datacodingin the study database patients will be identified onlythrough the unique randomisation number all data willbe stored with coded identification and no access to thepatients id patient identification will not be revealed intext files logbooks with identification numbers and therespective codes will be stored in a locked environmentat the local centre that is not accessible to personnel notinvolved in conducting the trialstatistical analysisdescriptive statistics will be performed to ensure comparability between data at baseline to evaluate the effectof the intervention mixed models or linear regressionmodels spss inc chicago il usa according to theintention to treat procedure and with an alpha level of will be used significance of main or interaction effects will be explored using the bonferroni posthoc multiple comparison test in the case of skewed distributionlogarithmic transformations or corresponding nonparametric statistics will be used to assess the effect of theinterventionimplementation processbecause the intervention design is intricate we will inaddition to testing effects of the canmore programmeon patientlevel outcome measures also observe andgather information on factors that might have influencedthe implementation of the programme the evaluation of the implementation process will be based on themedical research council guide for process evaluationof complex interventions the knowledge gainedcan also be used to offer recommendations on whichstrategies to use when implementing the canmoreprogramme in other clinical settings and on a largescalethe initial strategies for the process evaluation will include meetings with surgeons the head of the surgicalward and phc clinics discussions and involvementwith physiotherapists and nurses at the surgical wardand physiotherapists at phc clinics and education ofthe canmore programme and outcome measures tophysiotherapists who will be involved in the intervention a leaflet for the patients an extended educationalleaflet and a short education for the physiotherapistshave been developedto study what is delivered measures of fidelity doseadaptation and reach will be assessed fidelity relative tothe canmore programme will be evaluated as the extentto which the programme was delivered as expecteddose will be assessed as the quantity of the interventionthe canmore programme and the education of physiotherapists in phc implemented adaptation such aschanges done to fit different phc settings will be reported in a questionnaire reach will be assessed regarding how many eligible patients signed an informedconsent form and how many in the intervention groupfulfilled the canmore programme in addition adverseevents will be registerediethe extentcontext includes external factors that may act as abarrier or facilitator to both implementation itself andthe patient level effect assessing barriers and facilitators to programme implementation will also involveevaluating programme feasibilitytowhich patients and health care staff regard the canmore as satisfactory in terms of content and complexitydifficulty we plan to conduct an interviewstudy on patients experiences of the intervention inaddition we plan to collect information on possiblebarriers that might have influenced the implementation ofthe programme and facilitators that mighthave supported it at the various clinical sites bothqualitative structured observationfocus groups andindividual semistructured interviews and quantitativequestionnaires and enrolment files methods will beused to assess how the intervention was delivered aswell as experiences of the different stakeholders patients physiotherapists and other health care staff aswell as managersby using several sources for data collection triangulation can be achieved which supports the trustworthinessof the study the consolidated framework for implementation research cfir will be used in the currentstudy to guide the investigation of context ie potentialbarriers and facilitators of the implementation processconstructs that we believe specifically impact the implementation outcomes in the present study and guidelinesforand observation protocolsinterview questions 0cporserud bmc cancer page of published by cifr will be followed httpcfirguidetoolshtmldiscussionto our knowledge this is the first study to examine theeffects of individually targeted exercise in phc compared with traditional advice on home exercise trainingafter rarc moreover the study will include a processevaluation of factors thought to influence the implementation of the programmealthough there is evidence that exercise is beneficialto improve physical function physical activity hrqolreduce fatigue and perhaps reduce complications it isessential to design feasible and easy to implement interventions in a health care setting our intervention is individually targeted and designed based on currentglobal guidelines for physical activity and exercise strategies for behavioural support and adapted to fitwithin the phc structure the length of the interventionis based on exercise principles and what is feasibleto conduct within phc yet this study does not tell uswhether the length of the intervention is the optimallength for best health benefits nor if a booster sessionafter the intervention period is needed the dose of exercise has been previously tested in a pilot study andshowed a positive effect on physical function was safeand had no adverse events after the pilot study werevised the exercise programme to be individually basedbut still include aerobic and musclestrengthening exerto ourcises the addition of behaviourprogrammeselfmonitoring and feedback has been shown to be associated with increased physical activity behaviour setting graded taskseg goalsupportat discharge from the hospital the standard care forpatients includes information on the importance ofphysical activity in this study the control group receivea light intervention ie recommended daily walks andlegstrengthening exercises instead of the standard carethe exercise recommendation is low dose and not specific but can still affectthe results by producing asmaller difference between the groups because there isstrong evidence for the effect of physical activity we findit unethical not to give the control group any advice onphysical activity at discharge many patients are feeblebecause of surgery and the postoperative period at hospital which can also result in fear of movement thesepatients require supervised physical exercise as in theintervention group in this studythe process evaluation using measures offidelitydose adaption adherence and reach as well as patientperspectives and experiences of the programme can helpexplain the results in additionit can speed up theprocess of translating findings from research settings toclinically representative settings the programme fitswell within the healthcare system and the exercises aregeneric to those recommended for cancer survivors andthe tools for motivational support are generic for thewhole population if the programme is proven effectiveit should be generalisable to other patient groupsthatis notthere are some limitations first due to the difficultiesof doubleblinding we have a singleblind design inwhich a physiotherapistinvolved in theprogramme is conducting the measurements secondwe foresee a long recruitment period that can lead to achange in health care staffphysiotherapists in phc toensure quality we plan to have continued contact withthe clinics and a new educational structure for trainingif neededcystectomyin summary this proposed rct has the potential toprovide new knowledge within rehabilitation after radicalcancer theprogramme should be readily applied to other patientgroups undergoing abdominal surgery for cancer andhas the potential to change the health care chain forthese patientsfor urinary bladdersupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020071405additional file exercise programmeabbreviationscanmore cancer mobilisation rehabilitation cfir consolidated frameworkfor implementation research crf case report form cto clinical trials officehads hospital anxiety depression scale hrqol healthrelated quality of lifemct movement continuum theory ms meters per second phc primaryhealth care qol quality of life rarc roboticassisted radical cystectomyrct randomised controlled trial rr responsible researcher sbas stanfordbrief activity survey sec seconds spirit standard protocol itemsrecommendations for interventional trials 6mwt sixminute walk testacknowledgementsnot applicableauthors contributionsmh and ap conceived the idea for this study and designed it along with theother authors all authors ap pk er ma lh mnb and mh were involvedin drafting and revising the manuscript all authors will be involved in datacollection analysis or manuscript preparation as the study proceeds allauthors read and approved the final manuscri | Colon_Cancer |
s case reports duplicate studies and those with insufficient available data were excludeddata extraction and managementdata were independently extracted by two investigators yao xl and wu ww according to the same inclusionand exclusion criteria disagreements were adjudicated by a third reviewer qu kthe following data will be extracted from eligible literatures the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509¢¢¢study characteristics name of the first author year of publication and sample size of included studiesparticipant characteristics tumor stage and age of patientsinterventions intervening methods and dosage administration route cycles and duration of treatment of huaiergranule¢ outcome and other data overall response rate orr disease control rate dcr overall survival osdiseasefree survival dfs quality of life qol immune indexes [cd3 cd4 cd8 natural killer cellsnk percentage and cd4cd8 cell ratios] and adverse effects we will attempt to contact the authors to request the missing or incomplete data if those relevant data are notacquired they will be excluded from the analysisquality assessmentto ensure the quality of the metaanalysis the quality of the included randomized and nonrandomized controlledtrials was evaluated according to the cochrane handbook tool and methodological index for nonrandomizedstudies minrrs supplementary table s2 respectively types of outcome measuresmain outcomesthe primary outcomes in present analysis included shortterm and longterm clinical efficacy and adverse effectsaccording to response evaluation criteria in solid tumors recist criteria i shortterm clinical efficacy the shortterm tumor response included orr and dcr orr was defined as thesum of complete and partial response rates and dcr was defined as the sum of complete response partialresponse and stable disease ratesii longterm clinical efficacy year os the time from the date of randomization to death from any cause year dfs the time from date of random assignment to date of recurrence or deathiii adverse events gastrointestinal adverse effects myelosuppression and hepatotoxicity secondary outcomesi qol qol was evaluated using the qualityoflife improved rate qir and karnofsky score kpsii immune function indicators the immune function of breast cancer patients was assessed in terms of cd3cd4 cd8 nk cells percentage and cd4cd8 cell ratiosstatistical analysisstata stata corp college station tx usa and review manager nordic cochran centre copenhagendenmark statistical software were used for statistical analyses cochranes q test and i2 statistics were used to assessheterogeneity among the studies if p01 or i2 a fixed effects model was used for the metaanalysisotherwise a random effects model was used the mantelhaenszel method will be applied for pooling of dichotomous data and results will be presented as risk ratio rr with their confidence intervals cis inverse variancemethod will be used for pooling of continuous data and results will be presented as standardized mean differencesmd with their cis a twotailed p005 was considered statistically significantthe presence of publication bias was investigated using the funnel plots beggs and eggers test if or more studiesare included in the metaanalysis [] if publication bias existed a trimandfill method should be applied tocoordinate the estimates from unpublished studies and the adjusted results were compared with the original pooledrr sensitivity analysis was performed to explore an individual studys influence on the pooled results by deleting onesingle study each time from pooled analysisresultssearch resultsthe initial search retrieved a total of s of which were excluded due to duplication after title and review s were further excluded because they were noncomparative clinical trials n19 were the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure study selection process for the metaanalysisnot related to huaier granule n9 were nonpeer reviewed s n8 were literature review or metaanalysisn3 and were case report and series n5 leaving studies as potentially eligible after detailed assessment offull texts studies with breast cancer patients n5 trials with insufficient data n8 and inappropriate criteriafor the experimental or control groups n11 were excluded ultimately trials [] involving patients with breast cancer were included in the final analysis figure patient characteristicsall included studies were performed in different medical centers in china in total patients with breast cancerwere treated using conventional methods in combination with huaier granule while patients were treated usingconventional methods alone huaier granule was manufactured by qidong gaitianli pharmaceutical co ltd andgranted a manufacturing approval number issued by the chinese sfda z20000109 study and patient characteristics are summarized in table quality assessmentquality assessment of the risk of bias is shown in figure and table the results revealed that the literature retrievedfor the present study was of medium and high quality the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509table clinical information from the eligible trials in the metaanalysisincluded studieschen qj chen y dai yg guo fd han sj lei ss liang yq li zh lu mq2017lu y qun sx ren xb shan cy tan zd tang y wang mh wang w wu yb xiong y xu f yang z2017yin x iiiiiiiviiiiinotprovidediiiiiiiiiviiiiiiviivnotprovidediiiiiiiiiiiiiiiiiiiiiiiiiviiiiiiiiiiiinotprovidedtumorstagepatientsconexpage year control vsexperimentalintervening methods range median ct vs cthuaier granules vs ct vs cthuaier granulesoa meanoadosage ofhuaiergranulesduration oftreatments20gtime times monthcourse daycourses20gtime times weeks for a course daycourses months vs range vs vs meanmeanct vs cthuaier granules20gtime timesoadayct vs cthuaier granules20gtime times monthoadayct vs cthuaier granules20gtime times weeks for a course oadaycourses yearsnot providedct vs cthuaier granules20gtime timesoaday vs range vs vs meanmeanct vs cthuaier granules20gtime times monthsoadayct vs cthuaier granules20gtime times months for a course oadaycoursesct vs cthuaier granules20gtime times weeks for a course oadaycourses vs medianct vs cthuaier granules20gtime times weeks for a course not providedct vs cthuaier granules20gtime timesoadaycourses years mean vs vs vs meanmeanoadayct vs cthuaier granules20gtime times weeksoadayct vs cthuaier granules20gtime times weeks for a course oadayct vs cthuaier granules20gtime timesoadaycoursesnot providednot providedct vs cthuaier granules20gtime times monthoadaynot providedct vs cthuaier granules20gtime times months vs meanoadayct vs cthuaier granules20gtime times monthsoaday range mean ct vs cthuaier granules20gtime times monthsoaday vs rangect vs cthuaier granules20gtime times weeks for a course vs range vs vs meanmeanoadayct vs cthuaier granules20gtime timesoadaycoursesnot providedct vs cthuaier granules20gtime times weeks for a course oadayct vs cthuaier granules20gtime timesoadaycourses monthparametertypes1cid2 3cid21cid2 3cid25cid22cid22cid2 3cid25cid24cid2 5cid22cid2 3cid25cid22cid24cid2 5cid23cid2 4cid25cid24cid2 5cid21cid2 2cid24cid22cid2 3cid22cid2 3cid24cid25cid22cid23cid22cid22cid2 5cid25cid24cid2 5cid22cid22cid22cid21cid2 5cid22cid22cid2 3cid2zhang jg iiii vs rangect vs cthuaier granules20gtime times monthsoadayzhang y notprovidedzhao zw iiiiv vs range vs meanct vs cthuaier granules20gtime times monthsoadayct vs cthuaier granules20gtime times weeksoadayzhong sw zhou p iviiii74range median ct vs cthuaier granules20gtime times monthsoaday vs rangect vs cthuaier granules20gtime times monthsoadaynotes control group conventional treatments alone group experimental group conventional treatments and huaier granule combined group1cid2 overall response rate and disease control rate 2cid2 overall survival or diseasefree survival 3cid2 adverse events 4cid2 quality of life 5cid2 immunefunction indexabbreviations ct conventional treatments oa oral administration the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure risk of bias summaryreview of authors judgments about each risk of bias item for included studies note each color represents a different level ofbias red for highrisk green for lowrisk and yellow for unclearrisk of biastable quality assessment of nonrandomized comparative studiesnonrandomized studiesadditional criteria in comparativestudytotalabcdefghijklstudyguo fdhan sjlei ssren xbwu ybyin xzhang yzhongsw zhou pa a clearly stated aim b inclusion of consecutive patients c prospective collection of data d endpoints appropriate to the aim of the study eunbiased assessment of the study endpoint f followup period appropriate to the aim of the study g loss to follow up less than h prospectivecalculation of the study size i an adequate control group j contemporary groups k baseline equivalence of groups l adequate statistical analysesnotes the items are scored not reported reported but inadequate and reported and adequatetherapeutic efï¬cacy assessmentsorr and dcrfour clinical trials involving patients compared orr and dcr between the two groups as shown in figure the pooled results revealed that patients who underwent combination therapy experienced improved orr rr ci p002 and dcr rr ci p019 compared with those whoreceived conventional treatments alone although the dcr did not reach significant difference dcr p095 i2 was not heterogeneous among the studies therefore a fixedeffect model was used to analyze rr otherwise arandomeffect model was used the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of orr and dcr between experimental and control groupforest plot of the comparison of orr a and dcr b between the experimental and control group control group conventionaltreatment alone group experimental group conventional treatment and huaier granule combined grouplongterm survival1year 2year 3year and 5year oseleven clinical trials with breast cancer patients reported os figure metaanalysis revealed that the 2yearrr ci p002 3year rr ci p00001 and 5year os rr ci p0004 of patients in the combined treatment group were significantly prolongedcompared with the control group there was statistical heterogeneity in 1year os p009 i2 and 2year osp00001 i2 according to the heterogeneity test therefore a randomeffect model was used to pool thismetaanalysis otherwise the fixedeffect model was used1year 2year 3year and 5year dfsten clinical trials with breast cancer patients reported dfs figure metaanalysis revealed that the 1yearrr ci p0003 2year rr ci p000001 3year rr ci p000001 and 5year dfs rr ci p003 of patients in thecombined treatment group were all significantly prolonged compared with the control group there was statisticalheterogeneity in 5year dfs p005 i2 according to the heterogeneity test therefore a random effectsmodel was used to pool this metaanalysis otherwise the fixedeffect model was usedqol assessmentfour trials with participants evaluated qir and three trials including patients reported kps data figure results demonstrated that the qol of breast cancer patients in the combined group was significantly better than thatof the control group indicated by significantly increased qir rr ci p000001 and kpsrr ci p000001 qir p084 i2 was not heterogeneous among the studiestherefore a fixedeffect model was used to analyze rr otherwise a randomeffect model was usedimmune function evaluationimmune status of the patients was examined between the two groups in eleven controlled studies including patients figure the percentages of cd3 cd3 rr ci p005 cd4 rr ci p000001 and nk cells rr ci p00001 and cd4cd8 ratio rr ci p000001 in the combined treatment group were significantly increased compared with the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of os between experimental and control groupforest plot of the comparison of 1year a 2year b 3year c and 5year os d between the experimental and control groupcontrol group conventional treatment alone group experimental group conventional treatment and huaier granule combinedgroup the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of dfs between experimental and control groupforest plot of the comparison of 1year a 2year b 3year c and 5year dfs d between the experimental and control groupcontrol group conventional treatment alone group experimental group conventional treatment and huaier granule combinedgroup the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of qol between experimental and control groupforest plot of the comparison of qir a and kps b between the experimental and control group control group conventionaltreatment alone group experimental group conventional treatment and huaier granule combined groupthose in the conventional treatment alone group whereas the proportions of cd8 rr ci to p033 did not differ significantly between the two groups a randomeffect model was used to pool thismetaanalysis due to significant heterogeneityassessment of adverse eventsas shown in figure patients treated with huaier granule and conventional methods exhibited lower incidences ofmyelosuppression rr ci p0001 and hepatotoxicity rr ci p005 whereas analysis of gastrointestinal adverse effects rr ci p014 leukopeniarr ci p006 nausea and vomiting rr ci p052 andalopecia rr ci p020 did not differ significantly between the two groups there wasstatistical heterogeneity in gastrointestinal adverse effects p006 i2 according to the heterogeneity testand a random effects model was used to pool this metaanalysis otherwise the fixedeffect model was usedpublication biasas shown in figure the funnel plots beggs and eggers regression tests results showed that there was publicationbias in cd4cd8 ratio begg egger to determine whether bias affected the pooled risk ofcd4cd8 ratio a trimandfill analysis was performed the adjusted rr indicated a trend similar to the resultsof the primary analysis before p00001 after p00001 reflecting the reliability of the primary conclusionsparameters discussed less than papers were not conducted publication bias analysessensitivity analysisas figure signified the results revealed that no individual studies significantly affected the primary indicatorscd4 and cd4cd8 ratio which indicated statistically robust results parameters discussed less than paperswere not conducted sensitivity analysesdiscussionhuaier granule the active ingredient of huaier extract appears as a lightyellow powder through hotwater extractionethanol precipitation deproteinization and lyophilization procedures as a type of tcbp huaier granule hasbeen clinically applied as an effective adjuvant drug in cancer treatment for decades although several studies havereported that addition of huaier granule could be beneficial to patients with advanced breast cancer but the the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of immune function between experimental and control groupforest plot of the comparison of immune function indicators including cd3 a cd4 b cd8 c and nk d cells percentage andcd4cd8 ratio e between the experimental and control group control group conventional treatment alone group experimentalgroup conventional treatment and huaier granule combined group the random effects metaanalysis model inverse variancemethod was used the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of adverse effects between experimental and control groupforest plot of the comparison of adverse effects including gastrointestinal adverse effects a myelosuppression b hepatotoxicity c leukopenia d nausea and vomiting e and alopecia f between the experimental and control group control groupconventional treatment alone group experimental group conventional treatment and huaier granule combined group the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure funnel plot of cd4 a and cd4cd8 bexact therapeutic effects have yet to be systematically evaluated thus indepth knowledge of the efficacy and safetyof huaier granule is needed this systematic review will provide a helpful evidence for clinicians to formulate thebest postoperative adjuvant treatment strategy for patients with breast cancer and also provide scientific clues forresearchers in this fielddata from trials [] including patients with breast cancer were included in our metaanalysishuaier granule in all of the included studies was manufactured by qidong gaitianli pharmaceutical co ltd thedosages of huaier granule were g per day via oral administration the pooled results revealed that the combinationof huaier granule and conventional treatment for breast cancer achieved more beneficial effects compared withthose treated solely with conventional therapy compared with conventional treatment alone huaier granule couldsignificantly improve orr and qol in patients with breast cancer p005 the study also assessed whether huaiergranule could prolong the longterm survival rates of breast cancer patients and the results showed that the and 5year os and and 5year dfs of patients were all significantly prolonged compared with the controlgroup these results indicated that using huaier granule could improve the short and longterm curative effects ofconventional treatment for breast cancert lymphocyte subsets cd3 cd4 cd8 cell subsets and cd4cd8 ratio and nk cells play an importantrole in antitumor immunity studies have shown that patients with advanced cancer showed decreased immunefunction and nk activity and exhibiting imbalance of t lymphocytes percentage many studies have reportedthat huaier granule can enhance the ability of the bodys immunity and resistance to tumors our analysisdemonstrated that the percentages of cd3 cd4 and nk cells and cd4cd8 ratio were all significantly increasedin breast cancer patients treated with huaier granule indicating that immune function of breast cancer patients wasimproved after huaier granule adjuvant therapysafety is the top priority of clinical treatment seven clinical trials with breast cancer patients reported adverse events according to world health anization standards metaanalysis revealed that patients who underwent huaier granule plus conventional treatment demonstrated a lower risk for myelosuppression and hepatotoxicitycompared with conventional treatment alone whereas analysis of other toxic side effects did not differ significantlytherefore huaier granule appears to be a safe auxiliary antitumor medicine for individuals with breast cancerthere were some limitations to our analysis currently five clinical trials table in which breast cancer are being treated by huaier granule in conjunction with conventional regimens have been registered onclinicaltrialsgov nct02615457 and nct02627248 and chinese clinical trial register chictr1800015390chictroic16007737 and chictrtrc11001250 however except for two studies most of the includedtrials were not registered before the first participant enrolled second as an important chinese patent medicinehuaier granule was mainly applied in china which may bring an unavoidable regional bias and subsequently influence the clinical application of huaier granule worldwide third different trials evaluated the treatment efficacy withdifferent outcomes resulting in a reduction in the size of the statistical sample making it difficult to summarize theresults at the same scale fourth several results demonstrated significant heterogeneity among the included trialswhich may be due to the different tumor stage tumor subtypes ages of the breast cancer patients and duration oftreatment however based on the currently available literature there are insufficient data to perform more statistical analysis to evaluate correlations in addition the efficacy of monotherapy of huaier granule in the treatment of the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure sensitivity analysis for cd4 a and cd4cd8 b the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509table search results of clinical trial registrationregistrationnumbernct02615457nct02627248chictr1800015390chictroic16007737chictrtrc11001250titlephaseconditionsinterventionslocationshuaier granule intreating women withtriple negative breastcancerneoadjuvantchemotherapy with orwithout huaiergranule in treatingwomen with locallyadvanced breastcancer that can beremoved by surgeryhuaier granule forstage ii and iii triplenegative breastcancer with lymphnode metastasis amulticenterrandomizeddoubleblindplacebocontrolledclinical triala multicenter doubleblind randomizedplacebocontrolledstudy on stage iiiiitriplenegative breastcancer with lymphnode metastasistreated by huaiergranulesextract of fungi ofhuaier used for triplenegtive breastcancera prospectiverandomized controlledtrialivivtriple negative breasthuaier granuleqilu hospital ofcancershandong universityjinan shandong chinabreast cancerhuaier granule otherqilu hospital ofchemotherapyshandong universityjinan shandong chinaivtriple negative breasthuaier granulecancerthe first aï¬iliatedhospital of amusouthwest hospitalchongqing chinaibreast cancerhuaier granuleivtriple negative breasthuaier granulecancersouthwest hospital thethird military medicaluniversity chongqingchinasouthwest hospital thethird military medicaluniversity chongqingchinabreast cancer also needs highquality evidence to verify however up to now huaier granule is mainly combinedwith radiotherapy chemotherapy or surgery and other conventional treatment methods for breast cancer we willkeep paying close attention to upcoming highquality clinical trials in our later studies and carry out further analyseson studies conducted huaier granule monotherapy against breast cancer finally publication bias was exists in someindicators which might because some authors tended to deliver positive results of s to editors therefore anyconclusions need to be made with cautionconclusionin summary findings of this metaanalysis indicate that the combination of huaier granule and conventional treatment is effective in treating patients with breast cancer the clinical application of huaier granule not only clearlyenhanced the therapeutic effects of conventional treatment but also effectively improved qol and immune functionin patients with breast cancer thus we anticipate that our study will provide valuable evidence for further evaluationof huaier granule on the other hand the low quality of some of the included publications increased the risk of biaswhich to some extent affects the reliability of this research therefore additional studies with highquality evidenceto verify the effectiveness of huaier granulemediated therapy for breast cancer are warrantedcompeting intereststhe authors declare that there are no competing interests associated with the manuscriptfundingthis study was supported by grants from national science foundation of china [grant numbers and ] the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc by 0cbioscience reports bsr20202509101042bsr20202509author contributionxi w and yao xl conceived and designed the methods yao xl and wu ww extracted the original data and drafted themanuscript yao xl wu ww and qu k performed statistical analysis xi w and yao xl interpreted results xi w and quk revised the manuscript all authors had full access to all data in the study and take responsibility for the integrity of the data andthe accuracy of data analysisabbreviationscbm chinese biological medicine database ci confidence interval cnki china national knowledge infrastructure csjdchinese scientific journal database dcr disease control rate dfs diseasefree survival kps karnofsky performance scoreminrrs methodological index for nonrandomized studies nk natural killer cells orr overall response rate os overallsurvival prisma preferred reporting items for systematic reviews and metaanalyses qir qualityoflife improved rate qolquality of life rct randomized controlled trial recist response evaluation criteria in solid tumors rr risk ratio sfdastate food and drug administration smd standardized mean difference tcbp traditional chinese biomedical preparationreferences ferlay j colombet m soerjomataram i mathers c parkin dm pi Ëneros m estimating the global cancer incidence and mortality in globocan sources and methods int j cancer 101002ijc31937 bray f ferlay j soerjomataram i siegel rl torre la and jemal a global cancer statistics globocan estimates of incidenceand mortality worldwide for cancers in countries ca cancer j clin 103322caac21492 watkins ej overview of breast cancer jaapa 10109701jaa0000580524957333d akram m iqbal m daniyal m and khan au awareness and current knowledge of breast cancer biol res 101186s4065901701409 reeder jg and vogel vg breast cancer prevention cancer treat res 1010079780387731612Ë waks ag and winer ep breast cancer treatment jama 101001jama201820751 peart o breast intervention and breast cancer treatment options radiol technol 535m558m quiz merino bonilla ja torres tabanera m and ros mendoza lh breast cancer in the 21st century from early detection to new therapiesradiologia 101016jrx201706003 greenlee h dupontreyes mj balneaves lg carlson le cohen mr deng g clinical practice guidelines on the evidencebaseduse of integrative therapies during and after breast cancer treatment ca cancer j clin 103322caac21397 wong ky tan ey chen jj teo c and chan pm the use of traditional chinese medicine among breast cancer patients implications forthe clinician ann acad med singapore zhu l li l li y wang j and wang q chinese herbal medicine as an adjunctive therapy for breast cancer a systematic review andmetaanalysis evid based complement alternat med wang w xu l and shen c effects of traditional chinese medicine in treatment of breast cancer patients after mastectomy ametaanalysis cell biochem biophys 101007s120130140348z mcpherson l cochrane s and zhu x current usage of traditional chinese medicine in the management of breast cancer a practitionersperspective integr cancer ther 1011771534735415607656 chen y wu h wang x wang c gan l zhu j huaier granule extract inhibit the proliferation and metastasis of lung cancer cellsthrough downregulation of mtdh jak2stat3 and mapk signaling pathways biomed pharmacother 101016jbiopha201802028 hu z yang a fan h wang y zhao y zha x huaier aqueous extract sensitizes cells to rapamycin and cisplatin through activatingmtor signaling j ethnopharmacol 101016jjep201603069 su d zhang x zhang l zhou j and zhang f a randomized doubleblind controlled clinical study on the curative effect of huaier onmildtomoderate psoriasis and an experimental study on the proliferation of hacat cells biomed res int 10115520182372895 wang m hu y hou l pan q tang p and jiang j a clinical study on the use of huaier granules in postsurgical treatment oftriplenegative breast cancer gland surg 1021037gs20191208 chen q shu c laurence ad chen y peng bg zhen zj effect of huaier granule on recurrence after curative resection of hcc amulticentre randomised clinical trial gut 101136gutjnl2018315983 zhang y wang x and chen t efï¬cacy of huaier granule in patients with breast cancer clin transl oncol 101007s1209401819594 zhao gs liu y zhang q li c zhang yw ren zz transarterial chemoembolization combined with huaier granule for thetreatment of primary hepatic carcinoma safety and efï¬cacy medicine baltimore e7589 101097md0000000000007589 zhou l pan lc zheng yg du gs fu xq zhu zd novel strategy of sirolimus plus thymalfasin and huaier granule on tumorrecurrence of hepatocellular carcinoma beyond the ucsf criteria following liver transplantation a single center experience oncol lett sun y sun t wang f zhang j li c chen x a polysaccharide from the fungi of huaier exhibits antitumor potential andimmunomodulatory effects carbohydr polym 101016jcarbpol201209006 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc by 0cbioscience reports bsr20202509101042bsr20202509 xie hx xu zy tang jn du ya huang l yu pf effect of huaier on the proliferation and apoptosis of human gastric cancer cellsthrough modulation of the pi3kakt signaling pathway exp ther med 103892etm20152600 moher d liberati a tetzlaff j and altman dg preferred reporting items for systematic reviews and metaanalyses the prisma statementj clin epidemiol 101016jjclinepi200906005 zeng x zhang y kwong js zhang c li s sun f the methodological quality assessment tools for preclinical and clinical studiessystematic review and metaanalysis and clinical practice guideline a systematic review j evid based med 101111jebm12141 slim k nini e forestier d kwiatkowski f panis y and chipponi j methodological index for nonrandomized studies minorsdevelopment and validation of a new instrument anz j surg 101046j14452197200302748x schwartz lh litiere s de vries e ford r gwyther s mandrekar s recist 11update and clariï¬cation from the recistcommittee eur j cancer 101016jejca201603081 jackson d white ir and riley rd quantifying the impact of betweenstudy heterogeneity in multivariate met | Colon_Cancer |
objectives paired box protein8 pax8 immunohistochemical expression can be used as a diagnostic marker for epithelial cells tumors this study aimed at investigating the immunohistochemical expression of pax8 among sudanese females diagnosed with cervical endometrial and ovarian cancers between december and may by studying their formalinfixed paraffin embedded blocksresults sixty patients diagnosed with female reproductive tract cancers were included who aged ± years range cervix was the most common cancer site in patients regarding cancer stage there was and of the study population had stage 3b and 2b respectively the histopathological diagnosis included and poorly moderately and well differentiated cervical squamous cell carcinoma scc as well as and endometrial adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma respectively pax8 was positively expressed in endometrial adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma poorly and moderately differentiated scc all patients diagnosed with well differentiated scc and metastatic adenocarcinoma showed no expression of pax8 a statistically significant was seen for pax8 expression and the different histopathological diagnosis p value keywords female reproductive cancer paired box protein8 immunohistochemical expressionintroductionpaired box protein8 pax8 is a member of the family paired box proteins paxs [ ] pax8 consists of amino acids with a molecular weight of approximately kilo dalton and its molecular properties are located on chromosome 2q13 [] pax8 is a transcription factor that regulates ans development during the embryonic period as well as to maintain normal cellular functions in some cells after birth [ ] during the embryonic period pax8 also plays a significant role correspondence nouh_saadoutlookcom alfarrabi college for science and technology khartoum sudanfull list of author information is available at the end of the in the development of genital ans derived from the mesonephric and the m¼llerian ducts [] in a previous experiment the deletion of the pax8 gene resulted in dysfunctional uterus absence of the endometrium and the vaginal opening also resulted in poor development of the myometrial tissue several studies have described the immunohistochemical utility of pax8 as a diagnostic marker for epithelial cells neoplasms of many glands and ans such as thyroid thymus and kidney as well as some female reproductive tract tumors [ ]in a healthy female reproductive tract pax8 shown to be overexpressed in the epithelial cells of the endocervix and the endometrium [] pax8 was found to be expressed among endometrioid carcinomas transitional the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cali a0et a0al bmc res notes page of undifferentiated cell carcinomas and the metastatic carcinomas at a range of and [ ] whereas for the ovarian carcinomas pax8 was under expressed considering that few studies have investigated the immunohistochemical expression of pax8 in carcinomas of the endometrium and uterine cervix in the different parts of the world but none from sudan yet [ ] this study aimed at investigating the immunohistochemical expression of pax8 among sudanese females diagnosed with cervical endometrial and ovarian carcinomasmain textmaterials and a0methodsstudy design and a0population characteristicsthis is a descriptive retrospective hospital based study conducted at different histopathology laboratories during the period from december till may in khartoum state sudan we retrieved archived formalin fixed paraffin embedded blocks previously collected from female patients with cervical endometrial or ovarian carcinomas the retrieved formalin fixed paraffin blocks represent all the female population admitted at the hospitals for reproductive malignancies diagnosis the participants demographic data was collected including age place of residence the clinical data including site of cancer cancer grade and the histopathological diagnosis were also collectedsections preparation for a0immunohistochemistry stainingtwo sections were cut using rotary microtome leica germany from each histopathological block then one slide was stained by hematoxylin and eosin staining technique the other slide was mounted onto 3aminopropyltriethoxysilane coated slides for immunohistochemistry to retrieve pax8 tissues antigen we treated the sections with citrate buffer at ° a0c for a0min in a waterbath then the tissue sections were rinsed first in distilled water and later with tris buffer saline tbs this was followed by treatment with peroxidase block hydrogen peroxide in methyl alcohol for a0min to quench endogenous peroxidase activity the slides were then placed in a humid chamber then the slides were drained and rinsed in two successive changes of tris buffer wash buffer for a0 min each nonspecific proteinprotein interactions were blocked by incubating and treating the tissue sections in a humid chamber with the power block casein in phosphate buffered saline for a0 min then the remaining solution was drained from the slides the sections were then incubated in the primary antibody pax8 antipax8 rabbit antihuman monoclonal antibody ab189249 abcam united kingdom at room temperature in the humid chamber according to the manufacture instructionsobserving the yellowishbrown or brown appearance of the nucleus was considered a positive result for the pax8 for the negative control we omitted the incubation with the primary antibody step instead we incubated the section in the phosphate buffer saline pbsresults interpretationsfor the interpretation of the results we depended on the intensity as well as the number of the cells that expressed the marker and the expression was graded into categories negative no staining less than of the cells were expressing the marker of the cells were expressing the marker more than of the cells were expressing the marker more than of the cells were expressing the marker the slides were interpreted and validated by two expert pathologists blindly of each other results photomicrographs were taken using olympus sp350 camera olympus imaging america inc usastatistical analysisthe statistical analysis of the results was done using ibm spss statistics vs the chisquared test was performed to compare the frequencies of categorical variables statistical significance level was defined as p value at confidence intervalresultscharacteristics of a0the a0study participantsthe study included patients diagnosed with female genital tract cancer patients aged ± a0years range a0years patients were grouped into age groups those aged a0 years constituted half of the study participants the remaining were and patients distributed across the remaining age groups of a0 years a0 years and a0 years respectively according to patients place of residence patients were originating from the four regions of sudan most of the patients were from western part of sudan followed by from the central part of sudanregarding the site of cancer the cervix was the most commonly involved patients there were and endometrial and ovarian cancer respectively based on the international federation of gynecology and obstetrics figo cancer grading the majority of the study population was diagnosed with stage 3b and 2b cancer and of the patients respectively the were and stage 4b 3a 2a 1b and 4a respectively 0cali a0et a0al bmc res notes page of no statistically significant association between figo staging and age group was found p value histologically there were squamous cell carcinoma scc all of which were cervical cancers and adenocarcinoma scc and adenocarcinoma were further classified into poorly differentiated scc moderately differentiated scc and well differentiated scc endometrium adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinomabased on age groups age group showed no statistically significant relationship with either patients place of residence cancer site cancer histological type figo staging and cancer histopathological type table a0immunohistochemical expression of a0paxthe immunohistochemical expression of pax8 was shown as a yellowishbrown or brown staining of the nucleus fig a0 based on site of cancer all endometrium carcinoma showed positive expression of pax8 with p value there were only patients who had positive expression of pax8 including adenocarcinoma and scc a statistically significant difference was noted for the pax8 staining and cancer type with p value the analysis of pax8 staining results based on the histopathological diagnosis showed that all patients who were diagnosed with well differentiated scc and metastatic adenocarcinoma had negative results for the pax8 expression while of the endometrium adenocarcinoma were found positive for the pax8 expression a statistically significan was t seen for pax8 expression and the different histopathological diagnosis p value table a0table classification of a0participants demographic and a0clinical diagnosis based on a0age groupage group no total no p value a0years a0years a0years a0yearsresidence of patient central sudan east sudan west sudan north sudansite of cancer cervix endometrium ovarycancer histological type scc adenocarcinomafigo staging stage stage 2a stage 2b stage 3a stage 3b stage 4a stage 4bhistopathological cancer grades well differentiated scc poorly differentiated scc moderately differentiated scc endometrium adenocarcinoma endocervical adenocarcinoma metastatic adenocarcinoma ovarian mucinous cyst adenocarcinomascc squamous cell carcinoma 0cali a0et a0al bmc res notes page of fig immunohistochemical expression of pax8 among the different histopathological cancer types and grades the immunohistochemical expression of pax8 is shown as a yellowishbrown or brown staining of the nucleus a well differentiated scc negative b metastatic adenocarcinoma negative c poorly differentiated scc positive d moderately differentiated scc positive e endometrium adenocarcinoma positive f ovarian mucinous cystadenocarcinoma positive g endocervical adenocarcinoma positive and h endometroid adenocarcinoma positive 0cali a0et a0al bmc res notes page of table association of a0clinical diagnosis and a0the a0immunohistochemical expression of a0pax8pax results no total no p valuepositivenegativecancer histological type scc adenocarcinomacancer site cervix endometrium ovaryfigo staging stage stage 2a stage 2b stage 3a stage 3b stage 4a stage 4bcancer histopathological grading well differentiated scc poorly differentiated scc moderately differentiated scc endometrium adenocarcinoma endocervical adenocarcinoma metastatic adenocarcinoma ovarian mucinous cyst adenocarcinomascc squamous cell carcinoma discussionprevious studies on the immunohistochemical expression of pax8 in the normal female reproductive tract showed that pax8 was expressed in the endometrial endocervical and ovarian epithelial cells as well as in nonciliated epithelial cells of the fallopian tubes [ ] this study investigated the immunohistochemical expression of pax8 in sudanese patients who were diagnosed with female reproductive tract cancers patients on the 5th decade of life were constituting half of the study participants with no statistically significant association between age group and the type of cancer however previous studies had suggested other risk factors which could contribute in the development of certain gynecological cancer [ ]regarding the place of residence the majority of patients coming from western sudan this result is in contrary with a previous study in sudan conducted by saeed et a0al in which they showed that the percentage of patients suffering from different types of cancers residing in central and northern sudan were higher compared to the other regions in sudan nevertheless these findings could suggest the involvement of environmental risk factors however the limited study samples size is insufficient to support this suggestion therefore further research with a larger samples size investigating the potential environmental risk factors is essential for strategic prevention and protection measuresthe reported number of female patients with cervical cancer was high compared to ovarian and endometrium cancer similar results were seen previously among sudanese females also the high frequency of stages 3b and 2b compared to the other stages were comparable to previous study conducted in sudan this similarity underscores a delayed response among sudanese females in seeking healthcare and urge the need for health promotion and education to encourage young sudanese females for the early signs detection and seeking healthcare as early as possible for a better treatmentregarding the classification based on the histopathological diagnosis most of the female diagnosed with scc this result was also similar to previous study investigated the prevalence of the different gynecologic cancer in sudan however the expression of pax8 among the studied samples was relatively low compared to previous studies [ ] this could be attributed 0cali a0et a0al bmc res notes page of to the site of cancer development while agrees with another study where pax8 was expressed only in patient interestingly a high frequency of pax8 expression was noted among females diagnosed with endometrium cancer compared to scc this finding is in contrary with a previous report where pax8 was expressed among only of the studied samples also the result was strongly in accordance with other studies [ ] besides that the lack of pax8 expression among those who were diagnosed with well differentiated scc and metastatic adenocarcinoma could play a significant role in either gynecologic cancer differentiation or in detection of endometrium adenocarcinoma progression to metastatic adenocarcinoma [ ]conclusionalthough pax8 showed a significant expression among adenocarcinomas lesions and negative expression was noted among those with well differentiated scc and metastatic adenocarcinoma pax8 might not be beneficial when used alone as a diagnostic marker for the tumors that occur in the female reproductive tractlimitations¢ the small sample size investigated in this study reduced the ability of using the expression of pax8 as a diagnostic marker therefore a largescale study is needed and it should include other types of malignant tumors encountered in the female reproductive systemacknowledgementsthe authors would like to acknowledge the medical staff for their interest and cooperation during the study and thanks to all who participated in completing this studyauthors contributionseta nsm and ees provided conceptual framework for the study guidance for interpretation of the data and performed data analysis eta ees irs lah and amm performed laboratory work nsm ees msm aay and aa performed the statistical analysis nsm msm ees and aa participated in the manuscript preparation revision and coordination all authors read and approved the final manuscriptfundingnot applicableavailability of data and materialsthe datasets used andor analyzed during the current study are available from the corresponding author on reasonable requestethics approval and consent to participateethical approval was obtained from the research ethics committee of the faculty of medical laboratory sciences university of khartoum sudan ethical approval no fmlsrec002042 all participant approved to participate by signing an informed consentconsent for publicationnot applicablecompeting interestsno competing interests to discloseauthor details department of histopathology and cytology faculty of medical laboratory sciences university of khartoum khartoum sudan department of histopathology and cytology faculty of medical laboratory sciences national university khartoum sudan alfarrabi college for science and technology khartoum sudan faculty of medicine sinnar university sennar sudan molecular biology department faculty of medical laboratory sciences nile university khartoum sudan faculty of dentistry ibn sina university khartoum sudan department of neurology mayo clinic jacksonville fl usa department of radiology mayo clinic jacksonville fl usa institute of endemic diseases university of khartoum khartoum sudan mycetoma research center university of khartoum khartoum sudan faculty of medicine nile university khartoum sudan received july accepted august references gruss p walther c pax in development cell mansouri a hallonet m gruss p pax genes and their roles in cell differentiation and development curr opin cell biol macchia pe lapi p krude h pirro mt missero c chiovato l souabni a baserga m tassi v pinchera a pax8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis nat genet vilain c rydlewski c duprez l heinrichs c abramowicz m malvaux p renneboog bt parma j costagliola s vassart g autosomal dominant transmission of congenital thyroid hypoplasia due to lossoffunction mutation of pax8 j clin endocrinol metab park s vk c genetics of congenital hypothyroidism j med genet dahl e koseki h balling r pax genes and anogenesis bioessays lang d powell sk plummer rs young kp ruggeri ba pax genes roles in development pathophysiology and cancer biochem pharmacol stoykova a gruss p roles of paxgenes in developing and adult brain as suggested by expression patterns j neurosci mittag j winterhager e bauer k grummer r congenital hypothyroid female pax8deficient mice are infertile despite thyroid hormone replacement therapy endocrinology bouchard m de caprona d busslinger m xu p fritzsch b pax2 and pax8 cooperate in mouse inner ear morphogenesis and innervation bmc dev biol mittag j winterhager e bauer k grummer rje congenital hypothyroid female pax8deficient mice are infertile despite thyroid hormone replacement therapy endocrinolog laury ar perets r piao h krane jf barletta ja french c chirieac lr lis r loda m hornick jl a comprehensive analysis of pax8 expression in human epithelial tumors am j surg pathol wong s hong w hui p buza n comprehensive analysis of pax8 expression in epithelial malignancies of the uterine cervix int j gynecol pathol ozcan a shen ss hamilton c anjana k coffey d krishnan b truong ld pax expression in nonneoplastic tissues primary tumors and metastatic tumors a comprehensive immunohistochemical study mod pathol bowen nj logani s dickerson eb kapa lb akhtar m benigno bb mcdonald jf emerging roles for pax8 in ovarian cancer and endosalpingeal development gynecol oncol 0cali a0et a0al bmc res notes page of ozcan a liles n coffey d shen ss truong ld pax2 and pax8 expression in primary and metastatic m¼llerian epithelial tumors a comprehensive comparison am j surg pathol distinguishing ovarian mucinous neoplasms from colonic and appendiceal mucinous neoplasm bmc res notes nesrin r kilic d risk factors for cervical cancer results from a hospital ozcan a liles n coffey d shen ss truong ldjtajosp pax2 and pax8 based casecontrol study int j hematol oncol expression in primary and metastatic m¼llerian epithelial tumors a comprehensive comparison am j surg pathol nonaka d tang y chiriboga l rivera m ghossein r diagnostic utility of thyroid transcription factors pax8 and ttf2 foxe1 in thyroid epithelial neoplasms mod pathol tacha d zhou d cheng l expression of pax8 in normal and neoplastic tissues a comprehensive immunohistochemical study appl immunohistochem mol morphol bowen nj logani s dickerson eb kapa lb akhtar m benigno bb mcdonald jfjgo emerging roles for pax8 in ovarian cancer and endosalpingeal development gynecol oncol k¶bel m kalloger se boyd n mckinney s mehl e palmer c leung s bowen nj ionescu dn rajput a ovarian carcinoma subtypes are different diseases implications for biomarker studies plos medicine 2008512e232 nonaka d chiriboga l soslow ra expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas am j surg pathol tong gx devaraj k hamelebena d yu wm turk a chen x wright jd greenebaum e pax8 a marker for carcinoma of m¼llerian origin in serous effusions diagn cytopathol laury ar perets r piao h krane jf barletta ja french c chirieac lr lis r loda m hornick jljtajosp a comprehensive analysis of pax8 expression in human epithelial tumors am j surg pathol tong gx devaraj k hamelebena d yu wm turk a chen x wright jd greenebaum ejdc pax8 a marker for carcinoma of m¼llerian origin in serous effusions diagn cytopathol chu pg chung l weiss lm lau sk determining the site of origin of mucinous adenocarcinoma an immunohistochemical study of cases am j surg pathol brunner ah riss p heinze g meltzow e brustmann h immunoexpression of pax in endometrial cancer relation to highgrade carcinoma and p53 int j gynecol pathol ozcan a shen ss hamilton c anjana k coffey d krishnan b truong ldjmp pax expression in nonneoplastic tissues primary tumors and metastatic tumors a comprehensive immunohistochemical study mod pathol aldaoud n erashdi m alkhatib s abdo n almohtaseb a graboskibauer a the utility of pax8 and satb2 immunohistochemical stains in saeed me cao j fadul b kadioglu o khalid he yassin z mustafa sm saeed e efferth t a fiveyear survey of cancer prevalence in sudan anticancer res saeed me cao j fadul b kadioglu o khalid he yassin z mustafa sm saeed e efferth tjar a fiveyear survey of cancer prevalence in sudan anticancer res mohamed keh ashmeig aaa cervical cancer our experience in sudan philadelphia aacr elhasan lme bansal d osman of enan k abd farag eab prevalence of human papillomavirus type in sudanese women diagnosed with cervical carcinoma j cancer res ther tacha d zhou d cheng ljai morphology m expression of pax8 in normal and neoplastic tissues a comprehensive immunohistochemical study appl immunohistochem mol morphol ord³±ez ng value of pax immunostaining in tumor diagnosis a review and update adv anat pathol gailey mp bellizzi am immunohistochemistry for the novel markers glypican pax8 and p40 δnp63 in squamous cell and urothelial carcinoma am j clin pathol yemelyanova a gown am holmes bj ronnett bm vang r pax8 expression in uterine adenocarcinomas and mesonephric proliferations int j gynecol pathol liang l zheng w liu j liang sx assessment of the utility of pax8 immunohistochemical stain in diagnosing endocervical glandular lesions arch pathol lab med wong s hong w hui p buza njijogp comprehensive analysis of pax8 expression in epithelial malignancies of the uterine cervix int j gynecol pathol de andrade dap da silva vd de macedo mg de lima ma de andrade vm andrade cemc schmidt rl reis rm dos reis r squamous differentiation portends poor prognosis in low and intermediaterisk endometrioid endometrial cancer plos one 20191410e0220086publishers notespringer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations ¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your ï¬eld¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold open access which fosters wider collaboration and increased citations maximum visibility for your research over 100m website views per year ¢ at bmc research is always in progresslearn more biomedcentralcomsubmissionsready to submit your research choose bmc and benefit from 0c' | Colon_Cancer |
the development of fungal fruiting bodies from a hyphal thallus is inducible under low temperature cold stress the molecular mechanism has been subject to surprisingly few studies analysis of gene expression level has become an important means to study genefunction and its regulation mechanism but identification of reference genes rgs stabilityunder cold stress have not been reported in famous medicinal mushroomforming fungi cordyceps militaris herein candidate rgs had been systematically validated under coldstress in c militaris three different algorithms genorm normfinder and bestkeeper wereapplied to evaluate the expression stability of the rgs our results showed that ubc andubq were the most stable rgs for cold treatments in short and long periods respectively rgs ubc and pp2a and rgs ubq tub and cyp were the suitable rgs for coldtreatments in short and long periods respectively moreover target genes twocomponentsystem histidine kinase genes were selected to validate the most and least stable rgsunder cold treatment which indicated that use of unstable expressed genes as rgs leadsto biased results our results provide a good starting point for accurate reverse transcriptasequantitative polymerase chain reaction normalization by using ubc and ubq in c militarisunder cold stress and better support for understanding the mechanism of response to coldstress and fruiting body formation in c militaris and other mushroomforming fungi in futureresearcha1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation liu yn liu by ma yc yang hl liugq analysis of reference genes stabilityand histidine kinase expression under cold stressin cordyceps militaris one e0236898101371 pone0236898editor shwet kamal icardirectorate ofmushroom research indiareceived april accepted july published august copyright liu this is an open access distributed under the terms of the creativecommons attribution license which permitsunrestricted use distribution and reproduction inany medium provided the original author andsource are crediteddata availability statement all relevant data arewithin the paper and its supporting informationfilesfunding this work was supported by the nationalnatural science foundation of china no and the natural sciencefoundation of hunan province no 2020jj5972the scientific research fund of hunan provincialeducation department china no 17k106 and18b167 the national key r d programs ofintergovernmental international science andtechnology cooperation no 2017yfe0108100introductioncordyceps militaris a famous traditional chinese medicine has been used as a healthy food fora long time in china the c militaris fruiting body has antiinflammatory antitumor antiinfluenza virus and radioprotection functions methods for commercial production of fruiting bodies of this fungus have been established in artificial media [ ] or withinsects such as silkworm bombyx mori pupae but the molecular mechanism of fruitingbody formation is not well understood the essential role of light in fruiting body development one 101371 pone0236898 august one 0cthe project of international cooperation base ofscience and technology innovation on forestresource biotechnology of hunan province no2018wk4008 and aid program for science andtechnology innovative research team in highereducational instituions of hunan province thefunders had no role in the study design datacollection and analysis decision to publish orpreparation of the manuscriptcompeting interests the authors have declaredthat no competing interests existstable reference genes in cordyceps militaris for cold stressin c militaris was demonstrated in previous study inactivation of a bluelight receptene white collar1 wc1 resulted in thicker aerial hyphae disordered development in c militaris further study showed that the fruiting body primordia could form in the drosophilaarabidopsis synechocystis and human dash type cryptochromes crys gene mutantstrains but the fruiting bodies were unable to elongate normally and crydash and cmwc1exhibited interdependent expression under light in c militaris compared to light temperature is another pivotal factor influencing fruiting body formation from a hyphal thallus which represents a transition from simple to complex multicellularity the fruiting body formation of various mushrooms depends on low temperaturecold stress induction for example the fruiting body development was induced by shiftingthe cultivation condition Ëc for mycelia to lower temperature Ëc in flammulina velutipes in pleurotus ostreatus the mycelia were cultured at Ëc for days in a sawdustmedium and then the temperature was lowered to Ëc to induce fruiting body development in c militaris after the mycelia were cultured under static conditions at Ëc on potatodextrose broth medium for days the mycelia were injected into pupae and then the inoculated pupae were kept at Ëc to induce the fruiting bodies however the molecular mechanism of mycelial development to fruiting body under cold induction has been subject tosurprisingly few studies which will seriously restrict the further development of commercialproduction of mushrooms fruiting bodiestwocomponent signal transduction is commonly used by eubacteria archea and eukaryotes as a stimulusresponse coupling mechanism to sense and respond to changes in many different environmental conditions especially temperature sensing they consist of ahistidine kinase hk and a response regulator the histidine kinase desk from bacillus subtilis is mechanistically the best understood after a temperature downshift desk gets autophosphorylated at the conserved histidine residue and donates the phosphate group to theconserved aspartate residue of desr the phosphorylated desr activates the transcription ofthe des gene however little is known the role of hk genes in cold stress response andfruiting body formation in c militaris and other mushroomforming fungidue to its high sensitivity specificity and reliable quantification realtime quantitativepolymerase chain reaction qrtpcr is an important tool to understand the complex signaling networks when an anism is submitted to different stimuli [ ] however the resultsof qrtpcr are often inaccurate because of the occurrence of errors in the primer specificitycomplementary dna cdna synthesis and pcr amplification therefore qrtpcrmust be normalized using reference genes that show a stable expression however validationof suitable rgs for expression analysis under cold stress have not been conducted in c militaris and many other mushroomforming fungi in this study we systematically identified candidate rgs in c militaris that were measured using qrtpcr three different algorithmsgenorm normfinder and bestkeeper were applied to evaluate the expression stability of the candidate rgs under cold treatment in c militaris moreover the relative expression levelsof twocomponentsystem hk genes were analyzed under cold stress conditions with threedifferent normalization strategies the rgs screened out in this paper could provide robustness to study the regulatory mechanism of cold induced fruiting body formation from myceliain c militaris and other mushroomforming fungimaterials and methodsstrains and culture conditionsa laboratory and commercial strain of c militaris cgmcc from china generalmicrobiological culture collection center was used the c militaris was cultured at Ëc in one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressdark with an artificial medium containing g rice g powder of silkworm pupae and25ml nutrient solution glucose g kh2po4 g mgso4 g ammonium citrate g peptone g vitamin b1 mg and 1000ml distilled watercold stressafter cultured for days at Ëc in dark the c militaris mycelia were transferred to Ëc indark for different treatments under cold stress for different time periods including short periods and 8hours and long periods and 8d after treatmentmycelia samples were immediately frozen in liquid nitrogen and stored at Ëc in a deepfreezer until rna isolation untreated mycelia samples were used as the control each treatment was repeated for timesrna isolation and cdna synthesisrna isolation and cdna synthesis were performed as previously described method briefly g mycelia were collected and subsequently were disrupted under liquid nitrogenconditions the rna isolation kit takara china was used to extract total rna treatedwith dnase i to avoid genomic dna contamination μg total rna of each sample wasadded to μl reverse transcription reaction system to synthesize cdna the cdna was subjected to 10fold serial dilution for determining the amplification efficiency of qrtpcr uponcold treatmentsrealtime quantitative polymerase chain reaction conditionsqrtpcr experiments were performed using a previously described method briefly allgenes were amplified by initial heating at Ëc for min followed by cycles of Ëc for s Ëc for s and Ëc for s at the final amplification cycle the specificity of pcr reactions was checked through the use of melting curve analysis Ëc in increments of Ëcevery s negative controls were included to ensure the suitability of the assay conditionseach experiment described above was repeated independently at least for timesselection of candidate reference genesin this study genes namely act tub ubc ef1α gapdh pp2a ubq pgk rpsfbox cyp and gtpb were selected as candidate rgs our gene panel contained traditional rgs such as tubulin tub actin act polyubiquitin ubq glyceraldehyde3phosphate dehydrogenase gapdh and translation elongation factor1α ef1α geneschosen on the basis of stable expression in several rna sequencing and quantitative pcrexperiments such as cyclophilin cyp and ubiquitinconjugating enzyme ubc and novel stable rgs in cold stress such as phosphoglycerate kinase pgk and serinethreonine protein phosphatase 2a pp2a details of these genes are provided in table these genes were annotated with a c militaris genome database national center for biotechnology information accession gse28001 and aevu00000000 and used to design primers the specific primers for twocomponentsystem histidine kinase hk genes were listed ins1 tabledata analysis to select the internal reference genethe expression stability of the candidate rgs was analyzed using genorm normfinder and bestkeeper as previously described one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stresstable details on primers used for quantitative reverse transcriptase polymerase chain reaction analysisgeneacttububcef1αannotationaccession noactin cytoskeleton proteinxm_0066695521tubulinxm_0066692031ubiquitinconjugating enzymexm_0066722771elongation factor 1alphaxm_0066659681gapdhglyceraldehyde 3phosphate dehydrogenasexm_0066696971pp2aubqpgkrpsfboxcypgtpbserinethreonine protein phosphatase 2axm_0066730331polyubiquitinxm_0066724691phosphoglycerate kinasexm_0066734081ribosomal protein s25xm_0066653991fbox proteinxm_0066728101cyclophilinxm_0066747781gtpbinding proteinxm_0066746481primer sequences fcaacaacttcctgacgggcrtccttgggcttctgctgacfatgtcgttcgtcgtgaggragagtggcgttgtagggtfaccattgacacgagccagttrgcccatgtaagcctcctcaftatcggaactgtgcctgtrcgttaccacgacggatttfcatccactcctacactgctacrctcaagacgaacagtcaggtfcctcctacagtcgtcatcagcragaaatgtcaaagcgagaftcaaagaagataatggtaacgrgtatgggttctcggaaaggtfgctcaagcccgtcgtttcrccctcctcctcaatgtggfaagtggtctaagggcaaggrttctcctccaggtcggtaafccgatgacaacgacagcgacrgtagttgaccgtggagatgtfttttccgccttattccaccrtccagagcatcaaatccctftaagaagcccaagaagaaaargtcccacaggttcagcgtf forward r reverse101371 pone0236898t001amplification size bpefficiency abbreviationsact actin cytoskeleton protein cdna complementary dna cq quantification cycle cvcoefficient of variation cyp cyclophilin ef1α elongation factor 1alpha fbox fbox protein gapdh glyceraldehyde 3phosphate dehydrogenase gtpb gtpbinding protein hkhistidine kinase m value measurement value pgk phosphoglycerate kinase pp2a serinethreonine protein phosphatase 2a rgs reference genes rps ribosomal protein s25qrtpcr realtime quantitative polymerase chain reaction sd standard deviation tubtubulin ubc ubiquitinconjugating enzyme ubq polyubiquitin vnvn1 pairwisevariationresultsexpression profiling of candidate reference genes in c militaris genes namely act tub ubc ef1α gapdh pp2a ubq pgk rps fbox cyp andgtpb were selected as candidate rgs to determine the most stable rgs under cold treatments we first calculated the pcr efficiency of twelve pairs of primers according to the previously reported method as shown in table qrtpcr efficiency of the genes rangedfrom cyp to ef1α which fell within the acceptable range the quantification cycle cq values of the genes exhibited a high variation ranging and shown in fig the cq values ranged from to and to of short periods fig 1a and long one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressfig the range of quantification cycle cq values of the candidate reference genes in c militaris under cold stress for short a and long periods b101371 pone0236898g001periods fig 1b respectively rps and pp2a was the most and least transcribed respectivelyacross all the tested samples fig 1a and 1b three different algorithms genorm normfinder and bestkeeper were used to analyze the expression stability of the genes in the nextsectiongenorm analysisthe genorm algorithm calculates the expression stability factor m the m value is defined asthe average pairwise variation of a particular gene with all other reference genes within a givengroup of cdna samples where a low value represents stable and a high value represents unstable expression as determined by genorm fig expression stability m values of the genes both in short and long periods were within the acceptable range m the mvalue ranged from pgk to ubc and pp2a in short periods fig 2a and pgk to tub and ef1α in long periods fig 2b the genes were ranked from thehighest m value least stable to the lowest m value most stable pgk act gapdh tubef1α fbox rps ubq cyp gtpb ubc and pp2a in short periods fig 2a pgk ubcact fbox rps gtpb pp2a cyp gapdh ubq tub and ef1α in long periods fig 2bfig genorm analysis of the average expression stability values m of the candidate reference genes under cold stress for short a and long periods b a higherm value indicates more unstable expression101371 pone0236898g002 one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stresstable gene expression stability under short time cold stress ranked by genorm normfinder and bestkeepergenesubcpp2agtpbcypubqrpsfboxef1αtubgapdhactpgkgenormnormfindermean rankabestkeeperm valuerank orderstability valuerank ordercvsd101371 pone0236898t002normfinder analysisnext the expression stability of the candidate rgs was analyzed using normfinder to confirm the genorm results in short periods ubc and ubq were the most stable their stabilityvalues were and respectively table in long periods cyp and ubq were themost highly ranked their stability values were and respectively table combining the results of genorm and normfinder analysis ubc followed by pp2a gtpb and ubqwere the most stable rgs in short periods table mean rank ubq followed by tub cypand gapdh were the most stable rgs in long periods table mean rankoptimal number of reference gene for normalization across theexperimental setsnext we determine the minimal number of genes for qrtpcr normalization by estimatedpairwise variation vnvn1 in genorm below which was a proposed cutoff vnvn1value adding an additional rg is not required according to this principle the vnvn1value was calculated in short periods the v23 value was therefore ubctable gene expression stability under long time cold stress ranked by genorm normfinder and bestkeepergenestubef1αubqgapdhcyppp2agtpbrpsfboxactubcpgkgenormnormfindermean rankabestkeeperm valuerank orderstability valuerank ordercvsd101371 pone0236898t003 one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressfig genorm analysis of the pairwise variation v of the candidate reference genes under cold stress for short a and long periods b the pairwise variation vnvn1 measured the effect of adding additional reference genes on the normalization factor for cold stress treatment in c militaris101371 pone0236898g003together with pp2a would be sufficient for purpose fig 3a and table in long periodscompared with v23 v34 value was suggesting that three rgs ubqtub and cyp were identified as available for normalization fig 3b and table bestkeeper analysiswe further used bestkeeper to analyze the stability of candidate rgs by calculating thestandard deviation sd and the coefficient of variation cv of their cq values it has beenreported that any studied gene showing a sd in expression lower than can be considered stable and the most stably expressed gene exhibiting the lowest cv as shown in table inshort periods the top two stable genes ubc and pp2a with lower cv values and respectively and the sd values of ubc and pp2a were and respectively in long periods the top three stable genes ubq tub and cyp with lower cv values and respectively and the sd values of ubq tub and cyp were and respectively table these bestkeeper results also suggested that ubcpp2a and ubqtubcyp were stable in short and long periods respectivelytaken together our results suggesting that two rgs ubcpp2a and three rgs ubqtubcyp were identified as available for qrtpcr normalization under cold incubation duringshort and long cold periods respectively in c militarisquantitative effects of best and least ranked reference genes on target geneexpressionto validate the utility of stable rgs on gene expression analysis three different strategies theleast stable rg pgk the best ranked rg and multiple stable rgs were selected to normalizethe expression of target gene in short and long periods treatments respectively using pgkubc and ubcpp2a for short periods rg and pgk ubq and ubqtubcyp for long periods rg hk genes in cold stress response were used as target genes based on the annotationwith c militaris genome database national center for biotechnology information accessiongse28001 and aevu00000000 genes cmhk19 s1 table were identified as hk homologous genes normalization of the hk genes using the three different strategies showed that asignificant increase in transcription of hk2hk3hk6 and hk1hk3 hk5 were observed one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressfig relative expression levels of hk genes during cold stress conditions using the least stable gene best rankedgene or multiple stable reference genes for normalization the least stable reference gene pgk best rankedreference gene ubc and multiple reference genes ubcpp2a were used for normalization to analysis of hk2 a hk3b and hk6 c expression levels in short periods the least stable reference gene pgk best ranked reference geneubq and multiple reference genes ubqtubcyp were used for normalization to analysis of hk1 d hk3 e andhk5 f expression levels in long periods the average ct value of multiple reference genes is used to analysis of hkgene expression levels in the strategy of multiple reference genes were used for normalization101371 pone0236898g004under cold incubation during short and long cold periods respectively fig the geneexpression levels of other hk genes were not shown when using the best ranked ubc or themultiple stable ubcpp2a as the rgs in short periods the upregulated trends for hk2 hk3and hk6 genes were consistent and the peak points were observed at h fig 4a4c whenusing the best ranked ubq or the multiple stable ubqtubcyp as the rgs in long periodsthe upregulated trends for hk1 hk3 and hk5 genes were consistent and the peak points wereobserved at d fig 4d4f respectively however using the least ranked pgk as the rg inshort periods the peak points were observed at h of hk2 hk3 hk6 respectively fig4a4c in addition when pgk was applied as the rg in our experiment the transcriptionpatterns of hk genes were higher than those using the best ranked rg and multiple stable rgsfig 4a4f which indicated that normalization using the least stable pgk as rg resulted inan overestimated relative expression level of the target genes thus our experimental resultssuggest that it is feasible to use any one or more of ubcpp2a and ubqtubcyp genes asthe normalization gene under cold incubation during short and long cold periods respectivelywith c militarisdiscussioncompared with microarray and rna sequencing qrtpcr technique has the advantages ofhigh accuracy high sensitivity good repeatability and low cost for quantifying gene expression one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stresstable the screening results of candidate reference genes of different species under cold stressgenepp2acyppgkef1αactgapdhubcspeciesananas comosuselymus sibiricusananas comosusbeauveria bassianahordeum vulgareatropa belladonnahemarthria compressa and h altissimamorchella sppbeauveria bassianaelymus sibiricusmorchella spparabidopsis pumilaarabidopsis pumila101371 pone0236898t004expression stability stableunstablerefexpression stability in this studystablestablestablestableunstablestablestableunstablestablestableunstablestablestablechen zhang chen zhou cai li lin stable short periodsstable long periodsunstable short and long periodsunstable short periodszhang unstable short and long periodszhou zhang zhang unstable short and long periodsjin jin stable short periods unstable long periods[ ] to ensure the accuracy of qrtpcr results it is necessary to analyze the stability ofinternal rg under a specific experimental condition a previous study in c militaris showedthat polymerase ii large subunit rpb1 gene was the best rg during all developmental stagesexamined while the most common reference genes actin and tub were not suitable internalcontrols in addition the actin also was not suitable rgs under developmental stagestrain and nutrient source in lentinula edodes however suitable rgs for expressionanalysis under cold stress have not been reported in c militaris in this study the expressionstability of candidate rgs in c militaris was systematically analyzed by genorm normfinder and bestkeeper under the cold treatment our results showed that ubc and ubq wereidentified as the most suitable for qrtpcr normalization under cold incubation during shortand long cold periods respectively while the act a traditionally rg was not suitable coldstress in c militaris therefore we suggest to use two or more rgs rpb1 ubc and ubq tostudy the expression levels of the fruiting body developmental genes in c militaris in the followup studya large number of studies have been conducted to identify the suitable rgs under coldtreatment conditions among different species but the most stable rgs are diverse table inpineapple ananas comosus l pp2a and cyp were the stable rgs under cold stress andin hulless barley hordeum vulgare l var nudum hook f pgk was not stable rg undercold stress which were consistent with our results however in hemarthria compressaand h altissima leaf tissue ef1α was the most stable gene under cold stress and inatropa belladonna pgk was a reliable gene for normalizing gene expression under cold stressconditions which were not consistent with our results in entomopathogenic fungusbeauveria bassiana both act and cyp were the most stably expressed gene sets under a variety of stress conditions including cold however in our study act was not stable inour experimental conditions in another mushroomforming ascomycota true morelsmorchella spp two candidate internal control genes act and gapdh were not reliablegene for normalizing gene expression under cold stress conditions which was similar toour findings an interesting result is that in arabidopsis pumila both ubc and gapdhunder cold stress were the most stable rgs table but in our result ubc and gapdhwere stable and not stable rgs in short periods respectively the similar results were alsofound in siberian wild rye elymus sibiricus that pp2a and act presented the highest degreeof expression stability for cold stress but in our result only pp2a was stable rg while one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressact was found to be unstable the more interesting result is that in our study ubc was thestable rg in short periods but not the stable rg in long periods all of these results indicatethat the expression stability of the same gene differs in different species moreover the expression stability of the same gene is also not the same under the same treatment at different timesin arabidopsis thaliana arabidopsis histidine kinase ahk2 ahk3 and coldinducibletype a arabidopsis response regulators arrs play roles in cold signaling in additionstresssensing in fungi also depends on a signaling cascade comprised of a twocomponentphosphorylation relay plus a subsequent map kinase cascade to trigger gene expression moreover twocomponent pathways are important determinants of pathogenicity in animalpathogens such as candida albicans cryptococcus neoformans and plant pathogensincluding fusarium oxysporum tomato monilinia fructicola brown rot of stone fruit and botrytis cinerea bean tomato and apple in this study the relative expressionlevels of twocomponentsystem hk genes were analyzed under cold stress conditions withthree different normalization strategies the results showed that hk genes were significantlyupregulated after cold stress using stable rgs in normalization however an overestimatedrelative expression level in the hk genes transcription was obtained from qrtpcr usingpgk in normalization fig the relative expression level of target gene is overestimated dueto use unstable gene normalization is also found in other studies [ ] these results indicated that using unstable rgs for qrtpcr normalization will lead to inconsistent resultsmoreover our results suggested that the upregulated hk genes participated in response tocold stress of c militaris however whether or how these hk genes involved in coldinducedfruiting body formation of c militaris needs further studyconclusionswe systematic validated candidate rgs under cold incubation during short and long coldperiods respectively in c militaris our results showed that two rgs ubcpp2a and threergs ubqtubcyp were identified as available for qrtpcr normalization under cold incubation during short and long cold periods respectively in addition our results indicate thatfailure to statistically validate rgs leads to inconsistent results moreover the role of upregulated hk genes in coldinduced fruiting body formation in c militaris needs further studyall in all our results provide a good starting point for accurate qrtpcr normalization in cmilitaris under cold stress and better support for understanding the mechanism of response tocold stress and fruiting body formation in c militaris and other mushroomforming fungi infuture researchsupporting informations1 table details on primers of twocomponentsystem histidine kinas used for qrtpcranalysisdocacknowledgmentsthe authors thank jing zhang yanghong zhang and feifei xue for their help during carryingout the experiments the authors thank xiaoxiao lu and jiashun zhang for their help in dataanalysis we would like to thank muling shi for english language editingauthor contributionsconceptualization yongnan liu biyang liu one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressfunding acquisition gaoqiang liuvalidation youchu ma gaoqiang liuwriting review editing hailong yangreferences won sy park eh antiinflammatory and related pharmacological activities of cultured mycelia andfruiting bodies of cordyceps militaris j ethnopharmacol 101016jjep200410009 pmid jin c kim g choi y induction of apoptosis by aqueous extract of cordyceps militaris through activation of caspases and inactivation of akt in human breast cancer mdamb231 cells lee hh park h sung gh lee k lee t lee i antiinfluenza effect of cordyceps militaris throughimmunomodulation in a dba2 mouse model of microbiology 101007s1227501443000 wos000339600200010 pmid jeong mh park ys jeong dh lee cg kim js oh sj in vitro evaluation of cordyceps militarisas a potential radioprotective agent international of molecular medicine 103892ijmm20141901 wos000344423800020 pmid xie cy gu zx fan gj gu fr han yb chen zg production of cordycepin and mycelia by submerged fermentation of cordyceps militaris in mixture natural culture appl biochem biotechnol 101007s1201000985672 pmid zheng zl huang ch cao l xie ch han rh agrobacterium tumefaciensmediated transformation asa tool for insertional mutagenesis in medicinal fungus cordyceps militaris fungal bioluk 101016jfunbio201012011 wos000289023800008 pmid hong ip kang pd kim ky nam sh lee my choi ys fruit body formation on silkworm by cordyceps militaris mycobiology 104489myco2010382128pmid pubmed central pmcid pmc3741563tiantian l caihong d tao y junde s effects of blue light on the growth and bioactive compoundproduction of cordyceps militaris mycosystema yang t guo mm yang hj guo sp dong ch the bluelight receptor cmwc1 mediates fruit bodydevelopment and secondary metabolism in cordyceps militaris appl microbiol biot 101007s0025301570476 wos000368103200019 pmid wang f song x dong x zhang j dong c dashtype cryptochromes regulate fruiting body development and secondary metabolism differently than cmwc1 in the fungus cordyceps militaris applmicrobiol biot 101007s0025301782767wos000402008300025 pmid krizsan k almasi e merenyi z sahu n viragh m koszo t transcriptomic atlas of mushroomdevelopment reveals conserved genes behind complex multicellularity in fungi p natl acad sci usa 101073pnas1817822116 wos000463936900040 pmid wu t hu c xie b zhang l yan s wang w a single transcription factor pdd1 determines development and yield of winter mushroom flammulina velutipes appl environ microbiol 101128aem0173519 pmid sunagawa m magae y isolation of genes differentially expressed during the fruit body development ofpleurotus ostreatus by differential display of rapd fems microbiol lett 101016jfemsle200504018 pmid bourret rb silversmith re twocomponent signal transduction annual review of biochemistry aguilar ps hernandezarriaga am cybulski le erazo ac mendoza d de molecular basis of thermosensing a twocomponent signal transduction thermometer in bacillus subtilis embo vandesompele j de preter k pattyn f poppe b van roy n de paepe a accurate normalization of realtime quantitative rtpcr data by geometric averaging of multiple internal control genesgenome biol 37research0034 101186gb200237research0034 pmid pubmed central pmcid pmc126239 one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stress bustin sa benes v nolan t pfaffl mw quantitative realtime rtpcra perspective j mol endocrinol 101677jme101755 pmid huggett j dheda k bustin s zumla a realtime rtpcr normalisation strategies and considerations genes immunity lu x liu y zhao l liu y zhao m selection of reliable reference genes for qrtpcr during methyljasmonate salicylic acid and hydrogen peroxide treatments in ganoderma lucidum world j microbiolbiotechnol 101007s112740182476x pmid han bin yang zheng samma kaleem m systematic validation of candidate reference genes forqrtpcr normalization under iron deficiency in arabidopsis biometals 101007s1053401396235 pmid gutierrez n gimenez mj palomino c avila cm assessment of candi | Colon_Cancer |
"melatonin is a sleepregulating hormone created by the pineal glandand is released at night it has been found to have biological activityin almost all living anisms including plants animals and microbes itcan quickly enter cells through the bilipid bilayer and exhibit scavenging activity towards oxygen free radicals as well as antioxidant properties due to its low molecular weight and amphiphilic nature peripheral tissues have been found to show a high afï¬nity towardsthis hormone melatonin and hence act on receptors and bindingsites studies reported by tan show that melatonin can alsobe produced in the mitochondria and hence tissue melatonin levelsare more than that of serum levels hence can be used as a moleculethat targets mitochondria the main physicochemical and biologicalproperties of melatonin are sleepinducing effects antioxidant behavior [] antiammatory activity [] antiapoptotic effects and neuroprotective effects it also regulates various corresponding authoremail address renjithsbcollegeacin r thomas101016jmolliq2020114082 elsevier bv all rights reservedphysiological functions of the brain as melatonin can diffuse quicklythrough the bloodbrain barrier it is effectively used in the treatmentof brain injuries rapid eyemovement sleepbehavior disorder patients are managed by a combination treatment of melatoninand clonazepam [] the sensing of bacteria through tolllikereceptor4 and regulation of bacteria through altered goblet cells andantimicrobial peptides are all involved in the anticolitic effects of melatonin in ammatory bowel disease melatonin is involved in theaging process growth towards puberty and modulation of blood pressure this versatile compound blocks proangiogenic andantiangiogenic effects caused by docetaxel and vinorelbine which areantitumor drugs and it enhances their tumorï¬ghting behavior this molecule can modify the redox state of the rat pancreaticstellate cellmelatonin is an endogenous hormone that is involved in circadianrhythm control it is inexpensive and safe as it has a significant effectas an antioxidant and antiammatory melatonin chemically nacetyl5methoxytryptamine is a tryptophan derivative has multiplephysiological effects and can be used to treat many diseases related tovirus infections especially respiratory diseases in covid19 patientswith digestive complications melatonin has positive effects 0cn alzaqri of molecular liquids melatonin can thus be used as an adjunctive or even as a regular therapyas no antiviral treatment is currently available electrochemical measurements of melatonin overï¬ow demonstrate that melatonin secretiondecreases with age melatonin treatments result in the enhancement of essential oil production in salvia species in the context of the recent covid pandemic melatonin can beresearched as a potential molecule to control the dangerous effect ofthis disease rising patients' tolerance and decreasing the mortality infatal virus infections would control the innate immune response and reduce ammation during this period melatonin is a molecule with respective properties as it decreases the overreaction of the innateimmune response and overshoots ammation but also facilitatesadaptive immune function even though melatonin is a critical biomolecule few works havebeen reported on the electronic structure and reactivity of this moleculeexcept for a preliminary work reported by turjanski and coworkers in using semiempirical methods in this manuscript we describe a detailed investigation into the quantum mechanical propertiesof melatonin its spectral features reactivity preferences and the resultsof docking studies with three known structural protein receptors of thenovel coronavirus2 we found that melatonin docks strongly with thethree proteins we hypothesise that this compound can be used as anadjuvant medicine for the treatment of covid19 also significant restby a person peacefully sleeping in dark surroundings will enhance theproduction of this hormone which could help in the management ofcurrent patients or as a preventive measure in the vulnerablepopulation material and methodsthe melatonin molecule was optimised using the gaussian09 software package with the dftb3lyp functional and the 6311g 2dp basis set b3lyp is a commonly used functional and 63112dpbasis set is mediumsized basis set with diffused functions over heavyatoms and polarization functions to bring accuracy we performed frequency calculations to ensure that no imaginary frequency exists suchthat the geometry determined would correspond to a global minimumfor reaching the optimised geometry we used the same geometry for calculating frontier molecular analysis natural bonding orbitals and nonlinear optical studies for uvvisible spectrum simulation we usedtimedependent density functional theory tddft with longrangecorrected camb3lyp functionals with 6311g 2dp as the basis setbecause electronic transitions are timedependent phenomena tddftcalculations are done using the optimised geometry obtained fromb3lyp6311g2dp simulations the frontier molecular orbitals wereviewed from the checkpoint ï¬le generated during the optimisation calculations a wavefunction ï¬le was generated during a single point groundstate calculation job using which the subsequent analysis performedthe melatonin molecule has more than two reaction sites for examplemethoxy carbonylamide and purine ring reaction sites of melatonincalculated using the multiwavefunction software for calculatingtotal electrostatic potential average localised ionization energies and noncovalent interactions as it is reported that melatonincan be used as an adjuvant therapeutic material to ï¬ght covid19 we decided to dock the molecule with three ncov2019 protein's rcsb site melatonin is effective in critical care patients by decreasing vessel permeability anxiety use of sedation and increasing the quality ofsleep which may also be beneï¬cial to covid19 patients for improvedclinical outcome melatonin especially has a high health proï¬le significant data indicate that melatonin reduces virusrelated diseases and willpossibly also be effective in patients with covid19 the target proteinswere downloaded cleaned removed alien atoms and molecules andthen used for docking the energy received from the swissdock software and the score values received from patchdock as well as thedocked results collected from biodiscovery studio software arepresented results and discussion geometry of melatoninthe geometry of the molecule explains its rigid structure thestructure of melatonin can be explained based on its physical parametersof bond lengths and bond angles between important atoms or groups asshown in fig the bond lengths of and arebonds of 1c14n 7c14n and 12h14n respectively and the corresponding bond angles are1090343° ° and ° for 1c14n7c 1c14n12h and 7c14n12h respectively the bond angle of° for 4c25o26c having bond lengths of and for 4c\\\\25o and 25o\\\\26c respectively the bond lengths of 18c21n21n22h 21n23c 23c\\\\24o and 23c\\\\30c are and respectively with the corresponding bondangles of ° ° ° ° ° and° for 18c21c22h 18c21c23c 22h22n23c 21n23c24o21n23c30c and 24o23c30c respectively frontier molecular orbital fmo properties of melatoninthe frontier molecular orbitals are highly reactive orbitals of othermolecules and some chemical descriptors are shown in table the higher occupied molecular orbital homo lower unoccupied molecular orbital lumo and energy gap of melatonin are and kcalmol respectively the energygap is significant indicating that the molecule is inherently stable theionization energy electron afï¬nity hardness softness chemical potential electronegativity electrophilicity index and nucleophilicity indexof melatonin are and kcalmol respectively interactionof the melatonin with the biological target can be explained by the softness value the softness value is high kcalmol indicatingthat the molecule can positively interact with biological systems andshow the desired effect electron transition study and excitedstate properties of melatonin insolutionthe electron transition study explains electrontransfer excitedstates we used the tddft formalism using camb3lyp functionalsand 6311g 2dp basis sets in an implicit solvation atmosphere ofmethanol using the iefpcm model as transmission occurs some energyis also emitted melatonin electron transitions to homo having a pyrrole ring and oxygen in methoxy homo1 over the pyrrole ring andethyl carbons and homo2 over acetamide oxygen nitrogen andethyl carbons with energies of and ev respectively melatonin electron transitions to lumo which is over the pyrrolering lumo which is over the pyrrole ring oxygen in methoxyethyl carbons and acetamide nitrogen and carbon and lumo2 having acetamide carbons acetamide nitrogen and carbons with energiesof and ev respectively the electronic spectral datausing tddft simulations indicate a significant λmax of nm in amethanol solvent the transitions are due to the movement of electronsfrom homo1 to lumo and homo to lumo with an oscillator strength of the electronic transitions are due to chargetransfer transitions from one region of the molecule to another whichindicates its inherent stability due to electronic excitations nonlinear optical behavior of melatoninscientists and technologists working in the molecular electronics ï¬eldare continuously searching for compounds with substantial nonlinearoptical nlo activity such compounds ï¬nd immense application in electronic displays surveillance equipment and consumer electronic gadgetscomputationally the ability of a molecule to act as an nlo material can be 0cn alzaqri of molecular liquids fig geometry of melatonindetermined from the polarizability and hyperpolarizability data []the nlo properties of melatonin are shown in table this is an essentialbehavior of melatonin that has a light absorption nature movement ofelectrons or protons as compared with a standard nlo material such asurea the dipole moment of melatonin is d which is times greater than urea hyperpolarizability mean polarizabilityand anisotropy of the polarizability of melatonin are and esu and which are and times greater than urea respectively the compound is not centrosymmetric hence generates secondorder spherical harmonics and betahyperpolarizability functions this compound can hence be used as an anic nonlinear optically active substance in anic electronicappliances nature of nbo study of melatonina molecule especially one with profound biological activity may havemany intramolecular electron delocalisation and hyperconjugativestabilisation regions natural bond orbital analysis which is a quantummechanical method is useful for this type of study the molecular orbitaltable frontier molecular orbital properties for melatoninchemical descriptorshomolumoionization energy i Éhomo homoelectron afï¬nity a Élumo lumoenergy gap homo lumoglobal hardness η i a global softness s ηchemical potential μ i a electronegativity Ï Î¼electrophilicity index Ï Î¼2 2ηnucleophilicity index n Ïenergy in kcalmolproperties of melatonin for the occupancy of the natural orbitals wereperformed by the nbo suite embedded in the gaussian softwarefrom donorbonding orbital Ï c1c2 with occupancy is toacceptor antibonding orbitals Ï c3c4 Ï c5c6 and Ï c7c8exhibiting the transition the energies are and kcalmol respectively from Ï c3c4 with occupancy is to Ï c5c6 and the rydberg orbital r c30 with the energies are and kcalmol respectively from Ï c5c6 having an occupancy is to Ï c3c4 r and r h33 with the energies are and kcalmol respectively from Ï n21c23 has occupancy to rydberg orbital r c30 and r h33 with the energies are and kcalmol respectively from Ï c23o24 having the occupancy is to Ï c1c2 Ï c2c3 r c23 and r h33 withthe energies are and kcalmol respectivelyfrom Ï c23c30 has occupancy is to r h17 r c18 rh19 r c23 r o24 r c30 r h33 Ï c1c2 Ï c1c6Ï c2c3 Ï c26h27 Ï c30h31 Ï c30h32 and Ï c30h33 having the energies are and kcalmol respectively from Ï o25c26 having occupancy is to r c30 andÏ c1c2 with the energies are and kcalmol respectivelyfrom Ï c26h27 to Ï c1c2 having the energy kcalmol withthe occupancy is from Ï c30h31 with the occupancy is to r h17 r c18 r h19 r c23 r o24 r c30 rh33 Ï c1c2 Ï c2c3 Ï c23o24 Ï c30h31 and Ïc30h32 having the energies are and kcalmol respectively from Ï c30h32 with occupancy is to r h17 rc18 r h19 r c23 r o24 r c30 r h33 Ï c1c2 Ïc2c3 Ï c30h32 and Ï c30h33 having the energies are and kcalmol respectively and from Ï c30h33 with the occupancy is to r h17 r c18 r c19 r c23 r c24 ro25 r c26 r h28 r c30 r h33 Ï c1c2 Ï c1c6Ï c2c3 Ï c2c4 Ï c18h19 Ï c23o24 Ï c26h27 Ï 0cn alzaqri of molecular liquids table nonlinear optics property for melatoninnonlinear propertydipole moment μhyperpolarizability βmean polarizability α0anisotropy of the polarizability δαmelatonin d esu esu esuurea d esu esu esucomparison of melatonin with urea times greater than urea times greater than urea times greater than urea times greater than ureac30c31 Ï c30h32 and Ï c30h33 having the energies are and kcalmol respectivelyfrom core bonding orbital c c23 which has the occupancy electrons move to antibonding r c23 r c30 and r h33 withthe transition energies are and kcalmol respectively from c c23 with the occupancy is to r h33 Ï c2c8 and Ï c26h28 having the energies are and kcalmol respectively from c o24 to Ï c26h29 has the energy is kcalmol with occupancy is and from c c30 having the occupancy is to r h17 r c23 r c30 r h33 Ïc2c8 Ï c26h28 Ï c30h31 and Ï c30h32 with the energies are and kcalmol respectively from lone pair orbital n n14 with the occupancy is to Ï c7c8 with the energy kcalmol from nn21 has the occupancy is to Ï c23o24 having the energy kcalmol and from n o24 having the occupancy is to rc23 r c30 r h33 Ï c1c2 and Ï c2c3 with the energiesare and kcalmol respectively fromantibonding orbital Ï c1c2 having the occupancy to rc30 and Ï c2c3 with the energies are and kcalmolrespectively from Ï c5c6 has occupancy is to r c30 withthe energy is kcalmol and from Ï c23o24 has occupancy is to r c30 with the energy is kcalmol the inherentstabilisation offrom the series ofhyperconjugative interactions presented above these interactions canalso be between the melatonin and the surrounding solvent moleculeswhich reveals its stabilisation in biological medium and also betweenthe molecule and the target proteins used in the dockingthe molecule is evident total electrostatic potentials esp and average localised ionization energy alie of melatoninthe electrostatic potential explains how reactive sites canundergo nucleophilic or electrophilic addition or substitution reactionsof melatonin shown in fig within bohr and a color changefrom blue to red indicates charges on elements from to the blue color appears in both methoxyoxygen and acetamideoxygen these are electronrich sites and electrophiles can quickly attack them the red color appears on all of the hydrogens these areelectronpoor sites and nucleophiles can quickly attack themthe alie clariï¬es the stability of any molecule based on saturatedand unsaturated bond electron movements which are localised ordelocalised the number of the resonance structure is proportionalto the stability of the molecule the alie of melatonin shown in fig iswithin the range of ± bohr color is from indigo to red and the numerical value is from to the blue color of protons in themethoxy group three protons in the sixmembered ring in the indolegroup methyl protons and both adjacent carbons in the acetamidegroup are all sites that act as electrophiles the red color ofacetamidecarbon conjugated carbon with oxygen atoms and bothacetamideamide and methoxy groups are all sites that act as nucleophiles these blue and red regions represent saturated bonds thebluishgreen regions are on indole rings to methoxy carbons via oxygenand acetamide chains this indicates that delocalised electrons and unsaturated bonds lead to several resonance structures and explains thestability of melatonin the electrophilic and nucleophilic reactive centres identiï¬ed above interact with the covid virus proteins and providevarious electrostatic and noncovalent interactions and increases drugafï¬nity noncovalent interaction nci properties of melatoninnoncovalent interactions are a valuable biological property of molecules and are nonbonded directly but are bound by some forces suchas hydrogen bonding van der waals bonding andor steric constraintsnoncovalent interactions of melatonin are shown in fig plotted as agraph with energy plotted versus a reduced density gradient hydrogen bonds appear from to au between oxygenand protons from the acetamide group the van der waals force rangesfig electrostatic potentials of melatoninfig average localised ionization energy for melatonin 0cn alzaqri of molecular liquids fig noncovalent interactions of melatoninfrom to au between acetamideoxygen and its adjacentprotons in both methyl and methoxy groups the steric force rangesfrom to au between the indole ring the methyl groupand the carbonylamide group noncovalent interactions are a groupof interactions like hydrogen bond pistacking hydrophobic interactions van der waal's forces iondipole interactions and dipoledipoleinteractions responsible for the stabilisation of the molecule and thedocking between melatonin and the covid proteins molecular dockingmolecular docking is one of the essential functions of biologically active molecules this is the theoretical evidence to design the structureand reactivity relationship of a molecule at present the covid19 pandemic caused by a new strain of the coronavirus is creating havocthroughout the world we made efforts to dock the melatonin withthe three proteins isolated from the virus represented through thepdb id 6lu7 6m03 and 6w63 were deposited in the database as mentioned in the methodology sectionwith the rapid spread of the novel coronavirus globally the designof vaccines is of great importance sarscov2 is an enveloped nonsegmented and single stranded positive sense rna virus the bestdrug target among coronaviruses is the main protease mpro also called3cl protease this is a key coronavirus enzyme and plays a vitalrole in mediating viral replication and transcription it is identiï¬ed ashaving a mechanismbased inhibitor the main targeting protease protein pdb 6lu7 is widely studied a series of frontier molecular orbital based interaction analyseswere performed on the complex between the main protease ofcovid19 and the peptidelike inhibitor whose fundamental structure was obtained from the protein pdb 6lu7 anothertargeted protease protein in an apo form pdb 6m03 shows themost stable form after binding with the selected drug threonine residue with the help of several covalent bond interactionwith a kcalmol docking afï¬nity lasinavir brecanavirtelinavir rotigaptide 13bis2ethoxycarbonylchromon5yloxy2lysyloxypropane and pimelautide can be consideredas the main protease inhibitors of covid19 by docking them tothe binding cavities of apo pdb 6m03 and holo pdb 6lu7 another protease protein pdb 6w63 is a reversible inhibitorthe ï¬avonoid narcissoside is reported to have a high afï¬nity towards the protease protein pdb 6w63 according to moleculardocking studies thus these three protease proteins pdb 6lu7pdb 6m03 and pdb 6w63 can be included in the category ofnonstructural proteins in the structure of sarscov2 from table the result from swissdock explains the biological activity of melatonin with coronavirus proteins pdb id 6lu7 6m03and 6w63 in general the total δg is more than kcalmol isright active luckily melatonin has a total δg of and kcalmol with coronavirus2 proteins pdb id 6lu7 6m03 and6w63 respectively and the total δg is directly proportional to the fullï¬tness energy values which are and kcalmol respectively it can also be seen from this table theinterfull ï¬tness intrafull ï¬tness full solvent ï¬tness full surface ï¬tnessδg complex polar solvent δg complex nonpolar solvent δg proteinpolar solvent δg protein nonpolar solvent δg ligand polar solventδg ligand nonpolar solvent δg van der waals force and δg electricforce relationships between melatonin and coronavirus2 proteins asreferredthe result from patchdock are as follows the score values are and total surface interacting area and and the minimum atomic contact energies are and kcalmol for melatonin with coronavirus2 proteins pdb id 6lu7 6m03 and 6w63 respectively figs and s1show the skeletal structure and protein residue interactions betweenmelatonin and coronavirus2 protein pdb id 6lu7 6m03 and 6w63table explains what protein residues are interacting with melatoninand details the residue names labels hydrophobic values pka valuesaverage isotropic displacements secondary structures residue solventaccessibility sidechain solvent accessibility percent solvent accessibility and percent sidechain solvent accessibility values of coronavirus2proteinstable and fig s2 show the residue structure of the favorable nonbond interactions between melatonin and coronavirus2 proteinstable lists favorable nonbond interactions of 6lu7 having conventional hydrogen bonds carbonhydrogen bonds pidonor hydrogenbonds pisulfur and pialkyl with melatonin 6m03 has pisigmapipi tshaped and pialkyl with melatonin while 6w63 has pipi tshaped pialkyl and pialkyl with melatonin along with the bond distance from chemistry fig s2 and table show the residue structure 0cn alzaqri of molecular liquids table swissdock result for melatonin with coronovirus2 proteins pdb id 6lu7 6m03 and 6w63energysimple ï¬tnessfull ï¬tnessinter full ï¬tnessintra full ï¬tnesssolvent full ï¬tnesssurface full ï¬tnessextra full ï¬tnessδg complex solvent polarδg complex solvent nonpolarδg protein solvent polarδg protein solvent nonpolarδg ligand solvent polarδg ligand solvent nonpolarδg van der waals forceδg electric forcetotal δg6lu7 kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol 6m03 kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol6w63 kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmolof the unfavorable nonbond interactions between melatonin and coronavirus2 proteins protein 6lu7 does not have any unfavorablestericinteractions protein 6m03 having three unfavorable nonbond interactions and protein 6w63 having unfavorable bumpnonbond interactions fig s2 and table show unsatisï¬ed bonds within melatonininteracting with coronavirus proteins when interacting protein 6lu7has one hydrogen donor and one oxygen acceptor protein 6m03 hastwo hydrogen donors and two oxygen acceptors and protein 6w63has one hydrogen donor and two oxygen acceptors with melatonin table shows noncovalent interactions between melatonin and coronavirus2 proteins hydrophobic groups of protein 6lu7 residues areahis41 aleu141 acys145 ahis164 amet165 and aglu166those of protein 6m03 residues are ahis41 amet49 aphe140 aleu141 acys145 amet165 aglu166 and aleu167 and those ofprotein residues 6w63 are ahis41 acys44 amet49 aleu50 amet165 aglu166 aleu167 and agln189 with melatonin as shownin fig s2 and table the hydrophilic groups of protein 6lu7 residuesare ahis41 aasn142 ahis164 aglu166 ahis172 aasp187 aarg188 and agln189 those of protein 6m03 residues are ahis41 aasn142 ahis163 ahis164 aglu166 ahis172 and agln189 andthose of protein 6w63 residues are ahis41 ahis164 aglu166 aasp187 aarg188 agln189 and agln192 with melatonin as shownin fig s3 and table neutral groups of protein 6lu7 residues are atyr54 agly143 and aser144 those of protein 6m03 residues are agly143 aser144 and apro168 and those of protein 6w63 residuesare apro52 atyr54 aarg188 and athr190 with melatonin asshown in fig s4 and table acidic groups of protein 6lu7 residuesare aglu166 and aasp187 that of protein 6m03 residue is aglu166 and protein 6w63 residues are aglu166 and aasp187 withmelatonin as shown in table and fig s5 basic group interactions ofprotein 6lu7 residues are ahis41 ahis164 ahis172 and aarg188those of protein 6m03 residues are ahis41 ahis163 ahis164 andahis172 and those of protein 6w63 residues are ahis41 ahis164and aarg188 as shown in table and fig s6 tan etal has shownthat melatonin and derivatives has excellent biological responses likeacting against oxidative stress and free radical scavenging []our studies show that melatonin molecule can interact with differentproteins present in the ncov19 virus and inhibit their proliferationthese results need further clinical follow up and could assist in the management of covid pneumonia conclusionswe conducted a detailed quantummechanical investigation of thehormone melatonin and regulation of the sleepwake cycle naturalbonding orbital studies revealed the intensity of several intramolecularinteractions the various frontier molecular orbital data explain the nature and physical parameters of melatonin and the nonlinear opticalproperties are compared with urea which is a standard materialfig skeletal structure of interactions between melatonin and 6lu7 a 6m03 b and 6w63 c coronavirus2 protein residues 0cn alzaqri of molecular liquids table interactions between melatonin and coronavirus2 protein residuesnamelabelhydrophobicitypkapdbidsavg isotropicdisplacementsecondarystructureresidue solventaccessibilitysidechain solventaccessibilitypercent solventaccessibilitypercent sidechainsolvent accessibility6m03 histidine6lu7 histidineahis41amet49methionineatyr54tyrosinealeu141leucineaasn142asparagineagly143glycineaser144serineacys145cysteineahis164histidinemethionineamet165glutamic acid aglu166ahis172histidineaasp187aspartic acidaarg188arginineglutamineagln189ahis41methionineamet49phenylalanine aphe140aleu141leucineaasn142asparagineglycineagly143aser144serineacys145cysteineahis163histidineahis164histidinemethionineamet165glutamic acid aglu166aleu167leucineapro168prolineahis172histidineagln189glutamine6w63 histidineahis41cysteineacys44methionineamet49leucinealeu50prolineapro52tyrosineatyr54histidineahis164methionineamet165glutamic acid aglu166aleu167leucineapro168prolineaasp187aspartic acidarginineaarg188agln189glutamineathr190threonineglutamineagln192helixturnhelixcoilturnturncoilturnsheetsheetsheetsheetcoilcoilcoilhelixhelixcoilcoilturnturncoilturnsheetsheetsheetsheetcoilturnsheetcoilhelixcoilcoilcoilcoilhelixsheetsheetsheetcoilturncoilcoilcoilcoilcoilwavefunction studies gave information about electrostatic potentialsaverage localised ionization and noncovalent interactions these datahelped to predict the reactivity and identify the active site of the reactivity of the molecule melatonin docks with novel coronavirus proteinsand shows a variety of interactions with an excellent docking scorewhich leads to inhibition of the virus proteins leading to its destructionhence clinicians can consider incorporating melatonin also in thecovid19 treatment regime after further studiestable favorable nonbond interactions between melatonin and coronavirus2 proteinspdb idsdistance categorytypefromfrom chemistrytoto chemistry6lu76m036w63hydrogen bondhydrogen bondhydrogen bondhydrogen bond otherhydrophobichydrophobichydrophobichydrophobichydrogen bondhydrophobichydrophobichydrophobicconventional hydrogen bondconventional hydrogen bondcarbon hydrogen bondpidonor hydrogen bond pisulfurpialkylpisigmapipi tshapedpialkylconventional hydrogen bondpipi tshapedpialkylpialkylaglu166nunk0hunk0hacys145sgunk0amet165caahis41unk0unk0hahis41unk0unk0hdonorhdonorhdonorhdonor sulfurpiorbitalschpiorbitalspiorbitalshdonorpiorbitalspiorbitalspiorbitalsunk0oahis164oaglu166ounk0acys145unk0unk0acys145aglu166ounk0amet165amet165hacceptorhacceptorhacceptorpiorbitals piorbitalsalkylpiorbitalspiorbitalsalkylhacceptorpiorbitalsalkylalkyl 0cn alzaqri of molecular liquids table unfavorable nonbond between melatonin and coronavirus2 proteinspdb idsdistance categorytypefromfrom chemistrytoto chemistry6lu76m036w63nilunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorable bumpunfavorable bumpunfavorable bump carbon hydrogen bondunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpacys145sgaglu166ounk0hagln189caagln189caagln189cbagln189cgagln189cgagln189cgagln189cgagln189cdagln189cdagln189cdagln189cdagln189oe1agln189oe1agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189haagln189haagln189haagln189hg1agln189hg2agln189hg2agln189he21agln189he21agln189he21agln189he21agln189he22agln189he22agln189he22stericstericsteric hdonorstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericunk0cunk0caglu166ounk0cunk0hunk0cunk0cunk0hunk0nunk0hunk0cunk0cunk0hunk0hunk0cunk0hunk0nunk0cunk0ounk0cunk0hunk0hunk0hunk0cunk0hunk0hunk0hunk0cunk0hunk0nunk0cunk0cunk0hunk0cunk0cunk0hstericstericsteric hacceptorstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericsterictable unsatisï¬ed bonds in melatonin with coronavirus2 proteinspdb ids6lu76m036w63nameunk0hunk0ounk0hunk0hunk0ounk0ounk0hunk0ounk0oatomunsatisï¬ed typehohhoohoodonoracceptordonordonoracceptoracceptordonoracceptoracceptorrenjith thomas conceptualization formal analysis investigation methodology project administration resources softwaresupervision validation visualization writing review editingdeclaration of competing interestthe authors declare that they have no known competing ï¬nancialinterests or personal relationships that could have appeared to uence the work reported in this paperacknowledgementsresearchers supporting project number rsp202078 king saudcredit authorship contribution statementuniversity riyadh saudi arabianabil alzaqri cenceptualization funding acquisition tpooventhiran investegation methodology original draft alialsalme investegation original draft ismail warad resourcesreview methods athira m john methodology writing draftappendix a supplementary datasupplementary data to this article can be found online at 101016jmolliq2020114082table noncovalent interactions between melatonin and coronavirus2 proteinshydrophobicityhydrophilicityneutral groupacidic groupbasic grouppdbids6lu7ahis41 aleu141 acys145 ahis164 amet165 and aglu1666m03 ahis41 amet49 aphe140 aleu141 acys145 amet165 aglu166 and aleu1676w63 ahis41 acys44 amet49 aleu50 amet165aglu166 aleu167 and agln189ahis41 aasn142 ahis164 aglu166 ahis172 aasp187 aarg188 and agln189ahis41 aasn142 ahis163 ahis164 aglu166 ahis172 and agln189ahis41 ahis164 aglu166 aasp187 aarg188 agln189 and agln192atyr54 agly143 andaser144agly143 aser144 andapro168apro52 atyr54 aarg188 and athr190aglu166 andaasp187aglu166aglu166and aasp187ahis41 ahis164 ahis172 and aarg188ahis41 ahis163 ahis164 and ahis172ahis41 ahis164 andaarg188 | Colon_Cancer |
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 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| Colon_Cancer |
" medical practice variation in caesarean section rates is the most studied type of practice variation inthe field of obstetrics and gynaecology this has not resulted in increased homogeneity of treatment betweengeographic areas or healthcare providers our study aim was to evaluate whether current study designs on medicalpractice variation of caesarean section rates were optimized to identify the unwarranted share of practice variationand could contribute to the reduction of unwarranted practice variation by meeting criteria for audit and feedbackmethods we searched pubmed embase ebscocinahl and wileycochrane library from inception to march24th studies that compared the rate of caesarean sections between individuals institutions or geographicareas were included study design was assessed on selection procedure of study population data source casemixcorrection patient preference aggregation level of analysis maternal and neonatal outcome and determinantsprofessional and anizational characteristicsresults a total of studies were included most studies measured the caesarean section rate in the entirestudy population instead of using a sample national databases were most often used as information source casemix correction was performed in studies the robson classification was used in of thestudies following its endorsement by the who in the most common levels of aggregation were hospitallevel and grouped hospitals eg private versus public the percentage of studies that assessed therelationship between variation in caesarean section rates and maternal outcome was neonatal outcome determinants professional and anizational characteristics and patient preference s study designs of practice variation in caesarean sections varied considerably raising questions abouttheir appropriateness studies focused on measuring practice variation rather than contributing to the reduction ofunwarranted practice variation future studies should correct for differences in patient characteristics casemix andpatient preference to identify unwarranted practice variation practice variation studies could be used for audit andfeedback if results are presented at lower levels of aggregation and appeal to intrinsic motivation of physicians forexample by including the health effects on mother and childkeywords caesarean section medical practice variation study design characteristics correspondence mdhvinkvunl mdhvinkgmailcom1department health sciences faculty of science talma institute vrijeuniversiteit de boelelaan hv amsterdam the netherlands2department of obstetrics and gynaecology university medical centergroningen groningen the netherlandsfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cvink bmc pregnancy and childbirth page of the caesarean section has been the most performed surgical procedure worldwide for many decades it hasbeen extensively studied both to optimize treatment and to understand why deviations from optimal treatment occur this longterm popularity has not resulted in evidence based homogeneoustreatmentbetween geographic areas or healthcare providers [ ]the magnitude of the variation has raised questionsabout regional differences in quality of healthcare especially in countries with similar resources moreover years of study on practice variation shows no trend ofincreasing regional l one worldwide conformity variation in medical practice can be divided in warranted and unwarranted practice variation variationis warranted when it results from variation in need forexample due to varying rates of extreme premature deliveries extremely preterm deliveries are centralized atinstitutions with the highest expertise of neonatal careas it yields the most optimal outcome these institutions may deviate from the national average as theyserve a highrisk populationmedical practice variation is unwarranted if it cannotbe explained by patient characteristics or patient preference [ ] to identify unwarranted practice variationstudies should compare study groups that are comparable in terms of relevant patient characteristics or makethem comparable through careful casemix correction patient preference is important when both modesof delivery vaginal delivery and caesarean section arean acceptable option variation of caesarean sectionrates is desirable to allow for differences in patient preference across healthcare providers and random or unmeasured differences in need of having a caesareansection when a reported rate deviates more from an acceptable range differences may less likely be attributableto differences in patient preference as both over andundertreatment of caesarean sections harms mother andchild it is therefore likely that quality of healthcare formother and child can be improved by reducing unwarranted practice variation of caesarean sections sufficientevidence on risks and benefits of caesarean sections mayhelp to reduce variation higher quality evidence will result in better guidance on the optimal caesarean sectionrate for specific obstetric conditions subsequentlyuptodate clinical guidelines and clinical support systems may facilitate clinicians to implement recent evidence finally shared decision making should beincorporated in daily clinical practice to empower patients to decide what suits them best improving the process of generating evidence implementing clinical guidelines and incorporating shareddecision making requires healthcare professionals tochange their clinical behavior which is complex auditand feedback is a nonclinical intervention that supportschange of clinical behavior literature shows thathealthcare professionals may be encouraged with information relevant to them one example is performancefeedback on a low level of aggregation ie group or individual level another is to tap into the intrinsic motivation to do well for patients evidence shows thatunnecessary caesarean sections cause an increase in maternal death rates and may affect infant health negatively monitoring and reporting mother and child healthmay motivate change to reduce unnecessary caesareansections audit and feedback has been put forwardas a way through which research can contribute to thereduction of practice variation but it is unclearwhether current research designs of studies on medicalpractice variation of caesarean section rates can be usedfor this purposemedical practice variation in caesarean section rateshas been extensively studied studies started as earlyas and the interest for the topic has remainedstrong however unwarranted medical practicevariation of caesarean section rates has not been reduced the objective of this scoping review is to evaluatewhether studies on medical practice variation of caesarean section rates have used an optimal study design toidentify unwarranted practice variation and when identified can also be used for audit and feedback to contributereduction of unwarranted practicevariationto themethodsto evaluate the characteristics of all caesarean sectionvariation studies we opted for a scoping review wefollowed the prisma statement the researchprotocol was not publishedsearch strategywe searched studies that compared caesarean sectionrates between individual healthcare professionals hospitals groups of hospitals or geographic areas a comprehensive search was performed in collaboration with amedical librarian in the bibliographic databases pubmedembase ebscocinahl and wileycochrane libraryfrom inception up to march 24th the followingterms were used including synonyms and closely relatedwords as index terms or freetext words practice patterns caesarean section the freetext term caesarean section was only used in titles the meshtermpractice patterns includes several descriptions of practice variation the search strategy was performed without date orsearchlanguage restriction the full 0cvink bmc pregnancy and childbirth page of strategies for all databases can be found in additional filestudy selectionall studies that reported on any variation in caesareansection rates between individual healthcare professionals hospitals groups of hospitals or geographicareas were included we included any type of study designie crosssectional study designs and both prospective and retrospective longitudinal studiesexclusion criteriawe excluded studies that were not published in englishand studies that did not publish data on variation of caesarean section rates between healthcare professionalshospitals groups of hospitals or geographic areasprocess of study identification and selectiontitles and abstracts were downloaded and entered inendnote version x81 duplicates were removed tworesearchers mv and pdb screened titles and abstractsthe researchers independently decided whether to include the for full text screening disagreementwas resolved by consensus if no consensus could beachieved a third researcher made the decision evdhfull text screening was performed by two researcherswho decided independently whether to include the or not mv and pdb in case of disagreement thethird researcher evdh decided whether the metthe predefined inclusion criteria data on the designcharacteristics of the studies were extracted by one researcher mv these data were randomly crosscheckedby a second researcher pdbdata extractionto describe the variation of caesarean section rates wescored the minimum and maximum caesarean sectionrate when caesarean section rates of multiple yearswere reported the rate of the most recent year was usedas an indicator for the risk of selection bias we scoredthe selection of study population we differentiated between the use of a study sample or the entire studypopulation to estimate the caesarean section rate theuse of a study sample was considered as a high risk ofselection bias if the study lacked a description of thesampling frame the assessment of the caesarean sectionrate of the entire study population was considered as alow risk of selection bias to indicate the risk of information bias we differentiated between the use of electronic patientepf a national database orquestionnaires the use of epf and databases were considered as low risk of bias as the information was collected by attending healthcare professionalsfilesidentification of unwarranted practice variationto identify whether studies distinguished between warranted and unwarranted practice variation we scoredwhether casemix correction was performed and if patient preference was taken into accountno restriction was imposed on the method of casemix correction examples of casemix corrections include calculating an adjusted or expected caesarean section rate reporting stratified odds ratios by patientcharacteristics or using logistic regression to adjust forpatient characteristics we extracted which variableswere used for casemix correction and whether the robson classification was used the latter is the system proposed by the world health anisation who andthe international federation of obstetrics and gynaecology figo to classify caesarean section casemix no restriction was imposed on how patient preferencewas measured measuring patient preference requiresadditional data collection this could be unfeasible forlarge cohort studies unless a truly random sample of sufficient size is used we assessed whether all studies tookpatient preference into account registered the cohortsize and noted how patient preference was measured ifpatient preference was measured we assessed whether asample was used and whether it was randomusefulness for audit and feedbackto evaluate whether the studies were able to providehealthcare professionals feedback on their clinical behavior in order to reduce unwarranted practice variationwe extracted the aggregation level of analysis that wasused and differentiated betweenindividual physiciangroup of physicians hospital hospital category regionor country similarly we scored whether maternal andneonatal outcomes were measured as outcome reporting informs healthcare professionals on their clinicalperformance we extracted all reported variablesin addition we extracted several explanatory factorsthat might contribute to unwarranted practice variationincluding anizational characteristics ie profitstatusor teachingstatus of the hospital and physician characteristics ie physician gender and age furthermore wescored whether studies analysed financial consequencesof unwarranted practice variation of caesarean sectionratesresultsthe process of study identification and selection is schematically presented according to the prisma statementin fig a total of records were identified from pubmed from embase from cochraneand from cinahl after duplication we screened abstracts for eligibility in total s were 0cvink bmc pregnancy and childbirth page of fig prisma flowchartfig average of the reported minimum and maximum caesarean section rate per year is not presented in fig as only studies areonly included until 24th of march 0cvink bmc pregnancy and childbirth page of selected for fulltext screening we excluded studiesthe reasons for exclusion are presented in fig intotal studies met the inclusion criteria and were included the included studies and their design characteristics and reported variation in caesarean section ratesare listed in additional file the included studies were published between and the cohorts that were studied varied between and more than million women most studies analyzed variation of caesarean section rates in the unitedstates studies followed by brazil studies andaustralia studies the number of studies per country are shown in additional file a wide variation incaesarean section rates is reported some subsaharanregions perform caesarean sections in less than ofthe deliveries by contrast the reported caesarean section rate of some municipalities in brazil reached the variation of caesarean section rates did not decrease over time figure shows the average reportedminimum and maximum caesarean section rates peryear the outlier in is one study that reported variation between four hospitals in rio de janeiro in oneprivate hospital more than of the women deliveredby a caesarean section in a similar situation occurred only two studies were included both reportingon variation in indiain studies a sample of the study populationwas used to estimate the caesarean section rate the majority of the studies studies measured themode of delivery of the entire study population bothmethods were used in eight studies and the selection frame was unclear in nine studies an exampleof a study in which both methods were used was theanalysis of variation in caesarean section rates betweencountries some country estimates were based on a sample of their population while others were based on registries ofin the samplebasedstudies studies defined a selection frame designed to select a representative samplethe entire populationthe majority of studies used data from registries suchas national databases studies or electronicpatient files studies questionnaires were usedin studies eg the demographic and healthsurvey dhs that was used in studies suchquestionnaires were sent to a sample of households inorder to collect information on live births of the pastyears in studies multiple data sources wereused or the data source was not describedidentification of unwarranted practice variationcasemix correction was performed in studies the variables that were used for casemix correction areshown in additional file baseline patient characteristics were observed in studies some studies didnot describe patient characteristics per cohort but didperform a correction for maternal or neonatal characteristics many different maternal and obstetric variables were used as baseline characteristic or for casemix correction age parity gestational age birthweight and maternal education level are the characteristics that were used most often morethan half of the variables were only used in one or twostudiesto reduce this heterogeneity and to increase the quality of casemix correction the who in recommended to use the robson classification as the standardfor casemix correction for studies on caesarean sectionrates in the period out of studiesused the robson classification four of these studies did perform additional casemix correction byusing specific patient characteristics the who notesthat the robson classification should especially be usedwhen comparing caesarean section rates between healthcare facilities or within healthcare facilities over time in the period studies compared caesarean section rates between healthcare providers individuallevel andhospital category of which used the robson classification within the same period of the studiesthat compared caesarean section rates between geographic areas used the robson classification thepercentage of studies that corrected for casemix did notchange over time figure shows the number of studiesthat corrected for casemix by yearlevel group of physicians hospitalthe effect of casemix correction is shown in fig of the studies that corrected for casemix studies calculated an adjusted caesarean sectionrate figure shows these rates per study categorizedper aggregation level the remaining studies calculated an expected caesarean section rate reportedstratified odds ratios by patient characteristics or usedlogistic regression to adjust for patient characteristicsat provider level individual physician group of physicians and hospital level of the studies and at geographic level regional or national of the studiescorrected for casemix figure shows that at the provider level casemix correction had a substantial impacton the provider caesarean section rate at the geographic level the impact of casemix correction wascomparatively smallsix studies took patient preference into accountthese studies did not assess variation of caesarean section rates for a specific obstetric condition for instancepatients with a history of caesarean section in which acaesarean section and vaginal delivery are both an acceptable option all studies measured patientpreference by questionnairesthat were handed tomothers that gave birth between one day and years 0cvink bmc pregnancy and childbirth page of fig number of studies per year with and without casemix correction is not presented in fig as only studies are only included until24th of march rates was least often studied at the level of the individualphysician and group of physicians no clear timetrend was observed in the choice of aggregation level ortrend in observed variation based on the level of aggregation the largest variation in caesarean section rates is reported on both the lowest ie individual level and thehighest ie international level of aggregationneonatal outcomes were captured in andmaternal outcomes in of the studies all variables that were used to measure these outcomes arelisted in additional file many different variables wereused to measure neonatal and maternal outcomes neonatal mortality apgar score maternal mortalityandhaemorrhage are outcomes that were measured mostoften half of the outcome variables were used in justone single study table shows the numbers of studiesper aggregation level that took neonatal and maternaloutcome into account neonatal and maternal outcomeswere most often reported if variation of caesarean nicu admissionprior to the survey patient preference was assessed byposing a variety of questions on the reason of the caesarean section and perceived influence on decision makingno studies reported the quality of decision making in of the studies a sample of the study populationwas used to measure patient preference samples variedbetween and women and three studies used apredefined sampling frameusefulness for audit and feedbacktable shows the study characteristics by aggregation levelthe majority of the studies used one aggregationlevel a minority used two and only three studies used three aggregation levels healthcare providersindividual physicians group of physicians hospitals or hospital categories were compared in studies andgeographic areas national or regional in studies hospitals and grouped hospitals eg private vspublic hospitals were most often used as aggregation levelof analysis medical practice variation of caesarean sectionfig the effect of casemix correction on different aggregation levels 0cvink bmc pregnancy and childbirth page of table study characteristics by aggregation levelnumber of studiesaverage cohort sizemedian cohort sizeentire population measured instead of samplereported variation in caesarean section rate average ofstudiesreported variation in caesarean section rate median ofstudiescasemix correctionmedical outcomeindividuallevelgroup ofphysicians hospitallevelhospitalcategoryregionnational sections was studied atphysiciansthe level ofthe individualdeterminants and financial consequencesa limited number of studies explored determinants toexplain medical practice variation of caesarean sectionrates in an additional analysis hospital characteristics orphysician characteristics were used in studies to explain differences in caesarean section rates thevariables that were used are listed in additional file financial consequences of unwarranted variation of caesarean section rates were calculated in six studiesdiscussionalmost four decades have past and studies werepublished following opits first report on variation ofcaesarean section rates between geographic areas inaustralia clearly the issue raised by the first studies hasnot lost its sense of urgency among researchers nor forthe funders of research because the magnitude of unwarranted variation was considered problematic andremained stable over time while previous reviewsnarrowed their focus on measuring variation betweengeographic areas [ ] or studied the difference between public and private hospitals the focus of thisreview was on the presence of study characteristics thatmay help to reduce unwarranted variation of caesareansection ratesstrengths and limitationsa strength of our review was the systematic search andselection procedure which allowed us to identify almost all studies on medical practice variation that compared caesarean section rates this resulted in a largenumber of included studies a second strength is thehigh level of detail of our analysis the selection of theindividual variables that were used for casemix correction outcomes or determinants enabled us to present anindepth overview of study characteristicsseverallimitations should be addressed first weaimed to describe study characteristics of all relevantpublished studies ie irrespective of the quality in theenglish language and therefore did not perform a qualityassessment of the included studies second we were unable to retrieve all publications that were selected forfulltext screening in order to limit the number of missing s we contacted the authors of missing sby email or through researchgate however this wasnot successful for of the studiesinterpretationfirst we appraised whether studies were designed to distinguish between warranted and unwarranted practicevariation by performing casemix correction and analysing patient preference casemix correction is an essential aspect of the quality of studies on practice variationbecause without casemix correction it remains unclearwhat share of the variation is attributable to health differences between populations and thus to what extentthe variation is warranted our results show that only of the studies that compared caesarean section ratesbetween healthcare providers performed casemix correction casemix correction was performed by calculating adjusted rates expected rates stratified odds ratiosor logistic regression analysis over time we observed noimprovement in the performance of casemix correctionpatient preference another important aspect to identify unwarranted practice variation was only measured insix studies without the assessment of patient preference it remains unclear whether practice variation canbe explained by differences in the outcome of shared decision making this is especially important when bothmodes of delivery vaginal delivery and caesarean section are an acceptable option patients with a historyof caesarean section breech or twin delivery are examples in which information on patient preference is necessary to differentiate between warranted and unwarrantedmedical practice variation [] our results show 0cvink bmc pregnancy and childbirth page of that none of studies that assessed variation in these specific obstetric situations patient preference was analysedto improve the identification of unwarranted practicevariation future studies should not only measure patientpreference but should focus on the implementation ofshared decision making recent literature shows thatseveral shared decision making measures are availablewhich could be included in the study design of medicalpractice variation studies to improve comparability both within healthcare facilities and between them robson proposed what latercame to be known as the robson classification systemfor assessing monitoring and comparing caesarean section rates in the who and the figo jointly endorsed this classification as the international standardfor casemix correction [ ] our data show that following the publication of this guideline by the who in the robson classification was only used in of all studies comparing providers and in studies comparing regions literature shows that casemixcorrection can be improved even more if additional patient characteristics are considered only of thestudies that were published between and andused the robson classification did perform additionalcasemix correction ie age adjusted caesarean sectionrates within robson groups studies that performedcasemix correction with or without robson classification used a wide variation of maternal and obstetriccharacteristics to identify unwarranted practice variation we advise to at least use the robson classificationand to standardise variables for additional casemix correction a delphi procedure can help obstetricians andmidwives to reach consensus on which variables to use the necessity to which casemix correction is neededdepends on the level of aggregation the lower the levelof aggregation the more case mix correction contributesto a valid description of clinical performance health care providers often operate in a network andtreat a subset of the entire population that subset ismore likely to differ between providers as they get morespecialized and the casemix differences may justify differences in caesarean section rates this requiresthat studies aimed at lower levels of aggregation placemore emphasis on casemix correction reporting standards and appropriate small number statistics once casemix has been appropriately controlled forfuture studies should aim to decompose regional unwarranted variation into lower levels of aggregation thisdecomposition allows regional variation to be attributedto health care providers however disaggregation ofcontributions to lower levels of aggregation is not without major risk on its own as groups of physicians getsmaller their clientbase may diverge more from theregional averagefor example due to specializationreporting differences or chance within providers eghospitals further disaggregation to the physician levelcould help identify individual contributions while thelower number of observations may harm both validityand reliability they may also provide a stronger stimulusfor change if the information is interpreted intelligentlyand presented in a motivating environmentstimulus for change might be further enhanced whenoutcome reporting becomes integrated in future studydesigns healthcare professionals are intrinsically motivated to deliver the best healthcare for their patients reporting outcomes in casemix corrected feedbackinformation directly stimulates the intrinsic motivationto improve outcome for patients if it becomes visiblethat increased caesarean section rates do not yield improved maternal and neonatal outcomes professionalsmight be encouraged to adapt clinical behavior our results show that the wide variation in outcome variablesdemands consensus and standardization studies will become more comparable and better interpretable when astandard set of outcomes is used for maternal infectiousmorbidity outcomes after caesarean delivery a core outcome set cos is developed we encourage to develop a cos for neonatal outcomes after caesareansectionforty years of research on caesarean section rates havebeen unable to reduce unwarranted practice variationour study shows that most studies do not meet the criteria to identify unwarranted practice variation and cannot be used for audit and feedback to contribute to thereduction of unwarranted practice variation future studies should correct for differences in patient characteristics and patient preference present results at low levelsof aggregation and appeal to intrinsic motivation by including the health effects on mother and childsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12884020031693additional file search strategies additional file contains the searchstrategies that were used for the databases pubmed embase ebscocinahl and wileycochrane libraryadditional file list of included studies additional file contains a listof all studies that were included in this scoping review per study theresults are summarized we used the following definitions for theindependent variables used in the table year year of publication author first author of study included title title of study included study period years from which caesarean section rates were reported inexample if a researcher performed a questionnaire survey in andincluded deliveries from y prior to the survey we reported study period caesarean section rate unadjusted caesarean section rate ofmost recent year reported cohort size the cohort size from which thecaesarean section rate is calculated if caesareans section rates from 0cvink bmc pregnancy and childbirth page of multiple years were reported we noted specifically the cohort size of thecohort that was used to calculate the most recent caesarean section rate data source data source that was used by the authors to calculate thereported caesarean section rate it is reported as other if data source isunknown or multiple data sources are used casemix correction thestudy reported an adjusted caesarean section rate expected caesareansection rate reported stratified odds ratios by patient characteristics orused logistic regression to adjust for patient characteristics yn aggregation level aggregation level of analysis outcome outcomes maternal or neonatal were noted yn determinants anisational orphysician characteristics were used to explain reported difference in caesarean section rates between healthcare professionals hospitals groupsof hospitals or geographic areas ynadditional file studies per country additional file describes thenumber of studies on medical practice variation of caesarean sectionrates that were conducted in each count | Colon_Cancer |
"what clinicians know about the diagnosis treatment and prevention of disease originates from studies mostly done on male cells male mice and men1 historically for multiple reasons including the purported safety of women and their offspring women of childbearing age were excluded from clinical trials as a result medical research and care have been centred on male physiology the assumption was that male and female cells and animals were biologically identical and evidencebased medicine was defined by clinical trials done predominantly in men1 in the us national institutes of health nih mandated the inclusion of women in nihfunded clinical trials but many investigators did not follow this mandate and many of those who did include women did not analyse the results by sex23 minimising the effectiveness of this policy preclinical research and drug development studies have also predominantly used male animal models and cells4 it is not surprising that a us government accountability office report found that eight of the ten prescription drugs withdrawn from the market between and posed greater health risks for women than for men most funding agencies from europe and north america have implemented policies to support and mandate researchers to consider sex and gender at all levels of medical research8 still the field of sexbased biology and medicine is often viewed as a specialised area of interest rather than a central consideration in medical research essential for the success of clinical care and translational science is awareness by clinicians and researchers that the diseases they are treating and studying are characterised by differences between women and men in epidemiology pathophysiology clinical manifestations psychological effects disease progression and response to treatmentthis review explores the role of sex biological constructs and gender social constructs as modifiers of the most common causes of death and morbidity and articulates the genetic biological and environmental determinants that underlie these differences we aim to guide clinicians and researchers to better understand and harness the importance of sex and gender as genetic wwwthelancetcom vol august biological and environmental modifiers of chronic disease ultimately it is a necessary and fundamental step towards precision medicine that will benefit women and mensex as a genetic modifier of biology and diseasesex differences in disease prevalence manifestation and response to treatment are rooted in the genetic differences between men and women genetic sex differences start at conception when the ovum fuses with a sperm cell carrying an x or a y chromosome resulting in an embryo carrying either xx or xy chromosomes this fundamental difference in chromosome complement eg genes outside the testisdetermining sry gene generates ubiquitous sex differences in the molecular makeup of all male and female cells9 first the y chromosome carries genes that exhibit subtle functional differences from their xlinked homologues eg zfy vs zfx and uty vs utx and also carries genes with no homologue at all eg sry in addition in men the x chromosome carries only maternal imprints ie epigenetic modifications made by the parent in generating the sex cellswhich alter the expression of genes in the offspring as women have x chromosomes from both parents they carry maternal and paternal imprints which target a different set of genes random inactivation of one of the x chromosomes in female cells which prevents sex differences in x chromosome gene dosage causes another degree of sex difference in gene expression as some of these xlinked genes escape inactivation in women those genes are often expressed at higher levels in women than in men9 sexspecific gene expression due to genomic search strategy and selection criteriawe searched pubmed for papers published in english between jan and june using sex or gender and the name of the disease of interest as search terms although we tried to cite seminal studies when necessary because of space limitation representative reviews were often selectedlancet diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine and southeast louisiana veterans health care system medical center new orleans la usa prof f mauvaisjarvis md barbra streisand womens heart center cedarssinai smidt heart institute los angeles ca usa prof n bairey merz md national heart lung institute imperial college london london uk prof p j barnes md department of pharmacology and department of neurology college of medicine center for innovation in brain science university of arizona tucson az usa prof r d brinton phd department of medical epidemiology and biostatistics and center for gender medicine karolinska institutet stockholm sweden prof jj carrero phd channing division of network medicine and the division of pulmonary and critical care medicine department of medicine brigham and womens hospital harvard medical school boston ma usa d l demeo md neuroscience institute and department of biology geia state university atlanta ga usa prof g j devries phd department of psychiatry university of colorado school of medicine anschutz medical campus aurora co usa prof c n epperson md division of oncology department of medicine washington university school of medicine st louis mo usa prof r govindan md w harry feinstone department of molecular microbiology and immunology the johns hopkins bloomberg school of public health baltimore md usa prof s l klein phd department of biomedical metabolic and neural sciences university of modena andreview 0creggio emilia azienda ospedalierouniversitaria di modena ospedale civile di baggiovara modena italy prof a lonardo md department of psychiatry department of psychology and department of obstetrics gynecology university of illinois at chicago chicago il usa prof p m maki phd department of neurology mcgovern medical school university of texas health science center houston tx usa prof l d mccullough md berlin institute of gender medicine charituniversittsmedizin berlin berlin germany prof v regitzzagrosek md department of cardiology university hospital z¼rich university of z¼rich switzerland prof v regitzzagrosek center for womens health research divisions of general internal medicine and cardiology university of colorado school of medicine aurora co usaimprinting extends to autosomes as well and these imprinted genes exhibit sexspecific and tissuespecific expression in humans10 thus fundamental sex differences deriving directly from genetic heterogeneity between the x and y chromosome complements and parentoforigin inher itance exist at the molecular level in all human cells these sex differences persist throughout life and are independent of sex hormones figure arguably the greatest source of differences between men and women comes from the y chromosomal sry gene which directs the development of a testis in men the ensuing developmental surge of testicular testosterone permanently masculinises the reproductive tract and the anisation of brain circuits affecting male behaviour at puberty1112 in humans the first surge occurs at the end of the first trimester of pregnancy because it alters cellular gene expression and tissue structure in multiple ans of men via epigenetic mechanisms this testosterone surge is also paramount in programming sex differences in physiology and susceptibility to diseases that will manifest in adulthood after this initial testicular testosterone surge gonadal hormone concentrations remain low until puberty which triggers lasting sex differences in circulating oestrogens and testosterone concentrations after puberty cells with androgen or afemale sexbrandom x chromosomeinactivation and escapexxxxxxxxxxxxxxxxxxxy chromosome complementmale sexsryctesticular testosterone surgefetal testistestosteroneohogenetic diï¬erences of male and female cellsepigenetic programming of male cellsfigure genetic causes of sex differencesa genetic sex differences start with cells carrying either xx or xy chromosome complement eg genes outside the testisdetermining sry gene which generates ubiquitous sex differences in the molecular makeup of all male and female cells b random inactivation of one x chromosome in female cells causes another level of sex differences in gene expression some xlinked genes escape inactivation in female individuals and have a higher expression in female than male individuals c the y chromosomal sry gene directs the development of a testis in male individuals which produces a surge of testicular testosterone at the end of pregnancy the testosterone surge programmes cellular gene expression and tissue structure in multiple ans of male individuals via epigenetic remodelling the combination of these genetic and developmental events programmes sex differences in physiology and susceptibility to diseases that will manifest in adulthoodoestrogen receptors will be affected differ ently in men and women the combination of all genetic and hormonal causes of sex differences aforementioned culminates in two different biological systems in men and women that translate into differences in disease predisposition manifestation and response to treatment therefore sex is an important modifier of physiology and disease via genetic epigenetic and hormonal regulations figure gender as a determinant of patients and doctors behaviour and as a modifier of health disease and medicinegender according to the global health definition refers to the socially constructed norms that impose and determine roles relationships and positional power for all people across their lifetime13 gender interacts with sex the biological and physical characteristics that define women men and those with intersex identities13 gender is not a binary term it includes the understanding that in many people traits of masculinity or femininity coexist and are expressed to different degrees gender attributes are fluid more than two thirds of women and men report genderrelated characteristics traditionally attributed to the opposite sex1415 in transgender people gender identity differs with the sex they were assigned at birth so far transgender people have generally been underrepresented in clinical studies to date although this underrepresentation is changing gender is an equally important variable as biological sex in human health and influences the behaviour of communities clinicians and patients1415 gender roles represent the behavioural norms applied to men and women in society which influence individuals everyday actions expectations and experiences including diet perceived stress smoking and physical activity and affect health and disease susceptibility gender identity describes the fluidity of how a person perceives oneself as a woman or a man which affects feelings and behaviours gender relations refer to how we interact with or are treated by people on the basis of our ascribed gender institutionalised gender reflects the distribution of power between men and women in the political educational and social institutions in society and shapes social norms that define perpetuate and often justify different expectations and opportunities for women and men1617 as such the distribution of genderrelated charac teristics within populations of men and women can influence health differently than biological sex together these gender constructs determine access to health care helpseeking behaviours and individual use of the healthcare system being perceived as a man or a woman triggers different responses from clinicians who might diagnose and suggest interventions differently according to gender as such gender largely determines the use of preventive measures and referral for or acceptance of invasive therapeutic strategies genderrelated behaviours contribute to risk exposure and preventive behaviour in several wwwthelancetcom vol august review 0cdiseases this postulation is well exemplified in the cardiovascular field in which women often underestimate their risk compared with men and seek consultation later than men in the clinic for treatment of myocardial infarction1819 in the genesispraxy prospective study mortality year after an acute coronary event was more strongly associated with gender than with biological sex1415 similarly control of cardio vascular risk factors hypertension diabetes depres sive symptoms was better predicted by gender than by biological sex1415 therefore including a gender dimension in clinical studies and practice will contribute to the understanding of different clinical manifestations and outcomes of diseases in women and men1617 although beyond the scope of this review it is also important to consider that regarding health and disease gender intersect with race or ethnicity and age20 sex and gender are fundamentally and frequently reciprocally interrelated in biology and disease24 sex influences behaviours eg towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and pain and nutritional habits might produce epigenetic modifications that modulate gene expression and biological phenotypes figure summarises how sex and gender are interrelated in biology and diseasesex and gender differences in major chronic diseaseshaving established the importance of sex and gender in disease we will summarise their influences on the most common causes of death and debilitating diseases in the usa as an example figure note that these sex and gender disparities are relevant to other highincome countries as well as lowincome and middleincome countries where the burden of these diseases becomes increasingly like those in highincome countries26 in most diseases efforts to separate the effects of sex and gender are still incomplete so that we just refer to the differences among women and men because current knowledge about pathophysiology diagnosis and treatment of disease is primarily based on men as representative of the human species this review focuses on how women differ from men we discuss some key aspects regarding the dimensions of men in a dedicated sectionheart diseaseepidemiology pathogenesis manifestations and diagnosisheart disease is the leading cause of death in the usa in heart disease accounted for · of all deaths for men and for · of all deaths for women figure ischaemic heart disease and heart failure are major contributors to heart disease mortality and have important sex and gender differences for example heart failure disproportionately contributes to coronary heart disease mortality in women27 potentially due to undiagnosed ischaemic heart disease in women the strength of the association with cardiovascular risk factors differ by sex biological sexsex chromosomesepigeneticeï¬ectssex hormonesohohohobehaviour of patients and doctorssocietygender constructslifestylenutritional habitsexerciseperceived stresssmokingdiseasepathophysiologymanifestationresponse to treatmentdisease perceptionhelpseeking behaviouruse of health caredecision makingtherapeutic responsesex and gender diï¬erences in health disease and medicinefigure interrelation between sex and gender in health diseases and medicinebiological sex causes sex differences through genetic and hormonal influences in disease pathophysiology clinical manifestations and response to treatment sex also influences behaviours towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and nutritional habits produce epigenetic modifications that modulate the expression of biological sex gender constructs determine patients perception of disease helpseeking behaviour and individual use of health care gender constructs also influence decision making and trigger different therapeutic responses from providers biased by gendersystolic blood pressure and hypertension smoking and diabetes are associated with higher hazard ratios for myocardial infarction in women than in men28ischaemic heart disease is the most recognised example for integrating the concept of gender and sex which shape divergent or distinct disease outcomes compared with men women suffering from ischaemic heart disease are older this difference is historically believed to be due to the protection of endogenous oestrogens29 although contemporary study refutes this simplistic explanation30 and associations cannot be inferred to be causation still women suffering from ischaemic heart disease are underdiagnosed3132 and less likely to have a prehospital diagnosis of myocardial infarction33 the reasons for this disparity reflect the intersection between sex and gender first biological sex differences exist in the pathogenesis of ischaemic heart disease whereas men are more likely to be affected by obstructive coronary artery disease of large vessels than women coronary microvascular dysfunction36 leading to chronic myocardial ischaemia without obstructive coronary artery disease has a higher prevalence in women than men37 a metaanalysis reported that following acute myocardial infarction both sexes most often presented with chest pain but compared with men women were more likely to present with pain between the shoulder blades nausea or vomiting and shortness of breathprof j g regensteiner phd department of medicine department of paediatrics and department of neuroscience washington university school of medicine st louis mo usa prof j b rubin md center for the study of sex differences in health aging and disease geetown university washington dc usa prof k sandberg phd division of gastroenterology duke university medical center durham nc usa a suzuki md and durham va medical center durham nc usa a suzukicorrespondence to prof franck mauvaisjarvis diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine new orleans la usa fmauvaistulaneeduwwwthelancetcom vol august review 0cmale individualsother·heart disease·protection might disappear after menopause45 by contrast testosterone induces adverse cardiac remod elling in the male heart44chronic liver disease ·inï¬uenza and pneumonia ·suicide ·alzheimers disease ·type diabetes ·stroke ·cpd ·injuries ·female individualsother·septicaemia ·chronic kidney disease ·inï¬uenza and pneumonia ·type diabetes ·injuries ·alzheimers disease ·stroke ·cpd ·cancer·heart disease·cancer·figure percent distribution of the ten leading causes of death by sex usa adapted from heron25 cpdchronic pulmonary diseasesecond a gender bias appears to be responsible for the absence of recognition of ischaemic heart disease presentation in women38 men and women with ischaemic heart disease who score high on feminine roles and personality traits on questionnaires designed to ascertain aspects of gender are at an increased risk of recurrent ischaemic heart disease independent of female sex39heart failure affects of adults aged years and older and more women than men in absolute numbers4041 heart failure occurs at an older age and with less ischaemic causes in women than in men however hypertension and diabetes predispose older women to heart failure to a greater extent than men heart failure with preserved ejection fraction a form of heart failure with normal systolic function is twice as prevalent in women as in men by contrast heart failure with reduced ejection fraction affects more men than women women who have heart failure with preserved ejection fraction have smaller and stiffer hearts than men inflammation and the resulting fibrosis play a sexspecific role in the pathogenesis of heart failure with preserved ejection fraction under stress premenopausal womens hearts develop less inflammation resulting in less fibrosis than mens hearts4243 this difference is partially driven by sex hormones as oestrogens produce antiinflammatory actions on endothelial and immune cells and promote cardioprotective effects in premenopausal women44 this response to treatmentcompared with men women suffering from ischaemic heart disease are less likely to receive evidencebased treatment3132 and when suffering from acute myocardial infarction they are less likely to receive reperfusion33 an stelevation myocardial infarction registry revealed that compared with men women exhibit delayed reperfusion leading to higher mortality46 women suffering from acute myocardial infarction treated by male emergency physicians have a higher mortality rate than those treated by female physicians38 additionally male physicians are more effective at treating female patients with acute myocardial infarction when they work with female colleagues and when they have experience in treating female patients38 this treatment disparity between women and men can be corrected by improving emergency recognition of stelevation myocardial infarction in women and acceleration of percutaneous coronary intervention which equilibrates gender mortality47guidelines for the treatment of heart failure are similar for women and men24 however evidence suggests that optimal survival in women occurs with lower doses of β blockers angiotensin receptor blockers and angiotensin converting enzyme inhibitors than in men48 finally fewer women undergo heart transplantation than men although women are more frequently donors suggesting a referral bias could exist41cancersepidemiology pathogenesis manifestations and diagnosiscancers are the second leading cause of death dominated by lung cancer accounting for · of deaths in men and · of deaths in women figure more men develop cancer than women49 with few exceptions eg meningioma thyroid cancer lung cancer in nonsmokers nonreproductive cancers exhibit a male predominance though for some cancers oropharynx larynx oesophagus and bladder the male versus female incidence ratios can be higher than a male predominance in cancers that affect both sexes is evident around the world in all races and at all ages50 survival is also shorter for men than women across multiple cancer types the higher cancer risk in men is partially explained by gender constructs like dietary habits or risk behaviours such as smoking and alcohol consump tion51 it is unlikely to be the only cause after appropriate adjustment for these risk factors adult men still have a higher cancer risk than women52 moreover a similar male bias in incidence and survival is seen in paediatric cancers before puberty and the adoption of highrisk cancerpromoting behaviours eg smoking53the universal male predominance in cancer incidence and differential outcomes argues for a fundamental role wwwthelancetcom vol august review 0cin inutero tumour suppressors of sex in addition to gender in cancer biology sexspecific biology includes genetic differences xx vs xy chromosomes the incomplete xinactivation in female individuals which results in biallelic expression of xencoded female cells54 y chromosomeencoded oncogenes such as the rnabinding motif on y chromosome in male cells55 and the chromatin remodelling effects of testicular testosterone in male cells56 these mechanisms have an influence on several of the hallmarks of cancer57 including metabolism growth regulation58 angiogenesis and immunity which all contribute to cancer predisposition59 a crucial example is the male predisposition to glioblastoma which is the most common form of brain cancer in glioblastoma there is a cellintrinsic predisposition of male astrocytes a subtype of glial cell to malignant transformation60 after puberty sex hormones produce additional epigenetic and acute effects on cells that further influence sex disparities in cancer for example the increased frequency and aggressive phenotype of hepatocellular carcinoma in male individuals has been linked to the stimulatory effects of androgens in male individuals and the protective effects of oestrogens in female individuals61 importantly the biology of cancer is not the same across histological and genetic diagnostic groups or even within single histol ogical subtypes thus the interaction between sex gender and cancer mechanisms cannot be expected to be constant take colon cancer the second leading cause of cancerrelated death for example although women have a lower overall incidence of colon cancer than men they have a higher incidence of rightsided colon cancers which have the worst outcomes62 tumours from women with rightsided colon cancers exhibit a distinct molecular signature of energy metabolism compared with those of women with leftsided colon cancers63 this molecular signature is not observed when comparing tumours from men with rightsided colon cancers to men with leftsided colon cancers thus overall the male predisposition to cancer is probably the consequence of genetic program ming of male cells and the effect of sex hormones after puberty interacting with genderspecific behaviours to establish cancer risksresponse to treatmentin the future cancer prevention and treatment will be improved by sexspecific and genderspecific approaches for example immune checkpoint inhibitors can improve survival for men with advanced melanomas and nonsmallcell lung cancers more than for women64 the molecular subtyping of glioblastomas based on sexspecific transcriptomes has the potential to enhance chemotherapy in a sexspecific manner65 in colon cancer sex differences in xenobiotic metabolism regulatory networks might underlie greater treatment response in women than men and require modification of approaches for men with colon cancer66 other elements of cancer metabolism are also ripe for novel sexspecific targeting in treatment cellular nutrient partitioning is sexually dimorphic so approaches such as ketogenic diets or glutaminase inhibition might be associated with substantial sexspecific responses67 furthermore data from models of development ageing and cancer all indicate that molecular pathways such as the enzyme phosphatidylinositol 3kinase and tumour suppressors such as p53 and retinoblastoma protein are sexually dimorphic and require sexspecific targeting545968chronic pulmonary diseaseepidemiology pathogenesis manifestations and diagnosischronic pulmonary disease is the third leading cause of death for women · of deaths and the fourth for men · figure it is mostly accounted for by chronic obstructive pulmonary disease and to a lesser extent by asthma chronic obstructive pulmonary disease is characterised by irreversible airflow limitation and is associated with previous exposure to smoking or air pollutants women are overrepresented among individuals with chronic obstructive pulmonary disease especially among those with earlyonset disease or those who have never smoked69 women are also twice as likely to have chronic obstructive pulmonary disease with chronic bronchitis and women with severe chronic obstructive pulmonary disease have as much emphysema as men countering the misconception of emphysema as a male form of chronic obstructive pulmonary disease70 the female lung is more susceptible to chronic obstructive pulmonary disease than the male lung and women develop symptoms of the disease at a younger age with less tobacco exposure than men71 the genetic epidemiology of chronic obstructive pulmonary disease copdgene study72 suggests that earlyonset chronic obstructive pulmonary disease might originate in utero in susceptible women from alterations in lung development potentiated by maternal asthma and smoking genetic factors or hormonal influences future studies should focus on the contribution of maternally inherited factors such as mitochondrial and x chromosome genes to understand disease pathogenesis it is important to consider gender constructs as well smoking advertising campaigns targeting women rose in the 1960s and the resulting higher smoking rates influenced womens risk for developing chronic obstructive pulmonary disease73 from a disease severity vantage point chronic obstructive pulmonary disease exacerbation rates are also higher in women than men especially at a younger age74 additionally despite the burden of symptomatology and increased rates of hospitalisations and deaths women with chronic obstructive pulmonary disease are often misdiagnosed and disproportionally suffer from comorbid conditions including anxiety and depression therefore physicians should consider chronic obstructive pulmonary disease in the differential diagnosis of women with pulmonary symptoms regardless of tobacco or pollutant exposure historieswwwthelancetcom vol august review 0casthma characterised by variable airflow obstruction and chronic airway inflammation also affects men and women differently asthma is more prevalent in prepubertal boys than girls regarding asthma both male biological sex lung development and atopy and male gender constructs related to outdoor play and indoor pet exposure are factors contributing to the devel opment of asthma and sex versus gender contributions could be difficult to separate75 from puberty onwards more female than male individuals have asthma with an increased severity in middleaged women and a higher mortality rate76 this phe nomenon could be secondary to gender differences eg symptoms perception or healthseeking behaviours however biological sex plays a crucial role in asthma and sex hormones have a major impact on female asthma symptoms and severity after puberty75 worsening of asthma occurs in women before menstruation and is known as premenstrual asthma premenstrual asthma is more common in women with severe rather than mild asthma obesity rather than normal weight and a long rather than a short duration of asthma77 premenstrual asthma is hypoth esised to be due to a fall in progesterone and patients with a severe disease respond to progestogens78 during pregnancy approximately a third of asthmatic women exhibit a worsening of asthma a third show an improvement and the remainder are unaffected79response to treatmentthe menopausal transition represents a pivotal time of accelerated decline in lung function in women with chronic obstructive pulmonary disease and thus represents a sexspecific window for treatment intensification these observations also suggest that oestrogens protect from chronic obstructive pulmonary disease women exhibit greater expression of m2 over m3 muscarinic receptors and accordingly show greater improvements in lung function than men in response to the muscarinic anticholinergic bronchodilator ipatroprium80 pooled analyses of drug studies also suggest that women experience a greater improvement in quality of life than men after treatment of chronic obstructive pulmonary disease with a β2adrenoreceptor agonist combined with an anti cholinergic drug eg indacaterol and glycopyrronium81unlike chronic obstructiv | Colon_Cancer |
colorectal cancer crc is the second leading cause of cancer deathworldwide and most deaths result from metastases we have analyzed animal modelsin which apc a gene that is frequently mutated during the early stages of colorectalcarcinogenesis was inactivated and human samples to try to identify novel potentialbiomarkers for crcmaterials and methods we initially compared the proteomic and transcriptomicproï¬les of the small intestinal epithelium of transgenic mice in which apc andor mychad been inactivated we then studied the mrna and immunohistochemical expressionof one protein that we identiï¬ed to show altered expression following apc inactivationnucleosome assembly protein like nap1l1 in human crc samples and performeda prognostic correlation between biomarker expression and survival in crc patientsresults nap1l1 mrna expression was increased in mouse smallintestine followingapc deletion in a myc dependant manner and was also increased in human crcsamples immunohistochemical nap1l1 expression was decreased in human crcsamples relative to matched adjacent normal colonic tissue in a separate cohort of crc patients we found a strong correlation between nap1l1 nuclear expressionand overall survival in those patients who had stage iii and iv cancers nap1l1 expression is increased in the mouse smallintestine followingapc inactivation and its expression is also altered in human crc immunohistochemicalnap1l1 nuclear expression correlated with overall survival in a cohort of crc patientsfurther studies are now required to clarify the role of this protein in crckeywords colorectal cancer biomarkers apc nap1l1 prognosis survivaledited byboris zhivotovskykarolinska institutet ki swedenreviewed bygiovanna caderniuniversity of florence italygabriele multhofftechnical university of munichgermanycorrespondenced mark pritchardmarkpritchardliverpoolacukmarkpritchardlivacuk deceasedspecialty sectionthis was submitted tomolecular and cellular oncologya section of the frontiers in oncologyreceived december accepted july published august citationqueiroz cjs song f reed kralkhafaji n clarke arvimalachandran d miyajima fpritchard dm and jenkins jr nap1l1 a novel human colorectalcancer biomarker derived fromanimal models of apc inactivationfront oncol 103389fonc202001565frontiers in oncology wwwfrontiersinaugust volume 0cqueiroz introductioncolorectal cancer crc is the third most common cancer typeand the second leading cause of cancer death worldwide deaths usually result from recurrent and metastatic disease mostinternational guidelines recommend chemotherapy to reducethe risk of recurrence in stage iii tumors and to prolongsurvival in stage iv cancers conversely chemotherapy isgenerally not used in stage i and most stage ii tumors howeversome patients with lowrisk stage iii disease may respond wellfollowing courses of chemotherapy that are shorter than the6month standard schedule although the deï¬nition of lowrisk has not been well established in this scenario additionally almost of patients who have stage ii tumorsand who are therefore considered to have lowrisk diseaserelapse and eventually die from cancer progression thereis currently no accurate biomarker to better deï¬ne prognosiswithin stage groups therefore biomarkers for prognosticstratiï¬cation in crc have the potential ofimproving thetreatment decisionmaking process we hypothesized thatstudying molecular mechanisms that are known to be involvedin crc development might yield promising novel biomarkersfor this diseasethese modelsformation mirroring cancer developmentadenomatous polyposis coli apc inactivating mutationsare the earliest and most common genetic alterations in theadenomacarcinoma sequence thatleads to crc suchmutations result in the accumulation of catenin within cellsand activation ofthe wnt signaling pathway animalmodels of apc inactivation demonstrate epithelial transformationand tumorthe ahcreapcï¬ï¬ mouse one ofan animal bearing loxpï¬anked apc alleles and a crerecombinase transgene when this animalis exposed tonaphthoï¬avone crerecombinase transcription is activatedresulting in the deletion of loxpï¬anked apc alleles speciï¬callyin the smallthus causing an acuteactivation of the wnt pathway the apcmin mouse hasa germline mutation in one apc allele simulating a familialadenomatous polyposisfap patient and spontaneouslydevelops multiple intestinal neoplasms during its lifespan the myc gene is a wnttarget that plays an essentialrole in the development of malignant phenotypes upon apcinactivation we hypothesized that the analysis ofmouse models of apc and apcmyc deletion could lead to thediscovery of genes or proteins with potential clinical use ashuman crc biomarkersintestinal epitheliumisour group has previously published a proteomic evaluationof an animal model of crc based on acute apc deletionahcreapcï¬ï¬ mouse a 4plex itraq labeling analysisidentiï¬ed proteins that were significantly altered uponapc deletion up and downregulated and whichwere proposed as wnt targets we have now performed anadditional analysis of this dataset by comparing the proteinlist with the transcriptomic data generated using aï¬ymetrixarrays and intestinal tissues from the same mice thisstudy used apcdeï¬cient ahcreapcï¬ï¬myc and doubleahcreapcï¬ï¬mycï¬ï¬ micemutant apcmycdeï¬cientnap1l1 a novel crc biomarkerthatshowed congruent ï¬ndingsto identify myc dependant wnt pathway genes followingapc inactivation we investigated whether there were anygenesproteinsin bothanalyses according to strict criteria one protein nucleosomeassembly protein like nap1l1 wasidentiï¬ed thatmet our criteriawe therefore analyzed the expression of nap1l1 in apcdeï¬cient ahcreapcï¬ï¬myc and doublemutant apcmycdeï¬cient ahcreapcï¬ï¬mycï¬ï¬ mice to assess whether itsexpression was mycdependant and therefore a potentialbiomarker of wnt activation following conï¬rmation of ourï¬ndings we investigated the mrna and protein expressionof nap1l1 in tumor and adjacent normal mucosa samplesfrom patients with crc as well as colonic tissues fromunaï¬ected individualsmaterials and methodsanimal samplesmouse experiments were performed with uk home oï¬ceapproval with personal and projectlicenses and and according to arrive guidelines and followinglocal ethical review by the cardiï¬ university animal welfareethical review panel the transgenic mice that were used in thisstudy were generated and maintained at the university of cardiï¬as previously described in small intestinal epithelial cellextracts were generated from these mice following injection ofbetanaphthlaï¬avone as described by hammoudi intestinalqpcr rtpcrfor the mouse smalltissue samples and humancrc samples obtained in the united kingdom wales cohorts and total rna was used to synthesize ï¬rst strandcdna using a versotm cdna kit thermo scientiï¬c andanchored oligodt primers thermo scientiï¬c according tothe manufacturers instructions singlestranded cdna sampleswere ampliï¬ed in a polymerase chain reaction pcr usingsequencespeciï¬c primers eurogentec and probes from theuniversal probe library roche that were designed using theuniversal probelibrary assay design centre using pcr mastermix roche and a light cycler roche see supplementarytable for primers and probes usedfor the human crc samples recruited in brazil rnawas extracted using the rneasy rcid13 mini kit qiagen hildengermany cdna was produced using taqman rcid13 reversetranscription reagents kitapplied biosystems carlsbadca united states according to the manufacturers protocolquantitative pcr reactions were performed using the fastrealtime pcr system applied biosystems foster city caunited states see supplementary table for expression assayspeciï¬cationsproteomic and microarray comparisonanalysiswe set outto determine the subset of genesproteinswhich demonstrated upregulation of both protein and mrnafrontiers in oncology wwwfrontiersinaugust volume 0cqueiroz nap1l1 a novel crc biomarkerfollowing apc deletion but no increase in expression inahcreapcï¬ï¬mycï¬ï¬ mice the maxdviewprogram dataï¬les from the microarray analyses were used to calculate geneexpression fold changes in the intestinal tissues from the varioustransgenic mice these data were then combined with ourpreviously published proteomic proï¬le data in which weidentiï¬ed proteins which showed a greater than fold increasein expression following apc deletion using itraq analysisthe following features from the dna microarray analysiswere used to identify candidate myc dependant wnt pathwayproteins i a statistically significant p greater than fold increase in expression in apcdeï¬cient ahcreapcï¬ï¬mice compared to wildtype mice apcwt ii no significantincrease in expression in the doublemutant apcmyc deï¬cientahcre apcï¬ï¬ mycï¬ï¬ mice when compared to the wildtype mice a ratio value of with boundaries of and apcmycwt and iii a ahcre apcï¬ï¬ mycï¬ï¬ toahcreapcï¬ï¬ ratio with p apcmycapc andï¬ndings being conï¬rmed by at least three aï¬ymetrix probescorresponding to the proteinhuman samples and ethicsunited kingdom cohortstotal rna samples from crc tumor tissues and adjacentuninvolved colonic mucosa patients were obtained fromthe wales cancer bank wales cohort with the ethicalapproval and informed consent that is associated with thistissue bank and these samples were used in the initial geneexpression studies there were male and female patientswith samples from stage i from stage ii and fromstage iii crc and mean patient age was years anotherset of matched sample pairs was subsequently obtainedfrom the same tissue bank wales cohort and thesewere analyzed separately for validation of the ï¬ndings walescohort had samples with stage i samples with stageii and samples with stage iii crc and the mean patientage was yearsa tissue microarray tma was constructed using samplesfrom crc patients recruited at the countess of chesterhospital nhs foundation trust chester united kingdomagain with informed consent and local ethics committee approval12nw0011 cancer tissues were available for all cases caseswith stage i cases with stage ii cases with stage iii and caseswith stage iv crc whilst normal adjacent mucosa was onlyobtained for of them the analysis of this cohort was part of theimmunohistochemistry ihc validation study and the ï¬ndingsare presented in supplementary figure brazil cohortfresh frozen tissues from tumors removed from crc patientsand normal colonic tissues from normal individuals who hada normal colonoscopy on a bowel cancer screening programwere collected after informed consent was obtained and wereanalyzed in the gene expression studies the crc samples werefrom males and females with stage i ii iii and iv crc and the mean patient age was years the normalsamples were from males and females and the patients hada mean age of yearsfor the initial brazilian ihc study samples from patients were prospectively collected in cuiaba brazil betweenjanuary and august informed consent was obtainedfragments from the tumor and from the normal adjacent mucosaat least cm from the tumor were ï¬xed in buï¬eredformalin for h and then processed into paraï¬n blocks patientcharacteristics are described in the results section belowfor the ihc prognostic study samples were obtained for crc patients from the archives of two pathology laboratoriesin cuiaba brazil laboratorio são nicolau and the julio mulleruniversity hospital pathology lab inclusion criteria were i or more years since diagnosis ii presence of tumor tissue inthe paraï¬n block iii traceable patient survival information andiv survival for at least days after surgery we tracked thepatients current health service to obtain mortality informationalternatively if no clinical information was available we checkedthe brazilian electronic death database sistema de informacaode mortalidade which records all deaths and their causesoverall survival was recorded as the interval between diagnosisand death from any cause when death had occurred orthe date when the database was last checked when deathhad not occurredthis research was approved by the committee for researchethics of the hospital universitario julio muller federaluniversity of mato grosso cuiaba brazil and by the braziliannational commission for research ethics conep decisionnumber forimmunohistochemistry in the validationstudyfour µm sections from paraï¬n blocks were subjected toihc trisbuï¬ered salinetweenstandard protocols tbst was used for all washesin summary themain steps were dewaxing in xylene twice endogenousperoxidases block using hydrogen peroxide in methanolrehydration in decreasing concentrations of ethanol solutionsand ï¬nally distilled water heatinduced epitope retrievalusing mm citrate buï¬er ph in a microwave ovenat w for min block using normal goat serumdako ely united kingdom in tbst for min at roomtemperature primary rabbit antinap1l1 antibody cat numberab33076 abcam cambridge united kingdom in normal goat serum in tbst overnight at ¦c in a humidchamber biotinylated secondary goat antirabbit antibodydako ely united kingdom solution in normalgoat serum for min at room temperature avidinbiotinperoxidase complex vectastain elite abc kit peterboroughunited kingdom for min at room temperature visualizationusing 33cid48diaminobenzidine sigmafast dab tablets sigmagillingham united kingdom substrate solution applicationof hematoxylin counterstain dehydration in increasingconcentrations of ethanol clearing in xylene and mountingusing dpx mounting medium sigma and glass coverslipsstained sections were viewed and photographed using afrontiers in oncology wwwfrontiersinaugust volume 0cqueiroz nap1l1 a novel crc biomarkermicroscope and camera set leica biosystems milton keynesunited kingdom the same staining protocol was used forthe additional united kingdom patient cohort described insupplementary figure immunohistochemistry in the prognosticstudythe protocol adopted in this part ofthe research wasthe routine technique used in the são nicolau laboratorycuiababrazil a pathology lab with extensive expertise in ihcall branded solutions and buï¬ers were purchased from cellmarquetmsigmaaldrich rocklin ca united states fourµm tissue sections were dewaxed in xylene and rehydratedas previously described after a wash step in distilled waterslides were immersed in trilogytm pretreatment solution andincubated at ¦c for min for epitope retrieval after this theslides were washed in phosphate buï¬ered saline pbs peroxideblocktm solution was added and samples were incubated for min after another pbs wash the primary antibody solutionsame concentration as those described above was placed ontothe samples and incubated for min at room temperature afterwashes in pbs hidef detectiontm ampliï¬er secondary antibodysolution was applied to the slides for min after a furtherpbs wash the former step was repeated using hidef detectiontmdetector a horseradish peroxidase polymer solution finallycolor development was performed by incubating the slides withdab substratetm chromogen stained slides were counterstaineddehydrated and mounted as described above slides werephotographed using an axio scopea1 microscope coupled withan axiocamhrc camera zeiss oberkochen germany somesamples were stained using both the protocol described here andthat in the previous section and these conï¬rmed that the stainingpatterns were similar see supplementary figure tissues for cytoplasmic assessmentimmunohistochemistry scoring systemscoring was performed electronically using the software imagejpublicly available at rsbwebnihgovij imagej the imageswere initially edited in image j to exclude nonepithelialnoncancerousstromaltheplugin ihc proï¬ler was used based on the readingsproduced by this tool we calculated a modiï¬ed ihc proï¬lerscore [ of negative à ] [ of lowpositive à ] [of positive à ] [ of highpositive à ] with ï¬nalscores ranging from to for nuclear scoring the pluginimmunoratio was used it assesses the percentage of positivenuclei based on a threshold setting two microscopy ï¬eldsà containing at least stained epithelial in control casesor cancer cells each were analyzed per samplestatistical analysiscomparisons of continuous variables were carried out usingmannwhitney u test or kruskalwallis test followed by thedunnbonferroni test for post hoc comparison categorical datawere compared using the chisquare test or fishers exact test incase of less than ï¬ve expected counts per cell in the contingencytable for the survival analysis groups were assessed using thekaplanmeier method and survival curves were compared bylogrank tests when significant diï¬erences were observed coxproportional hazards model was used for multivariate analysistwosided pvalues were accepted as significant in theentire study all statistical analyses were performed using thesoftware ibm rcid13 spss rcid13 statistics version and r packagesresultscombination of proteomic andtranscriptomic datasetsour previously published proteomic data was integratedwith the dna microarray data from the doublemutant apcand mycdeï¬cient ahcreapcï¬ï¬mycï¬ï¬ mice as shown insupplementary table of the upregulated proteins fromthe itraq analysis only one also showed upregulation ofmrna in the intestine of ahcreapcï¬ï¬myc mice and nochange in the intestine of ahcreapcï¬ï¬mycï¬ï¬ mice using thecriteria described in the materials and methods section thisprotein was nap1l1qrtpcr wasevaluation of nap1l1 mrna expressionin mouse small intestinein order to validate whether nap1l1 mrna was upregulatedfollowing conditional apc deletion in the mouse intestinalepitheliumusing mrnacarriedoutintestinal epithelial cell extracts fromextracted from smallahcreapcï¬f mycï¬ï¬ ahcreapcï¬ï¬and ahcremycï¬ï¬mice days post induction we compared relative mrnaexpression in these transgenic mouse cohorts with the mrnaexpression levels in ahcreapcmyc wildtype miceresults are shown as fold change relative to the wildtypemice figure this analysis conï¬rmed that nap1l1 mrnaexpression was significantly increased following apc deletion ina mycdependent mannerevaluation of nap1l1 mrna expressionin human colorectal cancer samplesnap1l1 was then analyzed in three cohorts of human crcsamples we assessed the expression of nap1l1 ï¬rstly in mrnafrom crc tissues and matched normal mucosa from patientssupplied by the wales cancer bank nap1l1 demonstratedstatistically significant elevated mrna levels in crc samplesfigure wales cohort we next performed a conï¬rmatorystudy using a diï¬erent set of samples from the same tissuebank and we observed consistent results figure wales cohort in order to further validate the ï¬ndings we repeated theexperiment using a diï¬erent platform in terms of equipment andreagents and compared a cohort of normal colonic samplesindividuals without any colonoscopic evidence of colorectalneoplasia and crc samples from brazil figure brazilcohort once more significantly increased levels of nap1l1mrna were observed in crc specimens and this time thecomparison was with normal colonic tissue from patients whohad no evidence of colorectal neoplasiafrontiers in oncology wwwfrontiersinaugust volume 0cqueiroz nap1l1 a novel crc biomarkerdescribed in section materials and methods cancer tissues andthe matched unaï¬ected mucosa collected cm from the primarylesion were analyzed scoring was performed electronically usingthe software imagej publicly available at rsbwebnihgovij andthe plugins ihc proï¬ler for cytoplasmic scoring resulting in amodiï¬ed hscore ranging from to and immunoratiofor nuclear scoring ranging from to the sampleswere subdivided into two groups who had early stage samplesencompassing stages i and ii and late stage samples includingstages iii and iv crcwe initially assessed the expression of catenin to conï¬rmwnt pathway activation and to validate the electronic scoringmethods figure a clear and statistically significant increasein both nuclear and cytoplasmic localization of catenin wasobserved in cancer tissues compared to the adjacent mucosa asexpected based on previous literature thus validating ourscoring system using the same scoring methods we observed anopposite staining pattern for nap1l1 a clear and statisticallysignificant decrease in both the nuclear and cytoplasmicexpression of nap1l1 was seen in crc tissues relative to theadjacent mucosa figure no diï¬erence was detected betweenearly and late stage tumor groups for both markerswe also performed a conï¬rmatory analysis using a diï¬erentcohort of patients from the countess of chester hospitalnhs foundation trust chester united kingdom this analysisused a slightly diï¬erent manual scoring method as describedin supplementary figure the ï¬ndings were very similarto those demonstrated in the brazilian patients and againdemonstrated decreased nap1l1 expression in the crc samplessupplementary figure figure relative expression of nap1l1 mrna extracted from smallintestinal epithelial samples from induced ahcreapcmycahcreapcï¬ï¬ ahcre apcï¬ï¬ mycï¬ï¬ and ahcremycï¬ï¬ mouse modelsfour mice in each group showing the fold change in mrna expression foreach gene relative to ahcreapcmyc error bars standard error sep for ahcreapcï¬ï¬ compared to other groupsnap1l1 nuclear expression is a strongpredictor of survival in late stage crchaving demonstrated decreased nap1l1 immunohistochemicalexpression in crc samples we investigated whether theexpression pattern had any eï¬ect on patient outcome weanalyzed a further cohort of crc cases diagnosed between and median followup was months range months given the relatively small number of cases cancerstages were again combined into two groups early stage stages iand ii and latestage stages iii and iv immunohistochemistrywas conducted as described in section materials and methodstable describes the characteristics of the patients included inthis analysisinitially using mortality status as the binary event of interestroc curves were generated the area under the curve aucwas for the nuclear score and for the cytoplasmicscore cutoï¬s were determined either by manually assessing thebest balance between sensitivity and speciï¬city or electronicallyby the use of the software cutoï¬ finder and xtile all methods suggested the same cutoï¬ for nuclear staining of positive nuclei use of this threshold resulted in asensitivity of and a speciï¬city of for discriminatingmortality status for cytoplasmic staining sensitivityspeciï¬cityoptimization suggested a cutoï¬ of in a range from to yielding a sensitivity of and a speciï¬city offigure qpcr analysis of nap1l1 expression in crc tumors presentedas foldchange relative to normal tissues in different cohorts each columnrepresents the relative quantiï¬cation foldchange of nap1l1 mrnaexpression in crc compared to the respective normal control normal controlexpression mean wales cohort mean foldchange p paired colorectal cancer and adjacent normal colonic tissue samples walescohort mean foldchange p paired colorectal cancerand adjacent normal colonic tissue samples brazil cohort meanfoldchange p normal samples from healthy individualswithout colorectal neoplasia and colorectal cancer samplesmannwhitney u test error bars ± seconï¬rmation of differentialimmunoexpression of nap1l1 inhuman colorectal tissuesimmunohistochemistry for nap1l1 was then performed incolorectal tissue samples from a diï¬erent cohort of patients asfrontiers in oncology wwwfrontiersinaugust volume 0cqueiroz nap1l1 a novel crc biomarkerfigure staining patterns and scoring results for catenin nuclear and cytoplasmic staining of catenin increased in cancer tissues when compared to theadjacent mucosa no difference was observed between different stages of cancer p kruskalwallis test followed by post hoc dunnbonferroni test forpairwise comparisons error bars ± se sample numbers normal stages iii stages iiiiv magniï¬cation à the electronic tools suggested higher cutoï¬s and although these resulted in a higher speciï¬city the sensitivity was low despite these diï¬erences theprognostic results were similar so the cytoplasmic cutoï¬ of was selected to describe the resultsthe prognostic cohort was therefore divided into two groupswith lowexpression and highexpression of nap1l1 groupswere similar in terms of age gender stage and grade table shows the clinicopathological characteristics ofthe groupsaccording to the nuclear expression of nap1l1 similar balanceddistribution was also observed for cytoplasmic expressionusing the kaplanmeier method cumulative survivals for thetwo groups high and low nuclear expression were comparedinitially groups were assessed as a whole regardless of diseasestage figure 5a a clear diï¬erence in cumulative survival wasobserved according to nuclear nap1l1 staining p logrank test in the multivariate analysis including age genderstage and grade cox proportional hazards model the nuclearscore was independently associated with cumulative survival thehigh nuclear expression group exhibited a hazard ratio hr [95ci ] p denoting a reductionin cumulative mortality in this group as a result the estimated5year survival was in the low expression group and in the high expression group median duration of survivalwas months in the low expression group whilst it was notreached for the high expression cohort the only additionalvariable also associated with survival was tumor stage hr [95ci ] p an expected ï¬nding since stage isa known prognostic factor in crc these results strongly suggestan association between nap1l1 nuclear staining and survivalin crc patients conversely cytoplasmic nap1l1 staining wasnot associated with survival figure 5b or with any otherclinicopathological variablewe then analyzed survival according to nap1l1 nuclearexpression in diï¬erent stage groups figures 5cd for earlystage disease no significant diï¬erence in survival was found byfrontiers in oncology wwwfrontiersinaugust volume 0cqueiroz nap1l1 a novel crc biomarkerfigure staining patterns and scoring results for nap1l1 nap1l1 nuclear and cytoplasmic scores were decreased in the cancer groups when compared tonormal adjacent tissues no difference was observed between different stages of cancer p kruskalwallis test followed by post hoc dunnbonferronitest for pairwise comparisons error bars ± se sample numbers normal stages iii stages iiiiv magniï¬cation Ãcontrast a highly significant diï¬erence in survival was observedfor the cohort containing stages iii and iv tumors multivariateanalysis once again demonstrated that nap1l1 nuclear score wasan independent prognostic factor in crc patients the calculatedhr [95ci ] p was even more notablethan that observed for the entire cohort now suggesting a reduction in cumulative mortality the 5year survival advantagefor high expression tumors was also greater versus forlow expression cancers median survival was only months inthe low expression group and again was not reached in the highexpression cohortdiscussionthe discovery of novel crc biomarkers to assist in earlydiagnosis prognostic stratiï¬cation and prediction of responseto treatment remains an unmet medical need we hypothesizedthat the study of animal models of crc based on transgenic apcgene inactivation could lead to the discovery of novel useful crcbiomarkers in humansby combining transcriptomic and proteomic analyses of smallintestinal tissue from transgenic mice in which apc andor mychad been speciï¬cally deleted we identiï¬ed nap1l1 as the onlygeneprotein that showed significantly altered expression in apcand mycdependent manners in all analyses we conï¬rmed theseï¬ndings using qpcr in mouse small intestine and additionallydemonstrated that nap1l1 mrna expression was increasedin human crc it was unfortunately not possible to studywhether there was any altered nap1l1 expression in the colonthe ahcre mouse model as there is no cre mediatedofrecombination in the colon of these mice following injection ofnaphthoï¬avone and they have no colonic phenotype nap1l1 is a highly conserved histone chaperone proteinwhich is one of ï¬ve nap1like proteins in mammals it has been suggested to play a role in mediating nucleosomefrontiers in oncology wwwfrontiersinaugust volume 0cqueiroz nap1l1 a novel crc biomarkertable characteristics of the patients included in the prognostic analysischaracteristicsmean age rangegendermalefemalestageiiiiiiivgradewell differentiatedmoderately differentiatedpoorly differentiatedpatients n table clinicopathological characteristics according to nap1l1nuclear expressioncharacteristicslow nuclearexpressionn high nuclearexpressionn twosidedpvaluesmean agegendermalefemalestageiiiiiiivgradewell differentiatedmod differentiatedpoorly differentiatedno significant difference between groups was observed mean age was comparedby ttest categorical variables were compared by chisquare test or fishers exacttestformation and regulation of the h2ah2b complex as wellas nucleosome assembly cell cycle progression and cellproliferation it has also been linked to embryogenesis andtissue diï¬erentiation few researchers have previouslystudied nap1l1 expression in cancer celllines or tissuesdrozdov compared small intestinal neuroendocrine tumorsnets and normal enterochromaï¬n cell preparations andshowed a 137fold increase in nap1l1 expression in tumortissues however no analysis ofthe adjacent mucosawas performed kidd also suggested that nap1l1 wasincreased in nets but not in crcs line evaluatednap1l1 mrna expression in crc and adjacent tissues asa secondary endpoint in a study primarily aimed at ï¬ndingseroreactive biomarkers they showed that among cases of crc seven exhibited moderate increases in nap1l1expression ranging from to 93fold and eight cases showedexpression levels similar to those in the corresponding adjacentmucosa a recent paper has also demonstrated that nap1l1is a prognostic biomarker and contributes to doxorubicinchemotherapy resistance in hepatocellular carcinoma we acknowledge some limitations in this study althoughpositive and relevant ï¬ndings were observed the use of smallclinical sample sizes may have limited our observations thisfrontiers in oncology wwwfrontiersinaugust volume immunohistochemistry is used in routine clinical practice toassess the expression of proteins with prognostic or predictivevalue in other types of cancer such as breast and lungcarcinomas soft tissue sarcomas and lymphomas given the absence of a standard scoring method for nap1l1 weinitially decided to assess both the nuclear and the cytoplasmicexpression of the protein in our samples using electronic toolsour results showed that nap1l1 expression was decreased bothin the nucleus and the cytoplasm of crc tissues when comparedto the normal adjacent mucosathis was a somewhat unexpected ï¬nding given the increasedexpression of nap1l1 mrna in animal models and in humantissues such discrepancy between mrna and protein expressionhas however previously been demonstrated for other cancermarkers several processes could be responsible such asposttranscriptional modiï¬cations protein degradation secretionvia exocytosis or alterations in subcellular protein localizationfor example a recent paper has reported that nap1l1 undergoesalternative cleavage and polyadenylation in the more advancedstages of crc the full length isoform of nap1l1 wasoverrepresented in the cytoplasmic fraction of a crc cell linewhich had a more metastatic phenotype this may thereforerepresent one mechanism to explain the altered nap1l1subcellular localization thatis reported in crc specimensin our current manuscript counterintuitively our ï¬nding ofincreased gene expression in the initial screen may have been aresponse to reduced protein content and not the primary eventfurther research is required in order to clarify this issue qiao demonstrated that knock down of nap1l1 increasedcellular proliferation disrupted normal cell development anddistribution and caused global deregulation of gene expressionthese are classical hallmarks of cancer and of activated wntsignaling qiao also demonstrated that nap1l1 knoc | Colon_Cancer |
" accurate detection of patients with minimal residual disease mrd after surgery for stage ii coloncancer cc remains an urgent unmet clinical need to improve selection of patients who might benefit formadjuvant chemotherapy act presence of circulating tumor dna ctdna is indicative for mrd and has highpredictive value for recurrent disease the medocccreate trial investigates how many stage ii cc patients withdetectable ctdna after surgery will accept act and whether act reduces the risk of recurrence in these patientsmethodsdesign medocccreate follows the trial within cohorts twics design patients with colorectal cancercrc are included in the prospective dutch colorectal cancer cohort plcrc and give informed consent forcollection of clinical data tissue and blood samples and consent for future randomization medocccreate is asubcohort within plcrc consisting of stage ii cc patients without indication for act according to currentguidelines who are randomized into an experimental and a control armin the experimental arm postsurgery blood samples and tissue are analyzed for tissueinformed detection ofplasma ctdna using the pgdx elio¢ platform patients with detectable ctdna will be offered act consisting of cycles of capecitabine plus oxaliplatin while patients without detectable ctdna and patients in the control groupwill standard followup according to guidelinethe primary endpoint is the proportion of patients receiving act when ctdna is detectable after resection themain secondary outcome is 2year recurrence rate rr but also includes 5year rr disease free survival overallsurvival time to recurrence quality of life and costeffectiveness data will be analyzed by intention to treatcontinued on next page correspondence mkoopman6umcutrechtnlsj schraa and kl van rooijen are shared first authorrja fijneman gr vink and m koopman are shared last author1department of medical oncology university medical center utrechtutrecht university heidelberglaan cx utrecht the netherlandsfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cschraa bmc cancer page of continued from previous pagediscussion the medocccreate trial will provide insight into the willingness of stage ii cc patients to be treatedwith act guided by ctdna biomarker testing and whether act will prevent recurrences in a highrisk populationuse of the twics design provides the opportunity to randomize patients before ctdna measurement avoidingethical dilemmas of ctdna status disclosure in the control grouptrial registration netherlands trial register nl6281ntr6455 registered may wwwtrialregisternltrial6281keywords colon cancer circulating tumor dna ctdna adjuvant chemotherapy twics in patients with stage ii colon cancer cc the recurrence rate rr after surgery is approximately disease management after surgical resection in stageii cc is still under debate because the overall survivalos benefit of adjuvant chemotherapy act in thisgroup of patients varies between and only [ ]moreover offering act in a lowrisk population induces an important amount of overtreatment with unnecessary but sometimes severe toxicity and costsseveral prognostic characteristics of stage ii cc havebeen identified to provide better selection of patientsthat might benefit from act patients with presence ofat least one of the following characteristics are classifiedas being at high risk of disease recurrence poorly differentiated histology pt4 lesions inadequately less than sampled lymph nodes lymphovascular or perineuralinvasion or tumor presentation with perforation or obstruction in contrast patients with a deficient mismatch repair dmmr status in stage ii cc have a low risk ofrecurrence and act is not considered beneficial irrespective of the presence of other risk factors [ ]other known prognostic factors in cc like gene expression profiles or braf v600e and ras mutations have been investigated but do not adequatelyidentify the patients that will benefit from act []despite the definition of high and low risk subgroupsof stage ii cc patients retrospective analyses demonstrated that improved survival after administration ofact was not observed in high risk patients or exclusivelyin patients with a pt4 tumor [] therefore in thenetherlands act is currently only recommended in stageii cc patients with a pt4 tumor without dmmrunfortunately also pt4 is not an absolute predictorfor disease recurrence in stage ii patients in a retrospective analysis of stage ii cc patients with pt4tumors the 3year diseasespecific survival rate aftersurgery was in patients who received act and in patients who did not receive act whichmeans that of these patients are exposed to actunnecessarily considering nonpt4 stage ii patients a population registry analysis of patientsshowed that in this group of patients sufferedfrom recurrences these data demonstrate thatusing pt4 as a prognostic factor results in significantunder and overtreatmentminimal residual disease mrd is defined as the presence of tumor cells in the blood bone marrow or lymphnodes not detected by conventional staging procedures patients who have mrd after surgery are not completely cured and therefore at high risk of developingdisease recurrence development of a highly specific andsensitive biomarker testindicative for mrd wouldallow identification of the subset of patients likely to experience recurrence of disease thereby improving the selection of patients who may benefitfrom adjuvanttreatment in adjuvant trials this would solve problemsof high numbers needed for inclusion and dilution of effectiveness of adjuvant treatment by inclusion of manyalready cured participants cellfree circulating tumor dna ctdna has a strongpotential for being this sensitive and yet specific biomarker ctdna consists of smallfragments usually bp of tumorderived dna containing tumorspecific mutations which can be detected in liquid biopsies such as blood samples [] because of the shorthalflife of ctdna estimates ranging from to min the presence of ctdna in blood samples taken several days after surgery presumably reflects a state ofmrd [] patients with mrd have the highest riskfor disease recurrencerecently the presence of ctdna after tumor resection demonstrated a very strong prognostic value fordisease recurrence in stage ii cc with a 2years rrof versus in patients with and without detectable ctdna after surgery respectively in thisstudy the univariate prognostic value of ctdna was muchhigher than that of pt4 status hazard ratio of versus respectively there are several ongoing trials that usectdna in prognostication nct03637686 nct03737539nct03416478 nct03312374 nct02842203 nct0361 and treatment nct03748680 actrn12615000381 nrggi of nonmetastatic cc but to date thereare no results available of randomized controlled trialsrcts that use ctdna for selection of act treatment 0cschraa bmc cancer page of the accumulating evidence for the strong prognosticvalue of ctdna raises an important ethical dilemma forrandomization of patients when designing a conventional rct in which patients with detectable ctdna arerandomized into act treatment or standard of carefollowup while disclosing ctdna status to the controlgroup indeed the knowledge of having a very highchance of disease recurrence will be a big burden for patients with detectable ctdna in the control group andtheir caregivers as they are not being offered any additional therapy this warrants an innovative trial designlike the trialdifferent from the conventional rctwithin cohorts twics design [] the twics design enables an experimental group in which ctdna status is disclosed and a control group that is unaware oftheir ctdna statusthe medocccreate trial is designed as a multicenter twics study with two parallel groups in whichwe will investigate whether stage ii cc patients with detectable ctdna after resection are willing to receiveact and whether act reduces the rr in these ctdnapositive patientsmethodsdesignaimthis study investigates the willingness of patients to receive act after detection of ctdna postsurgery andthe effect of ctdnaguided act on the rr in stage iicc patientsstudy designthe medocccreate trial follows the twics designand is performed within the prospective dutch colorectal cancer cohort plcrc wwwplcrcnl plcrc is set up by the dutch colorectal cancer groupdccg and collects clinical data and patient reportedoutcome measures proms at baseline and at multipletime points during followup fig at enrollment patients give informed consent for use of their clinical dataand optionally for receiving quality of life questionnairesfig schematic presentation of medocccreate using the trial within cohort twics design a plcrc is a nationwide cohort study in thenetherlands with inclusion of crc patients all stages by optional informed consent regarding collection of biomaterials and futurerandomization observational as well as interventional trials can be performed within the cohort b nonmetastatic crc patients are included inmedocc when the patient signs informed consent for plcrc including additional blood sampling blood samples are withdrawn beforeresection days after resection and every months during the first years of followup c eligible stage ii colon cancer patients arerandomized following the twics design in the experimental group informed consent is being asked for immediate ctdna analysis of theblood sample obtained after resection if ctdna is detectable patients are offered adjuvant chemotherapy the control group is not informedabout medocccreate and will receive standard of care 0cschraa bmc cancer page of collection of biomaterials for research additional sequential blood sampling and for being approached forfuture studies conducted within the infrastructure ofthe cohort either in accordance with the twics design or notpatients with pt4 tumors will be offered act therefore we will include eligible patients with pt4 tumors without a recommendation for act according totheir treating physician and use pt4 status as a stratification factorpatient selection and recruitmentpatients will be recruited in both academic and nonacademic hospitals in the netherlands that are participating in plcrc nonmetastatic colorectal cancercrc patients that give informed consent for plcrcincluding consent for additional blood sampling at enrollment will be included in the observational plcrcsubstudy medocc molecular early detection of coloncancer before surgery the participants are eligible forthe current medocccreate trial if they meet the following criteria after surgery histopathological confirmed and radically resected stage ii cc age ¥ years informed consent for plcrc and medoccincluding consent for randomization in future trials anduse of tissue physical condition allows treatmentwith combination chemotherapy consisting of a fluoropyrimidine and oxaliplatin and no indication foract according to the treating physician andor multidisciplinary board patients who are pregnant have hadanother malignancy in the previous years except forcarcinoma in situ or patients with contraindications forfluoropyrimidines andor oxaliplatin will be excludedcurrently the dutch guidelines recommend act forpatients with pt4 tumors however there is large ageand hospital dependent variation in administration ofact in this group and in clinical practice not all stage iiblood sample collectionblood samples are collected before and days aftersurgery for all patients included in the medocc clinicalstudy predominantly comprising stage i ii and iii ccpatients table blood samples two tubes of mlper timepoint are collected in cell free dna streckblood collection tubes for various research purposesamong which the medocccreate trialrandomizationabout week after surgery when the histopathologicalreport is finished medocc patients who are eligiblefor medocccreate will be randomized to theintervention or control arm using slim an onlineplatform to manage patientinclusion including arandomizationgeneratedcomputerservice therandomization schedule is stratified by tstage and usespermuted blocks of random sizes allocation concealment will be ensured as the service will not release therandomization code only patients randomized to theintervention arm will be informed about medocccreate according to the twics design experimental armafter randomization only patients randomized to theexperimental arm will be asked separate informedtable standard protocol items for intervention trials spirit schedule of enrollment interventions and repeated measurementsact adjuvant chemotherapy ctdna circulating tumor dna qol quality of life intervention group only intervention grouponly if ctdna is positive 0cschraa bmc cancer page of consent for the immediate analysis of ctdna status ofthe postsurgery sample a small proportion of patientsestimated approximately will have detectablectdna in their blood these patients will be offeredact patients decide whether they accept or refuse thistreatment patients without detectable ctdna will receive routine standard of careact will consist of months of capecitabine and oxaliplatin capox or months of fluorouracil leucovorinand oxaliplatin folfox treatment starts preferablywithin weeks and not beyond weeks after surgeryduring and after completing act routine followupwill consist of regular visits at the surgical outpatient department blood withdrawals for analysis of carcinoembryonic antigen cea and imaging standard ultrasoundofthe liver according to current guidelines in thenetherlands no additional imaging will be performed toprevent detection biascontrol armin the control arm patients will not be informed aboutthe medocccreate trial and receive routine followup care consisting of cea tests every months for thefirst years and abdominal ultrasound or ct every months in the first year and once a year afterwards oneyear after surgery a colonoscopy is performed postsurgery blood samples will not be tested for ctdna immediately but will be analyzed batchwise after severalmonths without result disclosure to patients and theirtreating physiciansfollowupblood samples will be collected at 6monthly intervalsfor the first years after surgery for both patients in theexperimental arm and the control arm conform themedocc study protocol these samples will not be analyzed for ctdna immediately and results will not bedisclosed to patients and treating physicianstumor tissueinformed ctdna analysisafter surgery the local pathologist will send a formalinfixed paraffinembedded ffpetissue block to thecentral laboratory where dna will be isolated for further analysisthe postsurgery blood sample is drawn between and days after surgery the sample is not withdrawnbefore day to reduce the risk of falsenegative ctdnatests due to the relatively large amount of cell free dnacfdna released due to cell damage after surgery theblood is taken no later than days after surgery to beable to start chemotherapy within weeks after surgerysamples are kept at room temperature and sent by regular mail to the central laboratory within days wherectdna will be isolated for further analysistumor tissue dna will be analyzed by targeted nextgeneration sequencing of a panel of more than genes using the pgdx elio¢ tissue complete assay frompersonal genome diagnostics pgdx baltimore mdusa plasma ctdna will be analyzed by targeted nextgeneration sequencing of a panel of more than genesusing the pgdx elio¢ plasma resolve assay from pgdxbaltimore md usa both panels include the mostcommonly mutated genes in cc including apc tp53kras and braf tumor tissue dna mutations are usedas input information for plasma ctdna mutation callingthereby increasing both sensitivity and specificity of thectdna testprimary endpointthe primary endpoint is the proportion of patients starting with act after detection of ctdna in the postsurgery samplesecondary endpointsthe most important secondary endpoint is 2year rr inpatients with detectable ctdna in their blood expressedas the proportion of patients that experience a recurrence within years after surgery detection of recurrences in months after surgery will occur by standardfollowup investigations including monthly bloodsampling of tumor marker cea and monthly imagingwith ultrasound liver or ct abdomen and when indicated by symptoms radiological andor histopathological evidence is used to confirm the recurrence thedate of the said investigation is considered the date ofrecurrencedata about followup recurrences and survival areroutinely collected within plcrc using the netherlandscancer registry ncr managed by the netherlandscomprehensive cancer anisation iknl to provideinsight in the characteristics and magnitude of cancer inthe netherlands other secondary endpoints include 2year rr in aperprotocol analysis 5year rr intentiontotreatand perprotocol analysis time to recurrence ttr and 5year disease free survival dfs rate and7year diseaserelated os rate and 5year rr inpatients with undetectable ctdna after surgery quality of life qol and costeffectiveness of the ctdnaguided strategytimetoevent outcomesos rate is expressed as proportion of patients that arealive and years after surgery dfs rate is expressed asproportion of patients that did not experience disease recurrence a second primary cc or death within and years after surgery ttr is expressed as time monthsbetween surgery and detection of disease recurrence 0cschraa bmc cancer page of patients will be censored at the last date of followup if adate of death is not recorded and at the date of death ifthe cause of death is not due to ccquality of lifeqol is measured within the cohort at regular intervalsin patients who gave consent to send questionnaires nationally and internationally validated questionnaires areused among which the european anisation for research and treatment of cancer quality of life questionnaire core and the colorectal cancer moduleeortcqlqc30 and cr29 the work ability indexwai the euro quality of life5 dimensions eq5dthe multidimensional fatigue inventory20 mfi20and the hospital anxiety and depression score hadscosteffectiveness of the ctdnaguided treatmentthe costeffectiveness analysis will be carried out from asocietal perspectiveincluding both direct health carecosts as well as indirect costs from productivity loss thehealth outcome measure in the costeffectiveness analysis will be the total quality adjusted life years qalyper group for analysis offactors related to qalysquestionnaires are used provided within plcrcsample size considerationsthe primary endpoint is the proportion of ctdna positive patients starting with act however 2year rr inthe ctdna positive patients after surgery is an importantsecondary endpoint and the power calculation is performed for this secondary endpoint we estimate thatsimilar to effectiveness in stage iii cc patients act inctdnabased highrisk stage ii cc patients will lead toa absolute reduction of recurrences within yearsafter surgery in the observational trial of patientswith detectable ctdna experienced disease recurrencewithin years after resection with a power of and an alpha of patientswith detectable ctdna need to be included in botharms assuming a prevalence of ctdna after surgery of and adjustment for loss to followup and rejection ofadjuvant therapy in the intervention arm of a totalsample size of patients is calculated in eacharm we expect few patients with detectable ctdna inthe intervention group to refuse act because patientsare selected upfront for being in a physical condition toreceive act and the established prognostic value of detectable ctdna is highwe assume that crossover from the control arm tothe intervention arm will not occur because only eligiblepatients randomly selected in the cohort and allocatedto the intervention arm will be informed about the trialand have the opportunity for immediate analysis ofctdna patients in the control group will not be informed about the trial or their ctdna statuswe assume that of patients in the interventionarm with detectable ctdna will be treated with actthe proportion of patients starting with chemotherapythe primary endpoint can in that instance be determined with a margin of error width of the confidence interval of we expect to complete recruitment of patients within years with more than participating dutchhospitalsdata analysisdata will be analyzed according to the intentiontotreatprinciple for the primary endpoint and the secondaryendpoint of 2year rr in patients with detectable ctdnaafter surgery in this analysis we expect to compare patients with detectable ctdna who received act inthe intervention arm with patients with detectablectdna in the control arm ie based on ctdna analysisperformed retrospectively at least months after surgery and not disclosed to patients and treating physicians the proportion of patients that experience arecurrence in both arms will be compared by means of achisquare test in addition for other secondary endpoints and exploratory analyses we will analyze timetoevent outcomes in patients in both arms with detectablectdna after surgery differences in timetoevent outcomes will be analyzed by standard survival methodseg kaplanmeier curves compared by logrank testscoxs proportional hazards models will be used for multivariable analysiscomparison of qol of the ctdna positive patients inboth study arms will be done using repeated measurements methods and including act as factor qol willalso be analyzed for the whole population in both armsof the study treatment differences at each qol assessment time point will be compared by means of thewilcoxon rank sum testa lifetime horizon will be applied forthe costeffectiveness analysis parametric survival functions willbe used to extrapolate dfs and os curves beyond yearsresponsibilitiesprotocol modifications will be submitted as amendmentto the medical ethical committee by the study coordinator the local principle investigator of each participating hospital is responsible for patient inclusion logisticsof biomaterials to the centrallaboratory and patientfollowup to ensure quality of data study integrity andcompliance with the protocol and the various applicableregulations and guidelines a data monitor of the iknlhas been appointed to conductto thesite visits 0cschraa bmc cancer page of participating centers and randomly check patient datathe study coordinator together with the principle investigator will have access to the final dataset and is responsible for publishing study results the results will besubmitted to a peerreviewed journaldiscussionmedocccreate is the first clinical trial using thetwics design to investigate ctdnaguided strategies instage ii cc taking an important step towards clinicalimplementation of ctdna in cancer diagnostics andcarewithctdnadetectablea few other trials with the aim to reduce recurrencesin cc by use of a ctdnaguided approach are in preparation or recently started the improveit trial adanish study started in october uses a classicalrct in stage i and ii crc patients randomizing between months of act or intensified followup for patientspostsurgerynct03748680 four hundred fifty stage ii crc patients are being included in the australian dynamicstudy and randomized to be treated according to thectdna result with to months of act or accordingto standard of care actrn12615000381583 thecobra study in the united states and canada has asimilar rct approach nrggi also several trialsin stage iii crc patients started recently dynamiciii actrn12617001566325 in the near future thesestudies will provide deeper understanding and lead toimplementation of ctdnaguided strategies in clinicalpracticein the current era of rapidly emerging new diagnosticand treatment strategies the classical rct is challengedbecause of inefficient and therefore timeconsuming recruitment of eligible patients main reasons for patientsto refrain from participation in rcts are preference forone ofthe treatment arms anxiety or aversion torandomization and difficulties understanding the concept of an rct resulting in a delay of availability ofpotential beneficial treatments modern trial designsare being adopted to avoid thistimeconsuming and costly way of conducting trials with highrates of unfinished studies therefore the medocccreate trial uses the modern twics design the twicsdesign has shown to have a positive impact on trialefficiency also by enrolling higher proportions of eligible patients generalizability to daily clinical practiceimproves inefficientthisseveralstudy design hasstrengths firstmedocccreate is nested within the large nationwide plcrc cohort study with currently almost included crc patients the infrastructure of this cohortin which clinical data and biomaterials are collected afterbroad informed consent of participating patients allowsinnovative and efficientcomprehensiveresearch incrc using this infrastructure the study can be quicklyimplemented in many participating hospitals savingcosts and complicated logistics several studies accordingto the twics design are performed within this or comparable cohorts therefore experience with this trialdesign has been gained and this will contribute to execution of the medocccreate study [ ]secondly a difficult ethical dilemma in an rct analyzing ctdna presence postsurgery is avoided by thetwics design with the current knowledge about thestrong association with recurrent disease disclosingctdna status to all participants would be a great burdenfor patients with detectable ctdna and their treatingphysicians in the control group because of disappointment bias in the control group we would expect highdropout and contamination due to crossover when aclassical rct design would be applied making accrualand interpretation of results unfeasible in thistwics study all participants already have blood withdrawn after surgery for research purposes and only theeligible patients allocated to the intervention arm willhave the opportunity to obtain a ctdna test result andact if ctdna is detected patients in the control armtreated according to current guidelines will not be informed about randomization and their blood sampleswill be analyzed at a later point in time beyond the window of act treatmentthis study has also potentiallimitations and challenges the twics design is potentially susceptible tolow statistical power and internal validity biases levelsof participants eligibility and consent should be substantial to achieve valid and reliable results and measurements taken in the control group should be sufficient foradequate comparisons to be made therefore thetwics design is not appropriate for every experimentalintervention in case of the medocccreate studywe argue that eligibility and also consent will be substantial because of the high incidence of cc the large cohortwith high inclusion rates and the assumption that eligible patients in the intervention group are willing toaccept act because of the very strong association of thepresence of ctdna with recurrent diseaseanother limitation is the small sample size for primaryoutcome analysis eventually only patients in botharms of the trial are expected to have detectable ctdnaafter surgery based on previous data relapses areexpected within years and with a high event rate smallnumbers are sufficient capecitabinewe recommend a 6month duration of act consistand oxaliplatin capox oring offluorouracilleucovorin and oxaliplatin folfox forpatients with detectable ctdna after surgery the firstadjuvant cc trials investigating the combination of a 0cschraa bmc cancer page of fluoropyrimidine and oxaliplatin reported results for month duration of act in the idea trialfound a large reduction in toxicity for months treatment compared to months treatment although thistrial could not confirm noninferiority for monthstreatment for all patients treated with capox or folfox in stage iii crc the small difference limits clinicalrelevance besidesit did show noninferiority of theshorter regimen in patients treated with capox consequently dutch guidelines recommend months of actfor cc since however among patients withhighest risk of recurrence t4 n2 or both superiorityof 6month duration of therapy was found additionalideafrance resultsthe esmocongress showed the worst prognosis for ctdnapositive patients who only received months of act therefore in this study we recommend 6monthsact for patients with a very high risk of disease recurrence due to the presence of ctdna after surgerypresentedatliquid biopsy ctdna detection has become a promising technology with multiple putative clinical applications including its potential use as a biomarker for earlydiagnosis prognosis prediction and monitoring of treatment response driven by the excitement of its possibilities the field of technology of ctdna detection andanalysis is rapidly evolving yet the clinical utility ofctdna testing still needs to be proven when to applywhat technology to address which unmet clinical need isa key question that remains to be addressed applying ctdna detection as a biomarker for mrd isa challenging task biologically only a very low amountof ctdna is present in postsurgery patients with mrdstochastically by looking at mutations in a panel ofgenes chances increase that in a given blood sample atleast in one of the genes a mutation can be reliably detected test sensitivity can be further increased by making use of dna mutation information from tumortissue because the stringency in the calling of plasmactdna mutations can be reduced once you know whatmutations to look for tissueinformed ctdna analysisalso increases the ctdna test specificity recent observations showed that ctdna mutation detection can beconfounded by mutations that are present in clonalhematopoiesisincluding mutations in genes that arecommonly affected in cc such as tp53 these confounding mutations can be filtered by applying tissueinformed ctdna analyses as suchtechnically themedocccreate trial makes use of a ctdna test thatis wellsuited for mrd detection clinically howeverthe medocccreate trial needs to resolvewhether a positive ctdna test also allows to select forpatients who truly benefit from act treatment a requirement for clinical implementation to further support clinical implementation of ctdna analyses in thenetherlands the dutch coin initiative aims to providea validation framework for clinicalimplementation ofctdna analyses in the netherlands zonmw projectnumber in conclusion the medocccreate study is thefirst study using the modern and innovative twics design to study ctdnaguided administration of act instage ii cc patients the study aims to answer the important clinical question whether ctdna has prognosticas well as predictive value if this study demonstrates asignificant and substantial difference in disease recurrence in the intervention group compared to the controlgroup ctdna analysis and ctdnaguided treatmentshould be implemented into clinical practice to improvethe prognosis of stage ii cc patientsabbreviationsact adjuvant chemotherapy cc colon cancer cea carcinoembryonicantigen crc colorectal cancer ctdna circulating tumor dnadfs disease free survival dmmr deficient mismatch repair eortcqlqc30 and cr29 european anisation for research and treatment ofcancer quality of life questionnaire c30 and colorectal cancer module eq5d euro quality of life5 dimensions ffpe formalinfixed para | Colon_Cancer |
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 |
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 |
during the last decade green synthesized cerium oxide nanops ceo2 nps attracted remarkable interest in various fields of science and technology this review explores the vast array of biological resources such as plants microbes and other biological products being used in synthesis of ceo2 nps it also discusses their biosynthetic mechanism current understandings and trends in the green synthesis of ceo2 nps novel therapies based on green synthesized ceo2 nps are illustrated in particular their antimicrobial potential along with attempts of their mechanistic elucidation overall the main objective of this review is to provide a rational insight of the major accomplishments of ceo2 nps as novel therapeutics agents for a wide range of microbial pathogens and combating other diseases keywords nanotechnology green synthesis cerium oxide nanops antimicrobial infections biomedicalintroductionnanotechnology has got a remarkable interest in every field of science and technology and is presently considered among one of the leading research avenues it has a multitude of applications in the field of electronics imaging industry and healthcare14 mostly in healthcare it has been exploited in diseases diagnostics treatment delivery and formulations of novel drugs14 it exploits nano size structures with size ranges from nm known as nanop nps these nano scale entities have unique physiochemical properties and have been utilized in various fields of physics biology and chemistry5among other nps cerium oxide ceo2 nps have been mostly exploited due to their unique surface chemistry high stability and biocompatibility67 it is mostly used in the fabrication of sensors cells catalysis therapeutics agents drug delivery careers and antiparasitic ointments figure presently ceo2 nps is mostly synthesized via two methods such as physical and chemical79 however these methods utilize toxic reducing solvents posing several threats to the biodiversity and ecosystem moreover the nps obtained with such approaches are toxic and unstable making them less efficient910 thus recently a safe less toxic method has been used by researchers known as green synthesis this method utilizes various biological resources such as plants microbes or any other biological derivative1115 these biological extracts have a rich source of phytochemicals international of nanomedicine nadeem this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomterms php and incorporate the creative commons attribution non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0cnadeem dovepresslatter is extensively utilized for its biomedical pharmacological and food applications due to their safe and biocompatible nature9 moreover features like high yield everlasting stability and better morphologies can be obtained using a greener approach79green synthesis from plantsgreen syntheses of ceo2 nps have been reported using plant extracts microbial and other biological derivatives plants in this regard have been the most efficient source due to their abundance safe nature and rich source of reducing and stabilizing agents2629 various parts of plants such as leaves flower and stem have been used for the synthesis of ceo2 nps163031 till date the majority of green synthesis studies have been conducted on leaves extracts as it is a rich source of metabolites11163233 a broad variety of metabolitesphytochemicals in plant extracts such as ketones carboxylic acids phenols and ascorbic acid are used as reduction and stabilizing agents figure plants based ceo2 nps are produced through a simple approach in which bulk metal salt is mixed with the extract and the reaction completes in minutes to a few hours in ordinary lab conditions282934 the metallic salt solution is reduced into respective nanops via the phytochemicals whose synthesis is confirmed firstly through color change from colorless to yellowish brownish or whitish and then characterized through various spectroscopic and imaging techniques162935leaf extract of moringa oleifera l was used to synthesize ceo2 nps with spherical morphologies and size of nm the synthesized nps showed potential antimicrobial and wound healing properties36 gloriosa superba leaf extract was used as a reducing and stabilizing agent in synthesis of ceo2 nps and has shown potential antibacterial properties37 hibiscus sabdariffa natural extract yielded crystalline ceo2 with a diameter of nm30 spherical shaped nanops of size nm synthesized from gel extract of medicinally important plant aloe barbadensis38 the resultant ceo2 nps showed high antioxidant potential green synthesis of ceo2 nanops was demonstrated using jatropha curcus leaf extract and a monodispersed shape of nm10 spherical shaped cerium oxide nanops are synthesized using leaf extract of oleo europaea with a size of nm having high antimicrobial activity against both gramnegative and positive strains of bacteria16 origanum majorana extracts were used to synthesize ceo2 nps having pseudo photocatalytic activity having high figure general applications of ceo2 nanopssuch asketones amines enzymes and phenols which are believed to be responsible for the reduction and stabilization of bulk salts into respective nanops nps1619to date various applications of green synthesized ceo2 nps have been reported such as antimicrobial anti cancer antilarvicidal photocatalysis and antioxidant therapies162022 among other biomedical applications the antimicrobial potential is certainly the most exploited previously it has been reported that ceo2 nps to display their antimicrobial actions through various mechanisms9 but mostly ceo2 nps kill microbes via triggering the production of an excess of reactive oxygen species in cells916 however further studies need to be conducted to fully elucidate the complete mechanism of action here in this review we aim to focus on the following topics arrays of biological resources have been exploited to date for the synthesis of ceo2 nps moreover the synthesis mechanisms along their biomedical applications are discussed with special emphasis on the antimicrobial activitysynthesis of ceo2 npsnanops are synthesized through various physicochemical methods5 however both methods require toxic solvents high temperature and pressure which pose threats to the environment2325 moreover higher cost laborious downstream processing lesser biocompatibility instability and low yield make them further inefficient710 there is a growing need to fabricate nanostructures which have the potential to solve these problems59 presently researchers have exploited the green method to overcome all these challenges5 for instance plants microbes and other biological products have been used as reducing and or stabilizing agents in the fabrication of ecofriendly nps25 ceo2 nps have also been synthesized using various physical chemical and biological methods9 the submit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem figure biosynthesis reduction stabilization and characterization of ceo2 nanopsspherical shape nm ftir confirmed that the reduction is attributed to the presence of different phenolic and flavonoids compounds in the extract18 ceo2 was synthesized using rubia cordifolia leaf fusions spectroscopic and microscopic analysis revealed hexagonal shaped nps having a size of nm the biogenic ceo2 nps also showed excellent anticancer potential33 nano rod size ranges from nm ceo2 nps resulted when pedalium murex l was added to the aqueous solution of salt at room temperature having high antibacterial activity32 china rose petal was used as a robust bio template for the facile fabrication of novel ceria nano sheet with a size of about nm39 the deviation in size and morphology noticed among the reported studies might be due to the different influence of reaction temperature ph time concentration of salt precursor or plants extracts and part of the plant being used13272840 moreover plants based ceo2 nps showed excellent stability at diverse conditions for instance green mediated ceria nps remain stable at liquid solution changes were observed1336 similarly biogenic ceo2 nps also showed high thermal stability at high temperature and remained stable for a longer period of time which indicates their long durability and everlasting stability27283341 until now various plants have been used in the biogenic synthesis of ceo2 nps and are shown in table physiochemical and no green synthesis from microbesmicrobes also have an intrinsic potential to synthesize nanops as they are a rich source of secondary metabolites23 among other nanops ceo2 nps with various shapes and sizes have been synthesized in recent years from microbes table green synthesis of ceo2 from microbial species is a simple reliable costeffective and ecofriendly approach42 microbial metabolites such as enzymes proteins and heterocyclic derivatives play a crucial role in reducing and stabilizing of ceo2 bulk salt into respective nps4243 moreover microbiogenic ceo2 nps exhibited improved stability water dispensability and showed high fluorescent properties and were less agglomerated43aspergillus niger extract yielded cubic fluorite nps with spherical morphology and an average size of nm ftir analysis revealed the presence of an hydroxyl group carboxylic group and phenol group which are supposed to be involved in the reduction of nps21 curvularia lunata extract has also been used to synthesize spherical shaped ceo2 nps with size ranges from nm color change from white to yellow brown indicated initial reaction the nps were tested against microbial pathogens and showed excellent antibacterial potential spherical shaped ceo2 nps of size ranges from nm were made using fusarium solani extract which showed effective growth inhibition and biofilm formation of pathogenic bacterial strains42 shadab ali khan observed the biosynthesis of spherical shaped nm ceo2 nps by using a thermophilic fungus humicola capping agent43 the resultant nps were characterized by uv xps pl spectroscopy tem ftir and xrd moreover these nps showed excellent potential in treatment of neurodegenerative disorders such as alzheimers and parkinsons diseases bacterial extract has also been exploited in the fabrication of ceo2 nps for instance bacillus shaped nps with an average size of nm the bacterial mediated nps also showed excellent antioxidant potential in vitro44 despite all these applications the microbial route of synthesis has certain shortcomings such as the high probability of pathogenicity laborious culturing and contamination issues however it offers a lot of promise in the field of nanotechnology and could become a leading avenue in subtilis extract yielded spherical international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepresstable ceo2 nanops made from various plants speciesplant namepartcharacterizationshapesize nmftir groupoh ceoh hoh ocoleafxrd xps tem ftir uv vissphericalflower hrtem sem xrd xps eds ftirceohleafleafleafsem xrd ftir tgasphericalxrd sem tem uv vis ftir tgasphericaluvvis psa ftir xrd xps hrtemrtesphericalco oh hhoh ch nooh ceoseedxrd uvvis ftir fesem tgasphericalceo oceo ch co []acalypha indicaleafxrd sem tem eds ftiraloe barbadensisleafxrd tem ftir psasphericalspherical oh ceoh ceocech co ch cf ch cclaloe veraleafftir xps hrtemsphericalch ccrubia cordifolialeafuv vis xrd xps sem ftir edaxhexagonalprosopis farctaaerialuvvis pxrd tem fesem ftirsphericalchina rosepetalfesem fetem afm xrd xpsnanosheetcentella asiaticauvvis dls sem hrtem edssphericaloh ceooh ceo and no__ walnutshellleafxrd sem tem eds ftiruvvis xrd xps fesem tem hrtem eds uvdrs ftirsphericalsphericalceo and ohceoc oh chstempxrd sem tem ftir plflakyoh coo and chs nogloriosa superbahibiscus sabdariffaoliveoleo europaeaprosopis jujliflorasalvia macrosiphon boissazadirachta indicaeuphorbia tirucallipetroselinum crispummoringa oleiferaref[][][][][][][][][][][][][][][][][][][][]lemon grassgrassxrd pl tem saedleucas asperaleafpxrd sem uvvis tem saedmicrosphereuvvis ir xrd sem temsphericalpeeluv vis ftir xrd hrtemspherical watermelonfruit juicecarrageenanpxrd ftir uvvis semirregularpxrd ftir fesem uvvis and tga dtaspherical___ceo ceoceceo co co ch ohso3 ceo hoh co []continuedsubmit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem table continued plant namepartcharacterizationshapeleafxrd fesem tem ftirsphericalxrd fesem eds vsmsphericalsize nmftir group__ceratonia siliquastevia rebaudianasalvadora persicamorus nigraannona muricatajusticia adhatodas nopxrd ftir uvvis tem fesem edsspherical0h ch cc ceo co cfruitfruittem xrd uvvisxrd ftir uvdrs fesemirregular_ ___leafxrd sem tem ftir uvdrssticklikeoh ch co no cc co cncjatropha curcusleafxrd tem uvvismonodispersed _origanum majoranapedalium murexelaeagnus angustifoliaeuphorbia amygdaloidesleaftem fesem xrd ftirsphericaloh ch coleafxrd ftir uvvis drs semnanorodoh ch co co cn cn ceoleafxrd sem tem ftirsphericalch cotem sem xrd uvvissphericalorangepeelxrd tem ftir uvviscubic structurecoh or corpiper betleleafxrd ftir sem eds xps temnh no cnref[][][][][][][][][][][][][]nanomedicine but is yet to be explored particularly these microbial based nps can be used in designing novel fertilizers fabricating sterile surfaces polymers and medical accessories moreover these biogenic nps can also be exploited in disease management drug synthesis and deliverytable ceo2 nanops synthesized from various fungus speciess nomicrobe namecurvularia lunatacharacterizationshapesize nmftir grouptgdta xrd raman pl ftir uvvis and temspherical_humicola spuvvis xps pls tem ftir xrdsphericalceo ceocefusarium solaniftir pl tgdta fesem xrd edax tem xps saed clsmsphericaloh cn cocaspergillus nigeruvvis ftir xps xrd tgdta pl temsphericalhoh oco ce oref[][][][]international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepressgreen synthesis from biological productsapart from the synthesis of nanomaterial from eukaryotes and prokaryotes anisms nps have also synthesized from biological derivatives23 they also play a crucial role in the reduction and stabilization of nps2325 in contrast to plants and the microbial approach bioproduct based ceo2 are much safer scalable and have shown excellent biocompatibility4547 for instance egg white protein was used in order to synthesize ceo2 nps having size ranges from nm with spherical morphologies48 these nps were characterized and confirmed by uvs ftir tgadta and pxrd ftir analysis revealed that the phenol ether hydroxyl and amide groups were responsible for the reduction of these nps it also showed a good in vitro cytotoxicity effect towards human periodontal fibroblasts cells agarose is a natural matrix and has been used as a stabilizing and capping agent for ceo2 nps nps obtained have spherical morphologies with a diameter of nm nps were characterized using various methods including uv fesem ftir tgadta pxrd and tgadta techniques as revealed by ftir analysis it was found that the hydroxyl ether phenol and amide groups were involved in biosynthesis45 starch has also been exploited as a novel source for the synthesis of nanoceria with the results revealing spherical shape nps with a diameter of nm12spherical shaped ceo2 nps with a size of nm were synthesized from dextran the resultant nps exhibited strong anticancer potential46 gum tragacanth reported by darroudi 49 was used in the biosynthesis of ceo2 nps these nps were monodispersed shape with an average size range from nm the ceo2nps exhibited very low cytotoxic effects on neuro2a cell lines making them suitable candidates for various biomedical and pharmacological applications49 some other biological products which have been used for synthesis of ceo2 nps are listed in table despite their biological applications these biogenic nps could be used as a promising candidate in diseases treatment drug delivery and packing food some other products have also been explored for the synthesis of ceo2 nps which are shown in table biological activity of greenmediated cerium oxide nanopsantimicrobial activity of greenmediated cerium oxide nanopsin the last few years nanotechnologybased therapies have been exploited in disease diagnostics treatment and table biomediated ceo2 nanops from different sources of biological productss nonamecharacterizationshapeegg proteinuvvis fesem ftir tgadta pxrdsphericalsize nmhoneyuvvis fesem ftir tgadta eds pxrdsphericalagaroseuvvis fesem ftir tgadta pxrdsphericalstarchdextranpolyethylene glycoluvvis pxrd temtem dls xps uvvishrtem tem uvvis slsdlschitosanxrd hrtem ftir tgadta uvvispectindls fesem edss xrd ftir nmr pl fesem edss uvvissphericalsphericalsphericalsphericalspherical ¤ ftir groupoh ceo ceoce ch nooh ceo ceoce ceoc nooh ceo ceoce no____oh ch co och3 coc no ceotannic acidftir xps xrd hrtem uvvisiblepolycrystalline _ref[][][][][][][][][]submit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem figure schematic representation of antibacterial activity of ceo2 nanops cell wall disruption cell membrane disintegration free radicals productions loss of protein peptides dna fragmentation vital enzymes inhibition loss of cellular fluids and disruption in electron transportformulations of novel drugs1 for instance the antimicrobial potential of nps has been mostly exploited and has showed substantial outcomes2324 presently ceo2 nps have attracted great interest as an antimicrobial agent in particular against bacterial pathogens151650 the exact mechanism of killing microbes is yet not clearly elucidated however it is proposed that ceo2 nps mostly kill microbes via a massive production of reactive oxygen species ros in cells as shown in figure the bactericidal potential of ceo2 nps is attributed to strong electrostatic properties distinctive morphologies small size and low band energy1652 due to strong electrostatic potential ceo2 nps interact with membrane proteins thiols groups which results in protein denaturation membrane impermeability eventually leads to microbial death4253 figure exposure to ceo2 nps kills microbes via membrane collapse by attachment with mesosomes malfunctioning of cellular compartments and bio anic molecules which ultimately lead to abnormal metabolism and physiology1642 similarly green mediated nps killpathogens in a similar fashion and various biological species have been exploited and tested against a wide variety of microbes1316 table however biogenic ceo2 nps unique morphologies small size and biocompatible nature were found to be more effcient and have the potential to range of pathogenic bacterial species1113151642 moreover it also has the potential to kill both grampositive and gramnegative bacteria but due to structural complexity of gramnegative bacterias membranes it is more sensitive against grampositive species1321374754 in antimicrobial in electrostatics between nps and the bacterial wall plant species and wall compositiondifference to differences treat a wide activity the is due international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepresstable various microbes tested against biogenic ceo2 nanopss nosourcemicrobes testedolea europaeafungus aspergillus flavus fusarium solani and aspergillus niger bacterial sps staphylococcus aureus escherichia coli pseudomonas aeruginosa and klebsiella pneumoniamoringa oleiferas aureus and e colicurvularia lunatastaphylococcus aureus streptococcus pneumoniae and bacillus subtilis pseudomonas aeruginosa proteus vulgaris and klebsiella pneumoniaeleucas asperaklebsiella aerogenes escherichia coli pseudomonas desmolyticum and staphylococcus aureusacalypha indicaescherichia coli and staphylococcus aureusannona muricataenterococcusfaecalis staphylococcus aureus klebsiella pneumonia and escherichia coligloriosa superbastaphylococcus aureus and streptococcus pneumonia ecoli proteus vulgaris klebsiella pneumonia shigella dysenteriae and pseudomonas aeruginosaaspergillus nigerstreptococcus pneumoniae bacillus subtilis proteus vulgaris and escherichia colifusarium solanistaphylococcus aureus pseudomonas aeruginosa escherichia coli and klebsiella pneumoniajusticia adhatodastaphylococcus aureus and escherichia colieuphorbia amygdaloidesp acidilacticipectine coli and b subtilisref[][][][][][][][][][][][]resistance to confer due to rapid evolution of the bacterial genome bacteria have evolved to antimicrobial agents5556 thus in quest of a new treatment biogenic ceo2 nps has shown promising results in treating multi drug resistance bacteria and could be a promising candidate against such refractory pathogenesis26 ceo2 nps along other conjugates have been amalgamated with various anic and inanic hybrids to enhance the antimicrobial response1746 similarly biomediated ceo2 nps kills fungi by producing a mass number of free radicals and ros which causes distorted structure and physiology and leads to fungus death16 however a few studies have only been conducted on fungi despite the increasing knowledge on the antimicrobial activity of ceo2 nps much remains unknown about their exact mechanism of encountering bacteria toxicity in vivo studies and environmental concerns which needs to be addressed moreover the low band energy potential of ceo2 nps could be used in fabricating sterile surfaces at hospital or lab settings and will diminish nosocomial and other acquired infectionsother potential biomedical applicationsbeside antimicrobial therapies ceo2 nps have also been used in management of other ailments3557 for instance siliqua showed high biogenic ceo2 nps have been mostly used in treatment of various cancers such as osteosarcoma colon cervical and breast cancers1820274652 results indicated that these nps exhibited strong anticancer potential and could be used as a chemotherapeutic agent thanks to their minimal toxicity capacity to induce apoptosis andor necrosis in cancer cells46 ceo2 nps synthesized from origanum majorana and ceratonia antioxidant activity183157 results showed higher expression of antioxidant enzymes which in turn eradicated free radicals and improved cellular functions31 furthermore antioxidant potential was higher when compared to commercial synthetic antioxidants18 ceo2 nps synthesized from fruit extract of morus nigra exhibited excellent antidiabetic activity on l6 cell lines the treatment was dosage dependent and nps with lesser size resulted in higher uptake of glucose in vitro35 though biogenic ceo2 nps have shown excellent pharmacological potential however the mechanism of action minimum inhibitory concentration and best possible delivery system should need to be determined moreover cytotoxicity and genotoxicity should be tested in vivo models to evaluate the compatibility in both in vivo and in vitro modelssubmit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem to their unique conclusions and future prospectsin this paper we have reviewed the recent trends and understandings of biogenic ceo2 nps and their pharmacological applications various sources such as plants microbes and other biological products have been discussed with the mechanism of synthesis and their biomedical applications due surface morphologies crystal small nature and biocompatible nature biogenic ceo2 nps have got phenomenal interest in biomedical and other fields for instance it has been used in treating various cancers antimicrobial and antioxidant therapies in particular the green synthesized nanops have shown significant antimicrobial potential against a wide range of bacterial species the mechanism of combating such pathogens have also been elucidated and supposed to be due to the mass production of reactive oxygen species and deactivation of scavenging enzymes the ros impedes the membranes disrupts the cellular compartments and disintegrates the bio anic molecules and hampers the associated functions and ultimately causes death it has also shown promising results against multidrug bacteria and could be a potential antimicrobial agent in future against such refractory pathogens however further studies should conduct in vivo models to reveal the full mechanism alongside any sideeffects moreover it has also shown excellent anticancer and antioxidant potential in vitro setups but their toxicity and dosage are yet unknown which needs to be addressed despite their role in various therapies their mechanism of synthesis needs to be optimized whereas in vivo evaluation as well as toxicity should be further screenedabbreviationsceo2 nps cerium oxide nanops dga differential thermal analysis dsl dynamic light scattering eds energydispersive xray spectroscopy fesem field emission scanning electron microscopy ftir fourier transform infrared spectroscopy hrtem highresolution transmission electron microscopy pl photoluminescence pxrd powder xray diffraction ros reactive oxygen species sls static light scattering tem transmission electron microscopy tga thermal gravimetric analysis uvvis uvvisible spectroscopy xps xray photoelectron spectrometry xrd xray diffractiondisclosurethe authors report no conflicts of interest for this workreferences kubik t boguniakubik k sugisaka m nanotechnology on duty in medical applications curr pharm biotechnol doi1021741389201053167248 bhushan b springer handbook of nanotechnology springer jianrong c yuqing m nongyue h et al nanotechnology and biosensors biotechnol adv doi101016j biotechadv200403004 smith dm simon jk baker jr jr jr applications of nanotechnology for immunology nat rev immunol doi101038nri3488 mohanraj v chen y nanopsa review trop j pharm res das s dowding jm klump ke cerium oxide nanops applications and prospects in nanomedicine nanomedicine doi102217nnm13133 he l su y lanhong j et al recent advances of cerium oxide nanops in synthesis luminescence and biomedical studies a review j rare earths doi101016s1002 walkey c das s seal s catalytic properties and biomedical applications of cerium oxide nanops environ sci doi101039c4en00138a rajeshkumar s naik p synthesis and biomedical applications of cerium oxide nanopsa review biotechnol rep doi101016jbtre201711008 magudieshwaran r ishii j raja kcn et al green and chemical synthesized ceo2 nanops for photocatalytic indoor air pollutant degradation mater lett doi101016jmatlet arunachalam t karpagasundaram m rajarathinam n ultrasound assisted green synthesis of cerium oxide nanops using prosopis juliflora leaf e | Colon_Cancer |
chronic rhinosinusitis crs characterized by dysfunctionalmucociliary clearancemcc with subsequent microbialcolonization is known as a multifactorial disease process wherebacterial infection may play a role in the commencement orprogression of the ammatory response ramakrishnan crs patients have complex sinus microbial communitiesthat incite persistent ammation and airway damage lee andlane despite the high density of bacteria that colonizethe airway nutrient sources that sustain bacterial growth in vivoand the derivation of those nutrients are not wellcharacterizedrecently our laboratory successfully created a rabbit model ofcrs by blocking the sinus drainage pathway cho 2017akey data generated from this model indicate a clear sequenceof events that augment our understanding of recalcitrant crspathophysiology blockage of sinus ostia generates a shiftin microbiota to a predominance of anaerobes and theshift from acute to chronic sinus ammation is subsequentlyassociated with a robust increase in pathogenic bacteria egpseudomonas how the two events are mechanistically related isunknown but critical to understanding disease pathogenesis andthe potential for interventionabundant nutrient sources are provided by airway mucus toliving anisms in the microenvironment and mucins are themajor macromolecular constituents of mucus that provide a largecarbon reservoir [eg short chain fatty acids scfa] flynn mucins are the main nutrient source for nichespeciï¬c microbiota of the gut and oral cavity flynn therefore mucin degrading microbes mdm primary mucindegrader are thought to modify the nutritional landscape ofthe microenvironment and stimulate the growth of secondarycolonizers kolenbrander it is wellknown that similarinteractions exist between the commensal gut microbiota and themucus layer of the human intestine cameron and sperandio although the common sinus pathogen pseudomonasaeruginosa cannot derive scfas endogenously flynn little is known regarding the degradation of airway mucinsas a source of scfas by these or other opportunistic pathogens inthe upper airwaydata from observational studies tunney andthe rabbit model cho 2017a indicate a shift in themicrobiota to predominately anaerobic bacteria with impairedmcc in rabbits production of bioavailable nutrients scfafor pathogenic bacteria follows and there was a subsequent shiftfrom acute to chronic ammation with robust generation ofpathogenic microbes eg p aeruginosa since p aeruginosacannot derive scfas eg acetate and propionate from the hostmucus through fermentation on their own we hypothesized thatanaerobic bacteria may ferment mucins into scfa forms usableby p aeruginosa this would provide a novel mechanistic basisfor recalcitrant crs pathogenesis following mcc disruptionthat occurs with acute respiratory infectionsammation andsubsequent compromise of the sinus ostia by edema crabbe thereforethis study is toevaluate the concentrations of scfa within the sinonasal mucusfrom rabbit and human and its contribution to the growthof p aeruginosathe objective ofmethodspao1 stock preparationpseudomonas aeruginosa pao1 strain was expanded fromglycerol frozen stock by inoculating ml of lysogeny brothlb followed by overnight growth at ¦c on a shaker at rpm cultures were streaked on lb agar plates accordingto the quadrant method and grown in a static incubatorat ¦c overnight at least twice to conï¬rm conformity ofcultures from the plate an isolated colony was grown in ml of lbmiller broth at ¦c on a shaker at rpmovernight cultures were diluted with fresh lbmiller broth toan inoculation concentration of to make a pao1 stockfor further experimentsanimal modelthis study was approved by the institutional animal careand use committee iacuc approval number at theuniversity of alabama at birmingham uab pasteurellafreefemale new zealand white rabbits kg were used forthe study before initiation rabbits were acclimatized to theanimal facility for at least week for any procedure rabbitswere anesthetized with [ketamine mgkg mwi boiseid dextomitor mgkg zoetis inc kalamazoo mibuprenorphine mgkg reckitt benckiser pharmaceuticalsinc richmond va and carprofen mgkg zoetis incfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crskalamazoo mi] in a warm room for comfort rabbits did notreceive any antibiotics before or during this studymucus collection from rabbit model ofsinusitisbased on our previous experiments mdms dominated on week after blocking the sinus drainage pathway in the rabbit andtherefore mucus samples were collected at week cho 2017a a total of four rabbits were used to create a rabbitmodel of acute sinusitis without providing exogenous bacteriaor pathogens as described previously cho 2017a asterile synthetic sponge merocel rcid13 medtronic minneapolismn was inserted in the left unilateral middle meatus naturaloutï¬ow tract of maxillary sinus of new zealand white rabbitsfor weeks and the sponge was removed on week toconï¬rm acute sinusitis rabbits were examined with microcomputed tomography microct scanning using spectctxspect system gamma medica northridge ca and nasalendoscopy [ mm 30degree endoscope karl storz tuttlingengermany] on week and mucus samples were collectedon week control and week sinusitis using a specialsuction catheter created by our laboratory to evaluate whethertargeting fermentative anaerobes halts the sinusitis progressionfrom acute to chronic metronidazole mgkg twice a dayfor days was administered to the acute sinusitis rabbits week microct scans were repeated at week between acute andchronic to assess for ct evidence of sinusitis opaciï¬cationsinus opaciï¬cation grading was performed using kerschnersrabbit sinus ct grading system [scoring each imaging studybased on estimated percent opaciï¬cation of the maxillarysinus for for for for for for for for for and for ] kerschner opaciï¬cation was measured using the imagej version 150i national institutesof health bethesda md by two blinded judgesin vitro coculturingto test whether mdms at week are able to generate metabolitesfrom mucin that could simultaneously stimulate p aeruginosagrowth mucus samples collected at week were cocultured withpao1 in a polystyrene culture tube fisher scientiï¬c companypittsburg pa a bottom agar plug was made by adding µl of agar inoculated with µl of mucus collected from rabbitsday or week or agar negative control n afterallowing this to solidify a top plug was made with µl of minimal media agar inoculated with the dilution of anovernight culture of p aeruginosa pao1 after solidiï¬cationcocultures were placed at ¦c for h agar plugs were thenremoved from the upper phase and homogenized by pipettingin ml of phosphate buï¬ered saline colony forming unitscfu per tube were determined by serial dilution and platingon lb agarmucus collection and culture from humanchronic sinusitisto understand the concentration of scfas in human crs withp aeruginosa mucus samples were collected from the middlemeatus this study was approved by the institutional reviewboard irb approval number at the universityof alabama at birmingham and all patients provided writteninformed consent subjects ¥ years of age visiting theuniversity of alabama at birmingham sinus clinic were recruitedfor the study patient eligibility criteria were designed to limitenrollment to healthy individuals and patients who clearly havecrs based on sinus and allergy health partnership criteriabenninger orlandi control patientswere enrolled based on endoscopic procedures for unilateralbenign tumors where the opposite side could be tested andother disease entities where sinus ammation was not presenteg csf leaks nasal septal deviation benign nasal tumor andturbinate hypertrophy demographic and clinical data wereprospectively collected deidentiï¬ed and stored in a securelyencrypted electronic database for cultures specimens wereobtained in the clinic or operating room or with eswabscopan diagnostics inc murrieta ca for hospital laboratoryculture culture swabs were endoscopically guided to the areaof interest with care taken to avoid contamination from thenares the mucosal surface and overlying mucus of the middlemeatus from frontal ethmoid andor maxillary sinuses wasaggressively swabbed for at least ï¬ve full rotations until fullysaturated culture swabs were sent to the hospital clinicalmicrobiology laboratory for aerobic and anaerobic culture forbacterial growth and isolation for metabolite quantiï¬cationmucus samples from the area of interest were collected in theclinic or or using a modiï¬ed catheter suction created by ourlaboratory cho 2017ab durmowicz metabolite quantiï¬cationtargeted quantiï¬cation of scfas was performed via highperformance liquid chromatography hplc the systemconsisted of a shimadzu scl10a system controller lc10at liquid chromatograph sil10af autoinjector spd10auvvis detector and cto10a column oven separationof compounds was performed with an aminex hpx87hguard column and an hpx87h cationexchange column biorad hercules ca the mobile phase consisted of nh2so4 set at a ï¬ow rate of ml min the column wasmaintained at ¦c and the injection volume was µl aminoacid and metabolite acetate and propionate quantiï¬cationfrom enrichmentsupernatants were performed by millisscientiï¬c inc baltimore md using liquid chromatographymass spectrometry and gas chromatographymass spectrometrygcms samples for amino acid quantiï¬cation were spikedwith µl of uniformly labeled amino acids cambridge isotopelabs and derivatized using accqtag reagent waters corp for min at ¦c a waters micromass quatro lcms interfacedwith a waters atlantis dc18 µm mm columnwas used reversephase lc was used for separation mobilephases a mm ammonium formate in formic acid bmethanol with a constant ï¬ow rate ml min and acolumn temperature of ¦c electrospray ionization was usedto generate ions in the positive mode and multiple reactionmonitoring was used to quantify amino acids samples ¼µl for acetate and propionate quantiï¬cation were ï¬rst dilutedfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crs µl water spiked with internal standards µl of ppm acetate [13c2] and ppm propionate [13c1] andacidiï¬ed using µl of 12n hcl after equilibration at ¦c for h carboxenpolydimethylsiloxane solid phase microextractionspme ï¬ber was used to adsorb the headspace at ¦c for min acids were then desorbed into the gas chromatographinlet for min a m mm id db624 column attached toa thermo electron trace gas chromatograph with helium carriergas ml min was used for separation of analytes a watersmicromass quatro gc mass spectrometer was used for detectionand quantiï¬cation of target ionseffect of scfa on pao1 growthto test the eï¬ects of scfa on p aeruginosa growth we incubatedpao1 strains with scfa at varying concentrations each scfamedium was prepared by adding individual scfa to m9 minimalsalts media at diï¬erent concentrations obtained from sigma stlouis mo pao1 seeding solution was prepared by adding µl pao1 stock to ml lbmiller broth µl of pao1 seedingsolution was inoculated into µl scfas media solutionson a 48well plate and incubated at ¦c for h the ï¬nalconcentration of each scfa was or mm based onin vivo hplc data optical density od of planktonic pao1growth was measured at nm using a microplate readeradditionally to assess the growth of pao1 strains in the presenceof all scfas the colony counts of pao1 were comparedbetween the single the most dominant scfa vs scfas afterincubating h in m9 minimal salts media repeated statistical analysisstatistical analyses were conducted using excel andgraphpad prism software la jolla ca with signiï¬cance setat p statistical evaluation utilized unpaired student ttestsor analysis of variance anova based on the characteristics ofanalysis data is expressed ± standard error of the meanresultsmucus samples from acute sinusitisrabbits support the growth of pao1in vitroto test whether the mucus samples from acute sinusitis inthe rabbit model enriched with mdms and scfas couldsimultaneously stimulate pao1 growth the mucus samples werecocultured with pao1 in a minimal mucin medium once thelower agar phase containing the mucus had solidiï¬ed pao1 wassuspended in buï¬ered agar medium without mucin ie nocarbon source and was placed in the upper portion of the tubethis experimental setup establishes an oxygen gradient allowinganaerobes to grow and restricts the movement of microbes butallows metabolites to freely diï¬use flynn underthese conditions p aeruginosa would be expected to achieve ahigher cell density if provided with diï¬usible growth substratesfrom the mucus lower phase coculture growth was monitoredover a 72h period after h a diï¬usible bluegreen pigmentpyocyanin characteristic of p aeruginosa growth was observedthroughout the coculture tubes contained with sinusitis mucusweek figure 1a by contrast no observable pigment wasproduced in the tubes contained with control mucus day or without any mucus colony counts of the upper phase weresigniï¬cantly higher in those tubes containing mucus samplesfrom week sinusitis cfutube ± n compared to tubes containing control mucus ± n or no mucus ± n with a magnitude 5fold increase p figure 1bmucus samples from acute sinusitisrabbits contain short chain fatty acidsscfato provide evidence of fermentative activity in vivo scfasacetate propionate and butyrate were quantiï¬ed using gcms within the mucus samples from rabbits on day controland week sinusitis scfas were found at millimolar or lessconcentrations in all mucus samples and we were able to detect allthree scfas figure acetate concentrations were signiï¬cantlyhigher in the mucus samples collected on week sinusitisrelative to day control ± vs ± mm p n propionate and butyrate concentrations werenot signiï¬cantly elevated in the mucus samples from sinusitiscompared to those from control [propionate ± vs ± mm p n butyrate ± vs ± mm p n ] even though there wasa trendhuman mucus samples from crs withp aeruginosa contain signiï¬cantly higherscfasto provide the evidence of fermentative activity in human crswith p aeruginosa we analyzed the presence of scfas in humanmucus samples from controls and crs with p aeruginosa ofthose human mucus samples collected in the clinic or or eightsamples were collected from controls and six from crs withp aeruginosa all crs patients with p aeruginosa presentedwith purulence in the sinus cavity without any recent use oforal or intravenous antibiotics for at least weeks of those crs with p aeruginosa mucus samples mucusdegradingmicrobes were present in ï¬ve samples from our hospitalclinical microbiology laboratory report table using gcms scfas acetate propionate and butyrate were quantiï¬edin human mucus samples collected from eight controls and crs patients with p aeruginosa with active purulent drainagescfas were found at mm concentrations in all mucus samplesfigure scfas from crs patients with p aeruginosa were alsosigniï¬cantly higher than those from controls acetate ± vs ± mm p propionate ± vs ± p butyrate ± vs ± p figure collectively datapresented here demonstrate the presence of signiï¬cantly higherquantities of fermentation metabolites in crs with p aeruginosascfas increase pao1 growth in vitroto understand the role of scfas in the growth of pao1 invitro the growth of pao1 with multiple concentrations offrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure mucus samples from rabbit sinusitis support the growth of pao1 in vitro a the mucus samples were cocultured with pao1 in an anaerobic minimalmucin medium after h a diffusible bluegreen pigment pyocyanin characteristic of p aeruginosa growth was observed throughout the coculture tubescontaining sinusitis mucus week b colony counts were signiï¬cantly higher in those tubes contained with the mucus samples from week sinusitis cfutube ± compared to tubes containing control mucus ± or no mucus ± with a magnitude increasep represents statistical signiï¬cance p frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure concentrations of short chain fatty acids scfa in mucus samples from rabbits acetate concentrations were signiï¬cantly higher in the mucus samplescollected on week sinusitis relative to day control ± vs ± mm p n propionate and butyrate concentrations were higher in themucus samples from sinusitis compared to those from control propionate ± vs ± mm p butyrate ± vs ± mm p but statistical signiï¬cance was lacking n all groups consisted of at least four experiments represents statistical signiï¬cance p table presence of mucin degrading microbes in p aeruginosa culturepositive patientsno age comorbiditieslocation ofculturemucin degradingmicrobes dmfrontal sinusstreptococcusintermedius asthma mannosebindingethmoid sinusenterobacter cloacaelectin protein deï¬ciency cf 01f508 01f508frontal sinuscutibacterium acnesenterobacter cloacae immunocompromisedmaxillary sinusnonepostkt dm prostate cancermiddle meatusklebsiella oxytoca dm copdethmoid sinusrothia mucilaginosastreptococcus salivariusdm diabetes mellitus cf cystic ï¬brosis kt kidney transplantation copd chronicobstructive pulmonary diseasescfas was monitored concentrations of scfas were chosenbased on the previous hplc ï¬ndings above figure asacetate concentrations were above mm in the rabbit mucussamples we utilized four diï¬erent concentrations of acetate and mm because propionate and butyratewere mm two diï¬erent concentrations and mmbelow mm were used normalized od values were comparedto the average od from control without adding scfas foracetate propionate and butyrate figure 4a in the presence ofacetate pao1 exhibited increased growth from to mmand statistical signiï¬cance was noted between and mmcompared to control [normalized od values at nm mm ± n mm ± n mm ± n and mm ± n p anova with posthoc tukeykramer] in thepresence of propionate statistical signiï¬cance was noted in bothconcentrations [normalized od values at nm mm ± n mm ± n p anova] however the growth increases of pao1 observedwith butyrate were not statistically signiï¬cant in the analysis ofthese two concentrations [ mm ± n mm ± n p anova] additionallyas acetate is the major scfa at least or 200fold higherthan propionate or butyrate in figure the growth of pao1strains was compared in the presence of acetate alone mmvs scfas acetate mm propionate mm butyrate mm colony counts were signiï¬cantly higher when pao1strains were grown with acetate alone or scfas compared tocontrols control ± cfuml n acetate ± cfuml n threescfas ± cfuml n anovap figure 4b even though there was a trend towardhigher colony counts when pao1s were treated with scfasstatistical signiï¬cance was lacking between the acetate alone and scfas tukeys multiple comparison test p targeting anaerobes halts theradiographic evidence of sinusitisprogressionto evaluate whether targeting fermentative anaerobes haltssinusitis progression from acute to chronic stages acute sinusitisrabbits week were treated with metronidazole for days atweek midpoint between acute and chronic the metronidazoletreatment group n had marked improvement ofopaciï¬cation compared to the control group without treatmentn figure ct scores were signiï¬cantly higher in rabbitswithout metronidazole treatment kerschner scale n ± compared to those rabbits treated with metronidazole n ± p at week radiographically theprogression to sinusitis was halted by metronidazole treatmentdiscussionin this study we investigated the role of scfas produced bymucin fermenting anaerobes in the growth of p aeruginosafrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure concentrations of short chain fatty acids scfa in human mucus samples scfas from crs patients with p aeruginosa pa n were compared tothose from controls n acetate ± vs ± mm p propionate ± vs ± p butyrate ± vs ± p [represents statistical signiï¬cance p p and p ]in rhinosinusitis to our knowledge this is the ï¬rst study toquantitatively assess the presence of scfas in the human mucusincluding patients with crs while pseudomonas ineï¬ectuallyutilizes mucins as a carbon source on its own flynn we determined that mucin fermentation by mdms canstimulate the growth of p aeruginosa moreover we revealed thatscfas were also abundant and available in crs patients withand without p aeruginosa together these results suggest thatthe high levels of utilizable metabolites present in sinus mucusmay be derived from bacterial mucin degradation by anaerobesin the sinus cavity which may contribute to the establishmentand progression of recalcitrant crs cho under normal oxygen conditions most bacteria preferentiallyoxidize glucose and other saccharides to pyruvate and shuttlepyruvate through the citric acid cycle both processes requireoxygen as the ï¬nal electron acceptor in anaerobic conditionsbacteria must choose an alternative route by using the energyof another biochemical reaction and thus bypassing oxidativerespiration zumft ragsdale and pierce the processof fermentation involves the release of energy from compoundswithout utilizing exogenous oxygen eg muscle cells duringexercise through the formation of lactic acid ghorbani cystic ï¬brosis cf airway disease is one conditionwhich gives rise to persistent bacterial colonization coupledwith an anaerobic microenvironment the cf airway includesa thick mucus layer rendered hypoxic through the metabolismof host immune cells and bacteria under these circumstancesbacteria can produce scfas through the fermentation processfermentation of carbohydrates within the mucus results in theformation of scfas eg propionate butyrate and acetatewhich can thus uence the progression and resolution ofinfection and ammation in the airways ghorbani scfas have been shown to downregulate immune cellammatory responses promote neutrophil chemotaxis induceammatory protein expression in epithelial cellsinhibitproliferation and strengthen epithelial tight junctions in thegastrointestinal tract ferreira vieira inthe large intestine scfas are found at concentrations rangingfrom to mm which is signiï¬cantly higher than theconcentrations found in the airway mortensen and clausen smith while high mm concentrations impairgrowth low mm concentrations mm boost the growthof potential pathogens eg pseudomonas and upregulate il8in cf primary airway epithelium ghorbani thesemolecules easily penetrate the airway tissue because of their lowmolecular weight and subsequently interrupt host cell activityand host defense systems by inducing apoptosis in ï¬broblastsand lymphocytes tonetti kuritaochiai sato it is also possible that scfas may uencep aeruginosa bioï¬lm formation which will be investigated infuture experimentsit is interesting to note that scfa levels were higher inrabbits than human samples in both controls and noncontrolsevery species appear to have diï¬erent microbial fermentationpatterns even if they have similar diets kroliczewska this is the ï¬rst report to our knowledge to evaluatescfa levels in rabbit sinonasal mucus and we will requiremore numbers to strictly deï¬ne normal scfa levels in rabbitsinuses however the high ï¬ber intake in the diet encouragesthe growth of species that ferment ï¬ber into metabolites asscfas and thus rabbits baseline could be higher than humansas previously shown in the gut cummings tomova furthermore for experimental conditions weintentionally created an anaerobic environment in the sinuscavity and the scfa levels were measured during the acutesinusitis phase in the rabbit model by contrast the humansamples were collected from the postoperative sinuses ormiddle meatus in crs patients therefore the scfas fromrabbits may have been generated at higher levels than observedin human samplesbased on our experiments all scfas were signiï¬cantlyhigher in crs with p aeruginosa our clinical results are alsoconsistent with cf bronchoalveolar lavage ï¬uid ï¬ndings in otherstudies ghorbani mirkovic flynn the ratio of propionate to acetate in our humansinusitis samples was comparable to flynn s experimentsin human cf sputumsaliva samples flynn asexplained by flynn only a small amount of propionate isgenerated in vivo or it may be used by microanisms in a crossfeeding relationship when we compared the cfus betweenfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure growth of pao1 with scfas a pao1 growths were compared to controls in the presence of different concentrations of acetate between and mm normalizzed od values at nm mm ± n mm ± n mm ± n and mm ± n in the presence of propionate statistical signiï¬cance was noted in both concentrations compared to controls normalized od values at nm mm ± n mm ± n however no statistically signiï¬cant growth of pao1 was seen with butyrate at these two concentrations [ mm ± n mm ± n p ] oneway anova ns no signiï¬cance b colony counts were signiï¬cantly higher when pao1 strainswere grown with acetate alone or three scfas compared to controls control ± cfuml n acetate ± cfuml n scfas ± cfuml n anova p p p and p frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure radiographic progression of sinusitis when targeting anaerobes a at week midpoint between acute and chronic the metronidazole treatment groupn had marked improvement of opaciï¬cation compared to the control group without treatment n b scores were signiï¬cantly higher in rabbits withoutmetronidazole treatment kerschner scale n ± compared to those rabbits treated with metronidazole n ± p at week p frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure model for the development of recalcitrant chronic rhinosinusitis this model demonstrates a sequence of events that augment our understanding ofrecalcitrant crs pathophysiology blockage of sinus ostia generates a shift in microbiota to a predominance of anaerobes mucin metabolites [short chain fattyacids scfas] produced by these anaerobes are abundant within the mucus during an acute anaerobic stage and the shift from acute to chronic sinusammation is associated with a robust increase in pathogenic bacteria eg p aeruginosathe acetate alone and three scfas an additional growth ofp aeruginosa could be clinically presumable in the presenceof multiple nutrients even though statistical signiï¬cance waslacking in this experiment when comparing the total scfaconcentrations before and after antibiotic treatment in patientspresenting with a cf pulmonary exacerbation the mean scfaconcentrations were signiï¬cantly lower after the treatmentghorbani our results suggest the role of hypoxia in recalcitrant crspathophysiology figure hypoxia due to sinus closure at theostiomeatal complex at the junction of the ethmoid and maxillarysinuses is widely considered a major pathogenic mechanismleading to the development of crs and is strongly supportedby proof that hypoxia is present at the surface epithelium indiseased sinuses of closed ostia aanaes targetingmucinfermenting anaerobes and their metabolites could be anovel therapeutic strategy for the treatment of crs by restoringthe microbial community in diseased sinuses to the originalnoninfected pristine statusthere are several limitations to this study our hypothesisis from the animal model not human subjects and this maynot reï¬ectthe human in vivo environment even thoughsome wellcontrolled microbiota studies using animal modelshave also shown interstudy variations due to confoundingfactors eg origin maternal eï¬ects environmental conditionsnguyen the advantages of the rabbit modelare numerous in that it enables us to control for a numberof variables that are inherent in human samples includinggenetics medical history environmental allergiespollutants andmedication use cho as this is a clinical pilotstudy to assess the concentrations of scfas in crs patientsthe size and homogeneity of the clinical samples limit thestudys generalizability in addition we did not include anymicrobiomerelated analyses or molecular based quantiï¬cationofthe microanisms that colonize the mucus layers ofhuman samples a much larger group of patients will berecruited for future studies additionally bacteroides one ofthe most common anaerobes in the rabbit model is not oneof the common anaerobes in human sinusitis and there couldbe other nutrients other than scfas which are derived byfermentation that sustain bacterial growth in humans thereforewe are planning to testthe capability of patientderivedanaerobic microbiota taxa streptococcus peptostreptococcuspropionibacterium fusobacterium and prevotella to support thegrowth of p aeruginosa isolates using an in vitro and in vivocrossfeeding coculture modelconclusiongiven that scfas are solely derived from bacterial fermentationour experiments propose a critical role for mucinfermentingbacteria in generating carbonsource nutrients for pathogenicfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsbacteria in the airway mucin fermenting anaerobes maycontribute to the development of recalcitrant crs by degradingmucinsfor potential pathogenslike p aeruginosathus providing nutrientsdata availability statementexperimentsfrom rh sr ws and bw concept ofand editing ds dl ht crw and sz carried out therabbit and in vitro experiments and contributed to samplepreparationjg performed statistical analysis all authorsprovided critical feedback helped shape the research analysisand manuscriptthe datasets generated for this study are available on request tothe corresponding authorfundingethics statementthe studies involving human participants were reviewed andapproved by institutional review board irb approval number at the university of alabama at birmingham thepatientsparticipants provided their written informed consentto participate in this study the animal study was reviewedand approved by institutional animal care and use committeeiacuc approval number at the university of alabamaat birmingham uabthis work was supported by national institutes of healthnihnational heart lung and blood institute r01hl13300605 to bw nationalinstitute of diabetes anddigestive and kidney diseases 5p30dk07248202 to srand nihnational institutes of allergy and infectious diseasek08ai146220john w kirklin research and educationfoundation fellowship award uab faculty developmentresearch award american rhinologic society new investigatoraward triological society career development award andcystic fibrosis foundation k08 boost award cho20a0kbto dycauthor contributionsacknowledgmentsdyc designed the study carried outthe experimentsand took the lead in writing the manuscript with supporta part of this manuscript was presented at north americancystic fibrosis conference in denver coloradoreferencesaanaes k rickelt l fjohansen h k von buchwald c presslert hoiby n decreased mucosal oxygen tension in themaxillary sinuses in patients with cystic ï¬brosis j cyst fibros doi 101016jjcf201012002benninger m s fergusonm b j hadley j a hamilos d l jacobs mkennedy d w adult chronic rhinosinusitis deï¬nitionsdiagnosis epidemiology and pathophysiology otolaryngol head neck surg129suppl3 s1 doi 101016s0194599803013974cameron e a and sperandio v frenemies signaling and nutritionalintegration in pathogenmicrobiotahost interactions cell host microbe doi 1 | Colon_Cancer |
the molecular heterogeneity of renal cell carcinoma rcc complicates the therapeutic interventions for advancedmetastatic disease and thus its management remains a significant challenge this study investigates the role of thelncrna cdkn2bas1 and mir1413p interactions in the progression and metastasis of kidney cancer human renalcancer cell lines achn and caki1 normal rptec cells tissue cohorts and a series of in vitro assays and in vivo mousemodel were used for this study an overexpression of cdkn2bas1 was observed in rcc compared to normal samplesin tcga and our inhouse sfvamc tissue cohorts reciprocally we observed reduced expression of mir141 in rcccompared to normal in the same cohorts cdkn2bas1 shares regulatory mir141 binding sites with ccnd1 andccnd2 genes direct interactions of cdkn2bas1mir141cyclin d1d2 were conï¬rmed by rna immunoprecipitationand luciferase reporter assays indicating that cdkn2bas1mir141cyclin d1d2 acts as a cerna network in rccfunctionally attenuation of cdkn2bas1 andor overexpression of mir141 inhibited proliferation clonogenicitymigrationinvasion induced apoptosis in vitro and suppressed tumor growth in xenograft mouse model furtheroverexpression of cdkn2bas1 is positively correlated with poor overall survival of rcc patients expression of mir141also robustly discriminated malignant from nonmalignant tissues and its inhibition in normal rptec cells induced procancerous characteristics cdkn2bas1 attenuation or mir141 overexpression decreased ccnd1ccnd2 expressionresulting in reduced rac1ppxn that are involved in migration invasion and epithelialmesenchymal transition thisstudy for the ï¬rst time deciphered the role of cdkn2bas1mir141cyclin d axis in rcc and highlights this networkas a promising therapeutic target for the regulation of emt driven metastasis in rccintroductionrenal cell carcinoma rcc is one of the most commoncancers in the usa accounting for nearly deathsand new cases in surgery is the ï¬rst line oftreatment resulting in successful resection and longtermdiseasefree status with an overall survival rate of morecorrespondence rajvir dahiya rdahiyaucsfedu1department of urology veterans affairs medical center san francisco anduniversity of california san francisco san francisco ca usa2department of surgery university of miami miller school of medicine miamifl usafull list of author information is available at the end of the these authors contributed equally pritha dasgupta priyanka kulkarniedited by e candithan however in approximately of localizedrcc cases recurrence occurs with distant metastasis2the obstinate nature of rcc to current treatment regimens is a primary cause of poor prognosis in patients withmetastatic recurrence lack of sensitivity to both chemotherapytherapeuticoptions difï¬cult3 it is therefore of utmost importanceto improve our understanding of rcc pathogenesis byidentifying new biomarkers that lead to better predictionand therapeutic intervention of aggressive rcc6and immunotherapy makesemerging lines of evidence suggest that cancer aggressiveness is associated with epithelialmesenchymal transition emt7 it is a wellorchestrated process involved in 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 0cdasgupta cell death and disease page of tumor invasion and metastasis comprising characteristicphenotypic changes through transition from polarizedimmotile epithelial cells to motile mesenchymal cells8emt changes in cellular morphology and migratoryproperties are governed by numerous factors9 increase inmesenchymal properties accompanied by augmentedexpression of mesenchymal markers like ncadherbronectinvimentin and matrix metalloproteinasemmps and decreased expression of epithelial markerslike ecadherin αecatenin claudin etc10 are common emt phenomena often the progression of cancerthrough emt is significantly induced by the interaction ofcyclind with its binding partner cdk4 which act astranscriptional regulators controlling cell proliferationand migration14 it is well known that cyclind regulates the ratelimiting step in cell cycle progression fromg1 to s phase accumulating evidence also suggest thatabnormal cyclindcdk4overexpression promotestumor growth and metastasis17 but how this correlateswith tumor metastasis or controls cell adherence andinvasion is poorly understoodreports show that noncoding rnas are involved in thefactors involved in emt18 micrornasregulation ofmirnas a naturally occurring class of small noncodingrna molecules of nucleotides long19 are known toregulate gene expression via both translational inhibitionand mrna degradation20 whereas long noncoding rnaslncrnas with more than nucleotides can also actas regulators for tumorsuppressive mirnas in differentcancers21 recently a class oflncrnas have beencategorized as competing endogenous rna cernawhich involves crosstalk among lncrnas mrnas andtheir shared mirnas thus a novel regulatory mechanismis hypothesized suggesting that lncrnas and mrnascommunicate with each other by competing for commonmirna response elements24in this context we describe the novel role of lncrnacdkn2bas1 and mir1413p mir141 in the regulation of cyclind to govern the metastatic progression ofrcc to our knowledge this is the ï¬rst report to directlydemonstrate that cdkn2bas1mir141 interaction is acrucial component in rcc progression and metastasisthrough the cyclindrac paxillin pathwaymaterials and methodscell lines and cell culturethe normal rptec atcc number crl4031 andrenalcancer achn atcc number crl1611and caki1 atcc number htb46 cell lines were purchased from the atcc manassas va these humanderived celllines were authenticated by dna shorttandem repeat analysis by atcc cell line experimentswere performed within months of their procurementresuscitation achn cells were cultured in mem mediaofï¬cial of the cell death differentiation associationcaki1 cells in and mccoy 5a medium and rptec cellsin dmemf12 medium atcc® ¢ all mediawere supplemented with fbs and 1x antibioticspenicillin and streptomycin cell lines were maintainedat °c and humidiï¬ed atmosphere of co2mirnasirna transfectionsto induce overexpression or knockdown cells were transiently transfected with either mirvana mirna mimic nmoll or antimir mirna inhibitor nmollthermo fisher scientiï¬c and nmoll of sirna sigmaaldrich using lipofectamine rnai max thermo fisherscientiï¬c according to the manufacturers protocol toverify transfection efï¬ciency mirvana mirna mimicnegative control mirna inhibitor control and sirnacontrol were used respectively in each transfection experiment at the same concentration all transfection experimentswere carried out for hclinical specimensformaldehydeï¬xedparafï¬nembedded ffpe tissue specimens from patients undergoing radical nephrectomy wereobtained from the san francisco veterans affairs medicalcenter sfvamc written informed consent was obtainedfrom all patients and the study was in accordance withinstitutional guidelines irb approval no allpatient samples were pathologically conï¬rmed for clear cellrcc ccrcc and slides were reviewed by a boardcertiï¬edpathologist for the identiï¬cation of tumor foci and adjacentnormal tissue apart from sfvamc cohort tcgakirctcgakich tcgakirp icgc and geo cohorts forrcc from online databases were also used to check theexpression levelsrnamirna extraction and quantitative realtime pcrqrtpcrtotal rna was extracted from microdissected ffpetissues and cell lines using mirneasy ffpe and mirneasymini kits qiagen respectively in accordance to manufacturers instructions mature mirna and mrnas wereassayed by qrtpcr using quantstudio flexreal timepcr system applied biosystem using fast sybr®green master mixtaqman universal pcr master mixprobes and primers applied biosystems inc foster cityca usa following manufacturers protocol humangapdh and rnu48 were used as endogenous controlsand relative expression of rnamirna were calculatedusing comparative ct threshold cycle primer sequencesare provided in supplementary table t1dna methylation analysis insilico in cell lines and 5azacdr treatmentdna hypermethylation of the mir141 promoter innormal and rcc samples was ï¬rst conï¬rmed in the 0cdasgupta cell death and disease page of tcga database using wanderer software27 in order toconï¬rm the methylation status of the mir141 promoterin rcc cell lines we extracted dna from achn andcaki1 using dneasy tissue kit qiagen sodium bisulphite modiï¬cation was done using ez dna methylationgold kit zymo research orange ca usa followingthe manufacturers protocol bisulï¬tetreated dna wasanalyzed by methylationspeciï¬c quantitative polymerasechain reaction msqpcr with primer pairs speciï¬c formethylated and unmethylated regions of the mir141promoter msqpcr was performed as described earlier28 for each sample the percent of methylation wascalculated by the difference of ct in methylated samplectm and ct in unmethylated sample ctu the primers sequences are mentioned in supplementary table achn and caki1 cells were treated daily with μmoll5azadeoxycytidine 5azacdrfor h29 and total rna was isolated using a mirneasy minikit qiagen to check mir141 expressionsigmaaldrichcell viability clonability migratory invasion andapoptosis assayscell viability was measured at and h using acelltiter aqueous solution cell proliferation assay kitpromega madison wi following the manufacturersinstructions for colony formation assay cells were seeded at a low density cellsplate after h oftransfection and were allowed to grow until visible colonies were formed plates were then stained with giemsafollowed by crystal violet and colonies were countedculture inserts of 8µm pore size transwell costar wereused for migration and invasion assay inserts were coatedwith matrigel bd biosciences µgwell for invasionbrieï¬y h posttransfection cells were counted andplaced on inserts at à cellsml for migration and à for invasion in serumfree medium and wereallowed to migrateinvade for h at °c cellsmigrated or invaded through the pores were ï¬xed stainedwith crystal violet crystal violet was solubilizedwith methanol and quantiï¬ed at nm by a kineticmicroplate reader spectra max molecular devicesfacs analysis for apoptosis was done h posttransfection using annexin vfitc and 7aad kit beckmanin accordance with the manufacturerscoulter incinstructions cold pbs washed cells were resuspended in1x binding buffer and stained with annexin vfitc7aad viability dye after min of incubation at roomtemperature in the dark stained cells were analyzed usingbd facsverse bd pharmingendualluciferase reporter assaythe wild type wt and offtarget ot luciferasereporter constructs were made by ligating annealed custom oligonucleotides containing putative target bindingofï¬cial of the cell death differentiation associationsites and corresponding nontarget mutant sites into thepmirglo reporter vector luciferase constructs µgwere cotransfected into achn and caki1 cells along with nmoll mir141 mimic or controlmir using transfection reagent jetprime polyplustransfection illkirchfrance luciferase activities were measured using thedualluciferase assay promega madison wi h posttransfection relative luciferase activity was calculated bynormalizing fireï¬y luciferase to renilla luminescencerna immunoprecipitation rip assaysimultaneous binding of mir141 to lncrna andmrna was conï¬rmed by rip assay an imprint rip kitwas used following the manufacturers protocol sigmaaldrich st louis mo usa igg control and ago2antibodies were used forimmunoprecipitation theimmunoprecipitated rna fraction was reverse transcribed to cdna using high capacity cdna reversetranscription kit thermo fisher fold enrichment oflncrna and mrna to ago2 with respect to igg wascalculated using quantitative rtpcrwestern blot and immunoï¬uorescence analysistotal protein extraction was performed as describedpreviously18 proteins were then separated by nupage bistris protein gels invitrogen and subsequentlytransferred onto nitrocellulose membraneresulting blots were blocked using odyssey blockingbuffer licor and subsequently probed with primaryand secondary antibodies blots were scanned using anodyssey infrared imaging system scan and quantiï¬cationwas carried out with the licor odyssey® scanner andsoftware licor biosciences the primary antibodiesused are listed in supplementary table for immunoï¬uorescencetransfected achn andcaki1 cells were ï¬xed in paraformaldehyde for minfollowed by blocking 1x pbs5 normal goat serum triton x100 for h at room temperature cellswere then incubated overnight in fold diluted primary antibody at °c cells were then reprobed with fold diluted secondary antibody for h and counterstained with µgml of ²6diamidino2phenylindoledapi for min cells were then mounted on a slideusing prolong gold antifade reagent images were captured using zeiss microscope model axio imagerd2transientlyrenal cancer xenograftswe studied the antitumorigenic effects of mir141 inestablished tumors using a renal cancer xenograft nudemouse model as previously described630 male nude mice weekold n charles river lab were subcutaneously injected with à caki1 cells oncepalpable tumors were formed mice were randomized intwo groups for the treatment and control groups ï¬ve in 0cdasgupta cell death and disease page of each synthetic mirna mir141 mimicmircon of μg was complexed with μl siportamine transfection reagent ambion in μl pbs and deliveredintratumorally in 3day intervals tumor volume wascalculated according to the formula x2 y2 where x yx width y length experiments were terminated days after the last treatment day tumor measurements and statistical analysis were performed byresearchers who were blinded for the control and treatment groups all animal care was in accordance withinstitutional guidelines iacuc approval no statistical analysisall quantiï¬ed data represents an average of at leastthree independent experiments or as indicated statisticalanalyses were performed using graphpad prism andmedcalc error bars represent ± standard error meansem the mannwhitney u test was used to assessthe difference between mirna expressions in tumornormal adjacent tissue significant differences betweenthe groups were determined using the students ttest alltests were performed either one tailed or two tailed andresults were considered statistically significant at p ¤ receiver operating curves roc were performed toevaluate the potential of mir141 to differentiate betweenmalignant and nonmalignant samples using medcalcsoftware showing area under curve auc and conï¬dence interval kaplanmeier analyses for overall survival with respectto mir141 methylation levels intcgakirc cohort were generated using softwareezrhttpsdoi101038bmt2012244 tumormeasurements and statistical analyses for all experimentswere performed blindly for the control and treatment groupsresultslncrna cdkn2bas1 is oncogenic and is a direct target ofmir141initially we found cdkn2bas1 is an oncogeniclncrna in rcc based on tcga fig 1a icgc andgeo databases fig s1 tcga cohort also revealed thathigh cdkn2bas1 expression increases from lower gradeand stage to higher grade and stage fig 1b moreoverhigher expression is significantly p correlated tooverall survival fig 1c in agreement with these datacohorts significantly higher cdkn2bas1 expression wasalso seen in rcc cell lines achn caki1 as compared tonormal rptec fig 1d and sfvamc cohort fig 1epatient and tumor characteristics are summarized insupplementary table t3 roc analysis shows an auc of p ci fig 1f suggesting the diagnostic potential of cdkn2bas1 to discriminate between normal and tumor tissues we usedcomputational algorithms and identiï¬ed putative mir141binding sites in the cdkn2bas1 sequence fig 1g toofï¬cial of the cell death differentiation associationexamine potential mir141cdkn2bas1 interactionexperimentally we performed luciferase reporter assayboth achn and caki1 cells cotransfected with mir141and cdkn2bas1 wild type binding site revealed a consistent reduction ofluciferase activity suggesting thatmir141 directly interacts and regulates cdkn2bas1fig 1h thus all these data suggest that clinicallyimportant cdkn2bas1 is an oncogenic lncrna in rccand is a novel target of mir141cdkn2bas1 inhibition by sirna suppressestumorigenicity in rcctransient transfection of achn and caki1 cells withcdkn2bas1 sirnas for h showed significant reduction in cdkn2bas1 expression fig 2a cdkn2bas1knockdown in both cell lines significantly inhibited cellproliferation figs 2b s2a and clonogenic survivalfigs 2c s2b with a significant increase in apoptosis fig2d e decreased cell migrationinvasion figs 2f s2c dwith simultaneous changes in emt markers such as anincrease in epithelial markers αecatenin and claudinand decrease in mesenchymal markers vimentin andï¬bronectin were also observed figs 2g s3expression of mir141 and its clinical importance in renalcarcinomaand samples lowersince our results conï¬rmed cdkn2bas1 as a directtarget of mir141 we examined mir141 status andclinical importance in rcc expression of mir141 wassignificantly downregulated in rcc cell lines fig 3aand in tumor samples fig 3be compared to normal celllinesignificantlydecreased with increasing grade stages and in metastaticcompared to nonmetastatic tumors fig 3ce patientand tumor characteristics are summarized in supplementary table t3 roc analysis showed an auc of p ci fig 3f suggesting thatmir141 can be used as a potential diagnostic parameterto discriminate between normal and tumor tissuesexpressionsepigenetic regulation of the mir141 locuswe identiï¬ed a genomic site rich in cpg island locatedupstream of the mir141 in chromosome 12p13 in thetcga cohort we observed hypermethylation of mir141promoter in tumor tissues as compared to normalfigs 3g s4a which is significantly associated with poorpatient survival fig 3h similarly rcc cell lines achnand caki1 also showed hypermethylation compared tonormal rptec cells fig 3i further we treated achnand caki1 cell lines with demethylating agent 5azacdrand observed decrease in methylation fig s4b withconcomitant increase in mir141 expression fig 3jindicating possible epigenetic regulation a significantdecrease in the expression of methylation regulatory 0cdasgupta cell death and disease page of fig lncrna cdkn2bas1 is oncogenic in renal cancer and is a direct target of mir141 a expression levels of cdkn2bas1 among kircnormal tumor kich normal tumor and kirp normal tumor patient samples in tcga cohort using wanderersoftware pvalue calculated by mannwhitney twotailed test b expression of cdkn2bas1 in tcgakirc cohort among different grades normal grade grade and stages normal stage iii and stage iiiiv c overall survival in tcgakirc cohort as performedby kaplanmeier analysis using ualcan software d relative expression levels of lncrna cdkn2bas1 in rcc cell lines achn and caki1 e expressioncdkn2bas1 in matched pairs of rcc tissue samples from sfvamc cohort pvalue calculated by mannwhitney twotailed test f receiver operatingcurve roc analysis on sfvamc cohort showing ability of lncrna cdkn2bas1 to differentiate between malignant and nonmalignant samples sfvamccohort g predicted binding sites of mir141 in cdkn2bas1 sequence h luciferase assays showing decreased reporter activity after cotransfection witheither wildtype wt offtarget ot cdkn2bas1 or luciferase control constructs ev with mirconmir141 in achn and caki1 cellsgenes such as dnmtl dnmt3a and dnmt3b werealso noted after 5azacdr treatment compared to control dmso in both achn and caki1 cell lines31mir141 overexpression phenocopies functional effectsobtained with cdkn2bas1 inhibition in vitro andsuppresses tumorigenicity and in vivowe sought to determine if cdkn2bas1 causes itsantitumorigenic effects through mir141 we checkedthe effect of mir141 overexpression in rcc cellstransient transfection of mir141 mimic in achn andcaki1 cells for h led to over expression of mir141compared to control mircon fig 4a also overexpression of mir141 significantly reduced cdkn2bas1 expression fig 4b indicating a reciprocal correlation between mir141 and cdkn2bas1 a significant decrease in cell proliferation over time fig 4cand marked decreasefig 4din clonogenicityofï¬cial of the cell death differentiation association 0cdasgupta cell death and disease page of fig knockdown of lncrna cdkn2bas1 reduces tumorigenicity in renal cancer a relative expression of cdkn2bas1 in achn and caki1 cellstransfected with cdkn2bas1 sirnas b cell proliferation assessed by mts assay after knockdown of cdkn2bas1 in both cell lines with sirna2 cgraphical representation showing knockdown of cdkn2bas1 with sirna2 significantly decreased colony formation in achn and caki1 cellsd e achn and caki1 cell lines showing significant induction of apoptosis early late compared to control after knockdown of cdkn2bas1f reduced migration and invasion in cdkn2bas1 sirna transfected cells compared to control treatment g changes in emt related proteins in bothachn and caki1after knockdown of cdkn2bas1compared to controls were also observed further westudied the therapeutic potential of mir141 in amouse xenograft model a significant decrease intumor growth was observed by intratumoral delivery ofmir141 mimic compared to control over the course ofexperiment average tumor volume in the controlgroup was mm3 compared to mm3 in micethat received mir141 mimic fig 4e in additionmir141 overexpression significantly induced apoptosis with a concomitant decrease in the viable population in both rcc celllines compared to controlfig 5a this proapoptotic role was supported by theinduction of cleaved caspase3 cleaved polyadpribose polymerase parp an increase in bax and adecrease in bcl2 at protein levels fig 5b a significantdecrease in migration fig 5c and invasion fig 5dwas also observed in both rcc cell lines with mir141overexpression we also examined emt markers aschange in migration and invasion are directly associated with emt our results showed an increase inepithelial markers αecatenin and claudin with concomitant decrease in mesenchymal markers ï¬bronectinand vimentin at both protein fig 5e and mrnaofï¬cial of the cell death differentiation associationfig s5 levels taken together overexpression of mir phenocopies the functional effects of cdkn2bas1 inhibition in vitro and tumor growth suppressioneffects in vivolike cdkn2bas1aremir141cdkn2bas1 interaction negatively regulatescyclind and its downstream effectors in rcccyclind1cyclind2alsooncogenic in rcc fig s6 and are direct targets of mir as discussed earlier lncrnas can act as cernas tocarry out their regulatory functions32 we observedthat cdkn2bas1 shared regulatory mir141 bindingsites with cyclind1cyclind2 fig 6a and therebysponges mir141 allowing cyclind1d2 to be expressedin tumors to determine potential mir141cdkn2bas1cyclind interaction experimentally we performedrip assay both achn and caki1 cells over expressingmir141 revealed significant enrichment of cyclind1cyclind2 and cdkn2bas1 with ago2 as compared toigg control fig 6b moreover decreased luciferaseactivity also conï¬rmed direct binding of mir141 tocyclind in mir141overexpressing achn andcaki1 cells compared to controls fig 6c we also found 0cdasgupta cell death and disease page of fig expression clinical signiï¬cance and epigenetic regulation of mir141 in renal cancer a mir141 expression levels in achn caki1 andrptec cells b expression levels of mir141 in kirctcga cohort normal and tumor c expression levels of mir141 in normal n nonmetastatic n metastatic n kirc patient samples in tcga cohort d expression levels of mir141 in kirctcga cohort amongdifferent grades normal grade grade and stages normal stage iii and stage iiiiv e relative mir expression in rcc tissue vs matched adjacent normal regions n among different grades normal grade grade andin different stages normal stage iii stage iiiiv as assessed by qrtpcr sfvamc cohort f receiver operating curve roc analysisshowing ability of mir141 to differentiate between malignant and nonmalignant samples g methylation for kirc patient samples in tcgakirccohort for probe cg02624246 h overall survival with tcgakirc methylation as performed by kaplanmeier analysis i methylation status of mir141promoter in rcc cell lines compared to nonmalignant rptec as assessed by msqpcr j expression of mir141 in 5azacdr treated and untreatedachn and caki1 cell lines results are representative of three independent experiments p value calculated by student t test bar mean ± standarderror mean semthat overexpression of mir141 or inhibition of cdkn2bas1 significantly decreased cyclind1d2 expression atboth the mrna fig 6d e and protein levels figs 6fhs8a b this effect was significantly attenuated by mir inhibitor fig s7 indicating that cyclind expressionis dependent on the interaction between mir141 andcdkn2bas1 we further observed a decrease in rac1 asmall gtpase and a reduction in the phosphorylation ofpaxillin a focal adhesion protein at mrna levelsfigs s3 s5 and protein levels figs 6g ij s8cf inboth mir141 overexpressed and cdkn2bas1 inhibitedrcc cell lines which in turn are involved in regulatingcellular migrationinvasion cumulatively these resultsindicate that suppression of cdkn2bas1 by mir141inhibits renal cell proliferation invasion and migration byinhibiting cyclind rac1 and phosphorylation of paxillinofï¬cial of the cell death differentiation association 0cdasgupta cell death and disease page of fig mir141 overexpression mimics the knockdown effect of lncrna cdkn2bas1 in vitro and reduces tumorigenicity in vivo a relativeexpression of mir141 in achn and caki1 cells transfected with mir141 mimic and control b significant decrease in cdkn2bas1 expressioncompared to control in both cell lines overexpressed with mir141 c rcc cell proliferation after transfecting with mir141 mimic and control asassessed by mts assay d colony formation and its graphical representation in mir141 overexpressing achn and caki1 cells compared to controlse pictures of excised tumors are taken at the termination of experiment day graph represents tumor volume after intratumoral injection ofcontrol or mir141 mimic into established tumors injection was started at day and was followed for days each mouse in both groups mirconand mir1413pmimic received a total of eleven injections intermittently data represent the mean of each group and error bars are sem results arerepresentative of three independent experiments p value calculated by student t test bar mean ± standard error mean semattenuation of mir141 exerts tumorigenic attributes innormal rptec cellswe next determined whether attenuation of mir141induces tumorigenic characteristics in normal rpteccells by targeting cdkn2bas1 and cyclind transienttransfection of mir141 inhibitor indeed showed a significant decrease in mir141 expression fig 7a and anincrease in cdkn2bas1 expression fig 7b along withother procancerous phenotypes such as increased cellproliferation fig 7c colony formation fig 7d migration and invasion fig 7e as compared to controlsadditionally a significant increase in cyclind1 cyclind2 rac1 and paxillin pxn expressions were observed inmir141 inhibited rptec cells fig 7f a noticeableincrease in prometastatic ï¬bronectin and vimentin with aconcomitant decrease in antimetastatic claudin andαecatenin genes were also observed in mir141 inhibited rptec cells compared to controls fig 7gdiscussionprior studies have shown the regulatory role of noncoding rnas in tumorigenesis especially in the emtofï¬cial of the cell death differentiation associationpathway leading to cancer aggressiveness cdkn2bas1also known as anril is located at chromosome 9p21cdkn2bas1 is reported to be upregulated in tumortissues and function as an oncogenic lncrna in pancreatic ovarian and laryngeal squamous cell carcinoma36 human mir141 is located at chromosome12p1331 and is transcribed from a mir200 familyclusterinterestingly expression of mir141 is controversial since it exhibits either oncogenic39 or tumorsuppressive roles42 in speciï¬c types of cancer theprime goal of the present study was to understand therole of cdkn2bas1mir141 interactions in regulatingrcc progression and metastasisin this study we identify cdkn2bas1 to be a crucialoncogenic lncrna that plays an important role in renalcarcinogenesis cdkn2bas1significantly overexpressed in rcc and the expression increases fromlower to higher grades and stages lncrna cdkn2bas1directly interacted with mir141 as it was found to be anovel target of mir141 in contrast to cdkn2bas1 weobserved significant attenuation of mir141 expression inrcc cell lines and tumor samples compared to normalis 0cdasgupta cell death and disease page of fig ectopic mir141 expression induces apoptosis and inhibits migrationinvasion in renal cancer cells a apoptosis assessed by ï¬owcytometric analysis of annexinvfitc 7aadstained achn and caki1 cells transfected with mirconmir141mimic graph represents totalapoptosis early late b immunoblots showing apoptotic proteins in mirconmir141mimic treated achn and caki1 cells with gapdh asendogenous control c migration assay and d invasion assay as seen in pictures and graphical representation of both achn and caki1 cells after mir overexpression compared to control e immunoblot assay showing emt related proteins in mirconmir141mimic treated achn andcaki1 cells with βactin as endogenous control graphs are average of three independent experiments p value calculated by student t testbar mean ± standard error mean semcell line or matched normal samples as it is known thatextensive dna hypermethylation of cpg islands is highlycorrelated to activation of cancerspeciï¬c genes45 wechecked the methylation status of mir141 in normal andrcc tissues interestingly insilico analysis showed thepresence of cpg island in the promoter region of mir and we also found hypermethylation of mir141 intcga samples as compared to normal this hypermethylation is also found to be significantly associatedwith poor survival of patients similar results were alsoobserved in rcc cell lines compared to a normal rpteccell line functionally inhibition of cdkn2bas1 andoroverexpression of mir141 significantly inhibits thetumorigenic characteristics such as cell proliferationclonogenicity migration and invasion whereas inducesanticancer apoptotic phenotype in rcc in vitro in vivodata show suppression of tumor growth by mir141overexpression conversely attenuation of mir141 innormal rptec cells induced precancerous characteristicsindicated by increased proliferation migration andinvasionfrom a clinical point of view noncoding rnas signatures are powerful tools for early cancer diagnosisofï¬cial of the cell death differentiation associationmaking them attractive candidates as diagnostic andprognostic biomarkers184647 our results revealed thathigher expression of cdkn2bas1 is positively correlatedwith poor overall survival probability of rcc patientsindicating its prognostic capability cdkn2bas1 canalso discriminate normal from tumor samples showing itsdiagnostic potential similarly mir141 expression canalso robustly distinguish between cancerous from noncancerous samples and hence has potential to be a diagnostic biomarker for rcc collectivelythese resultshighlight the biomarker potential of cdkn2bas1mir expression in rcc although it needs to be conï¬rmedin a larger independent sample cohortinterestingly we found tha | 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 |
" 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 |
breast cancer bc is the most common malignancy among women emerging studies have demonstrated that circular rna circrna zinc finger rna binding protein circzfr serves as a crucial regulator in many human cancers however the role and mechanism of circzfr in bc tumorigenesis remain unclearmethods the levels of circzfr mir578 and hypoxiainducible factor 1α hif1a were detected by quantitative realtime polymerase chain reaction qrtpcr or western blot cell viability colony formation apoptosis migration and invasion capacities in vitro were determined by using the cell counting kit8 cck8 standard colony formation flow cytometry and transwell assays respectively glucose uptake lactate product and adenosine triphosphate atp levels of cells in vitro were measured using the commercial human assay kits targeted relationships among circzfr mir and hif1a in bc cell lines were verified by dualluciferase reporter and rna pulldown assays animal studies were performed to assess the effect of circzfr on tumor growth in vivoresults our data indicated that circzfr was overexpressed in bc tissues and cells and the increased circzfr level predicted poor prognosis of bc patients circzfr silencing or mir578 overexpression repressed bc cell viability colony formation migration invasion and glycolysis and enhanced cell apoptosis in vitro circzfr silencing also hampered tumor growth in vivo mechanistically circzfr acted as a sponge of mir578 and circzfr silencing hindered bc cell malignant behaviors by mir578 hif1a was a functional target of mir578 in regulating bc cell viability colony formation migration invasion glycolysis and apoptosis in vitro furthermore circzfr modulated hif1a expression through sponging mir578 our findings first identified that the silencing of circzfr suppressed bc malignant progression in vitro via the regulation of the mir578hif1a axis providing evidence for the crucial involvement of circzfr in bc pathogenesiskeywords breast cancer bc circzfr mir578 hif1a malignant progressioncorrespondence qiuxinguang2020163com department of thyroid surgery the first affiliated hospital of zhengzhou university no1 jianshe east road erqi district zhengzhou henan chinafull list of author information is available at the end of the breast cancer bc remains the most commonly diagnosed cancer and the leading cause of cancerassociated death among females in although the therapeutic methods have greatly improved over the past two decades effective treatment against metastatic bc is still limited [ ] therefore a deeper understanding of what drives this disease is the first step to design innovative interventions the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cchen a0et a0al cancer cell int page of circular rnas circrnas are covalently closed endogenous rnas that have crucial noncoding functions in human physiologic and pathologic processes work in biological functions has demonstrated the roles of circrnas as microrna mirna sponges dysregulation of circrnas has recently implicated in the pathogenesis of bc for example yuan et a0al uncovered that hsa_circ_0068033 a downregulated circrna exerted a repressive impact on bc malignant progression via sequestering mir659 cao and colleagues demonstrated that hsa_circ_0087784 functioned as a potential promoter in bc development through sponging mir487a xu et a0al reported that circtada2as attenuated bc progression and metastasis by the regulation of mir203a3p moreover hsa_circ_001783 and circabcb10 were reported as oncogenic regulators in bc through functioning as specific mirna sponges [ ] as for circrna zinc finger rna binding protein circzfr hsa_circ_0072088 it has been identified as an oncogenic modulator in many human cancers such as renal carcinoma bladder cancer and nonsmall cell lung cancer [] nevertheless the biological roles of circzfr in bc tumorigenesis remain largely unknownmirnas modulate gene expression but are frequently dysregulated in human cancers including bc danza et a0al reported that mir578 was underexpressed in brcabc and it regulated tumor angiogenesis however the precise function of mir578 in bc progression is still undefinedhere we undertook to examine the biological effect of circzfr in the malignant progression of bc in a0 vitro and in a0 vivo we identified that circzfr a prominently upregulated circrna in bc controlled bc progression in a0vitro via targeting the mir578hypoxiainducible factor 1α hif1a axismaterials and a0methodsclinical tissues and a0cellsin this study we enrolled bc patients admitted to the first affiliated hospital of zhengzhou university from april to june the clinicopathological features of these patients were provided in table a0 seventy pairs of primary tumor tissues and matched healthy breast tissues were collected and stored at a0°c to detect the expression levels of circzfr mir578 and hif1a accession nm_1810543 these patients were followedup for at least a0 months and the followup information was obtained by telephone calls every a0 months the study was approved by the ethics committee of the first affiliated hospital of zhengzhou university and written informed consent was provided by all participantstable correlation and a0the a0clinicopathological features of a0bc patientsbetween a0circzfr expression characteristicsnumbercirczfr expressionphighlowage years ¥ distant metastasis present absenttumor size cm tnm stage i ii iii ivher2 status positive negativepr status positive negativeer status positive negativeher2 human epidermal growth factor receptor2 pr progesterone receptor er estrogen receptorp p bc cell lines mcf7 atcc ®htb22 bt549 atcc ®htb122 mdamb231 atcc ®htb26 and mdamb453 atcc ®htb131 american type collection culture atcc manassas va usa were cultured in rpmi1640 medium supplemented with fetal calf serum fcs and antibiotic solution all from cell line atcc ®crl10317 atcc was maintained in hyclone logan ut usa the immortalized mcf10a dulbeccos modified eagles mediumnutrient mixture f12 dmemf12 with fcs a0ngml epidermal growth factor a0 μgml hydrocortisone and a0 μgml insulin all from hyclone all cells were cultured in a co2 incubator at a0°cquantitative realtime polymerase chain reaction qrtpcrthe expression levels of circzfr hif1a and mirnas were gauged by qrtpcr complementary dna cdna synthesis was done using total rna a0 ng 0cchen a0et a0al cancer cell int page of isolated by isogen nippon gene tokyo japan from tissues and cell lines the levels of circzfr and hif1a were quantified using the taqman gene expression assays with the indicated primers and mature mirnas were assayed using the taqman microrna assays with taqmanspecific primer probes as recommended by the manufacturers applied biosystems rotkreuz switzerland qrtpcr was run in triplicate on the icycler iq5 device biorad munich germany using the pcr conditions as previously reported results were normalized to the expression of glyceraldehyde3phosphate dehydrogenase gapdh or u6 internal control using the δδct method primer sequences for circzfr forward ²atg gtc tgc agt cct gtg tg3² were and reverse ²tgg tgg cat gtt ttg tca tt3² for hif1a were forward ²ttc ccg act agg ccc att c3² and reverse ²cag gta ttc aag gtc cca tttca3² for mir578 were forward ²gtg cag ggt gtt agga3² and reverse ²gaa gaa cac gtc tggt3² for mir944 were forward ²gag tag gct aca tgt tat taaa3² and reverse ²gtg cag ggt ccg aggt3² for mir5323p were forward ²atc ctc cca cac cca agg ² and reverse ²gtg cag ggt ccg aggt3² for gapdh and u6 were described previously lentivirus transduction and a0transient transfectioncirczfr knockdown in mcf7 and bt549 cell lines was achieved by the transduction of corresponding lentiviruses expressing three different sequence shrnas specific to circzfr shcirczfr1 shcirczfr shcirczfr2 and shcirczfr3 fulengen guangzhou china and nontarget shrna lentiviruses shnc were used as the negative control vectortransduced cells were selected by puromycin solarbio beijing china at a final concentration of a0μgml at least a0h mir578 overexpression and knockdown cell lines were generated using the synthetic mir578 mimic a0nm ribobio guangzhou china and inhibitor antimir578 a0nm ribobio respectively with a corresponding nontarget oligonucleotide mirnc mimic or antinc ribobio as the negative control to elevate hif1a expression in bc cell lines a recombinant overexpressing plasmid for hif1a hif1a a0 ng ribobio or negative control plasmid vector ribobio was transiently transfected into cell lines using lipofectamine reagent invitrogen breda the netherlands as per the manufacturers protocols each experiment was performed in triplicatecell viability colony formation and a0apoptosis assaysshcirczfrinfected or shnctransduced cell lines were transfected with or without antinc or antimir578 and mcf7 and bt549 cell lines were carried out the indicated transfections followed by the incubation for a0h at a0°c cell viability assay was done using the cell counting kit8 cck8 abcam cambridge uk assay as per the manufacturers guidance cell colony formation was tested using standard colony formation protocols as previously reported cell apoptosis measurement was performed by flow cytometry using doublestaining with fluorescein isothiocyanate fitclabeled annexin v and propidium iodide pi based on the directions of manufacturers invitrogen events were analyzed by a flow cytometer and the apoptotic cells were determined by calculating the sum of early annexin vpi and late annexin vpi apoptotic cells all experiments were done in triplicatetranswell migration and a0invasion assayscell migration and invasion were detected by the transwell assay using modified boyden chambers in 24transwell plates a0 μm pores corning amsterdam the netherlands chambers of cell invasion assays consisted of matrigelprecoated membrane inserts corning after transfection bc cell lines were seeded into the top chamber of a 24transwell insert and medium containing fcs was used as a chemoattractant in the lower chamber a0h later the well was stained with crystal violet solarbio and the number of cells that had migrated or invaded to the basal side of the membrane was counted under a microscope nikon shinagawa tokyo japan at à magnification each experiment was performed in triplicatemeasurement of a0glucose uptake lactate product and a0adenosine triphosphate atp levelthe colorimetric glucose uptake assay kit llactate assay kit and atp assay kit all from abcam were used to determine the levels of glucose uptake lactate product and atp according to the recommendations of manufacturers briefly the lysates of transfected cells were prepared using the assay buffer and incubated with standard protocols for the indicated time point followed by the measurement of the absorbance with a microplate reader invitrogen at od a0nm for glucose uptake a0nm for lactate product and a0nm for atp level all assays were done in triplicate 0cchen a0et a0al cancer cell int page of animal studiesthe xenograft models were constructed to assess the role of circzfr on tumor growth in a0vivo animal studies were implemented in accordance with a protocol approved by the ethics committee on animal use and care of the first affiliated hospital of zhengzhou university twelve a0weeks balbc female mice shanghai animal laboratory center shanghai china were used and randomly divided into two groups n per group shnc and shcirczfr approximately à shncinfected or shcirczfrtransduced bt549 cell line was subcutaneously injected into the dorsal flank of the mice one week later tumor size was measured every week with callipers and tumor volume was calculated using the following formula volume à length à width2 all mice were sacrificed at a0days after implantation and tumor tissues were collected for weightbioinformatics dualluciferase reporter and a0rna pulldown assaysanalysis for the targeted mirnas of circzfr was performed using the online web circinteractome https circi ntera ctome nianihgovindex html and circbank httpwwwcircb ankcnsearc hcirc html the putative targets of mir578 were predicted by targetscan v7 online software httpwwwtarge tscan vert_71targeted relationships among circzfr mir578 and hif1a were confirmed by dualluciferase reporter and rna pulldown assays in dualluciferase assays the fragment of circzfr containing the mir578binding sites and hif1a ²utr were individually cloned into pmirglo vector promega madison wi usa to construct corresponding wildtype reporters circzfrwt and hif1a3²utrwt the takara mutanbest kit was used to construct the corresponding mutations circzfrmut and hif1a3²utrmut as per the insturctions of manufacturers takara dalian china each reporter construct a0ng and a0nm of mir578 mimic or mirnc mimic were cotransfected into bc cell lines cell line extracts were prepared with ripa buffer takara a0h posttransfection and the ratio of renilla to firefly luciferase was detected using the promega dualluciferase assay in rna pulldown assays cell lysates were incubated with the biotinlabeled mir578 mimic biomir578 ribobio or nontarget control sequence bionc ribobio for a0 h at a0 °c before adding to the streptavidin beads sigmaaldrich for a0 h the beads were collected and total rna was extracted for circzfr quantification each experiment was performed in triplicatewestern blot for a0hif1a expressionwestern blot was used to determine the expression of hif1a using standard protocols the preparation of cell line extracts was done using ripa buffer with proteinase inhibitors roche charente france proteins a0 μg were resolved on a sdspolyacrylamide gel electrophoretically blotted onto nitrocellulose membranes ge healthcare little chalfont uk and probed with antibody against hif1a ab51608 a0μgml abcam or gapdh ma515738 a0 μgml invitrogen following the incubation with horseradish peroxidasecoupled igg secondary antibody ab97051 a0 μgml abcam the signals were visualized by cheniluminescence ge healthcare as recommended by the manufacturers all experiments were done in triplicatestatistical analysisdata were shown as the mean ± standard deviation from at least three independent assays pairwise comparisons were done using a twotailed students t test mannwhitney u test or analysis of variance anova with spss version software spss chicago il usa for survival analysis the kaplanmeier survival curve and logrank test were used correlations among circzfr mir578 and hif1a expression levels in bc tissues were determined by the spearman correlation test all tests were considered statistically significant at pvalue resultsoverexpression of a0circzfr predicted poor prognosis of a0bc patientsas demonstrated by qrtpcr circzfr was significantly upregulated in bc tissues and cell lines compared with their counterparts fig a01a b to determine its clinical relevance we preliminarily examined the link between circzfr level and the prognosis of bc patients kaplanmeier survival curves showed that the patients in low circzfr level group had a longer survival time than those in high circzfr group fig a0 1c additionally circzfr expression was remarkably associated with the distant metastasis and tnm stage of these patients table a0silencing of a0circzfr hindered bc cell viability colony formation and a0enhanced apoptosis in a0vitro and a0weakened tumor growth in a0vivoto study the biological role of circzfr in bc progression the lossoffunction experiments were carried out using shrnas against circzfr shcirczfr1 shcirczfr2 and shcirczfr3 in contrast to the negative control the transfection of the three shrnas prominently reduced circzfr expression in both mcf7 and bt549 0cchen a0et a0al cancer cell int page of fig circzfr was overexpressed in bc and associated with poor prognosis circzfr expression by qrtpcr in pairs of tumor tissues and adjacent normal tissues a mcf10a mcf7 bt549 mdamb231 and mdamb453 cell lines b c analysis for the overall survival of bc patients in high n or low n circzfr level group divided by the median of circzfr expression in bc tissues using kaplanmeier survival analysis and logrank test p cell lines fig a02a b notably shcirczfr1 also named shcirczfr caused the most significant downregulation in circzfr expression so we used it for further analyses functional experiments data revealed that the silencing of circzfr led to a striking inhibition in cell viability fig a02c colony formation fig a02d e as well as a strong promotion in cell apoptosis fig a02f gto determine whether circzfr regulated bc tumor development in a0vivo we performed the xenograft model assays when we infected bt549 cell line with shcirczfr tumor growth was remarkably blocked compared with the shnc control fig a02h isilencing of a0circzfr suppressed bc cell migration invasion and a0glycolysiswe also asked whether circzfr regulated bc cell migration invasion and glycolysis in a0 vitro transwell assays showed that in comparison to the control group cell migration fig a0 3a and invasion fig a0 3b were significantly repressed by circzfr knockdown moreover in both cell lines circzfr silencing resulted in decreased glucose uptake fig a0 3c lactate product fig a0 3d and atp level fig a0 3e demonstrating the suppression of circzfr knockdown on cell glycolysiscirczfr directly interacted with a0mir by a0binding to a0mirto further understand the role of circzfr in bc pathogenesis we performed a detailed analysis for its targeted mirnas the two online algorithms circinteractome and circbank collectively revealed that circzfr harbored a putative complementary sequence for mir578 mir944 and mir5323p fig a04a the data of qrtpcr showed that the silencing of circzfr led to a striking overexpression in mir578 and mir5323p levels but mir expression was not affected by circzfr knockdown in the two bc cell lines fig a0 4b c previous work had reported that circzfr regulated colorectal cancer progression through acting as a sponge of mir5323p so we aimed to identify whether mir578 was a molecular mediator of circzfr in bc progression by contrast mir was prominently upregulated in the two bc cell lines transfected with mir578 mimic fig a04d we then cloned circzfr fragment containing the mir578binding sites into a luciferase plasmid and mutated the mir578binding sites fig a0 4e the elevated mir578 expression significantly reduced the activity of circzfr wildtype reporter circzfrwt fig a04f however the mutation of the target sites circzfrmut completely abrogated the effect of mir578 on reporter gene expression fig a04f indicating the validity of the target sequence for interaction additionally rna pulldown assays revealed that the enrichment level of circzfr was remarkably elevated by biomir578 in both cell lines fig a04goverexpression of a0mir restrained bc cell viability colony formation migration invasion and a0glycolysis and a0promoted apoptosis in a0vitroin bc tissues mir578 expression was significantly decreased compared to the normal control fig a05a and it was inversely correlated with circzfr level fig a0 5b moreover mir578 level was lower in bc cell lines than that of control fig a05c subsequently we analyzed the biological effect of mir578 on bc progression in contrast to the control group the increased expression of mir578 0cchen a0et a0al cancer cell int page of fig circzfr silencing suppressed bc progression in vitro and in vivo a b qrtpcr for circzfr expression in mcf7 and bt549 cell lines transduced with shnc shcirczfr1 shcirczfr2 or shcirczfr3 cck8 assay for cell viability c colony formation assay for cell colony formation d e flow cytometry for cell apoptosis f g in shncinfected or shcirczfrtransduced mcf7 and bt549 cell lines h i shncinfected or shcirczfrtransduced bt549 cell line was subcutaneously injected into the nude mice n per group followed by the measurement of tumor volume and weight and the capture of representative pictures p 0cchen a0et a0al cancer cell int page of fig circzfr silencing suppressed bc cell migration invasion and glycolysis transwell assay for cell migration and invasion a b corresponding assay kits for glucose uptake lactate product and atp level ce in shncinfected or shcirczfrtransduced mcf7 and bt549 cell lines p induced a distinct repression in cell viability fig a05d and colony formation fig a0 5e and a strong promotion in cell apoptosis fig a0 5f as well as a striking reduction in cell migration fig a05g invasion fig a05h and glycolysis fig a05iksilencing of a0circzfr regulated bc cell viability colony formation migration invasion glycolysis and a0apoptosis in a0vitro by a0upregulating mira crucial question was whether circzfr regulated bc progression by mir578 to address this we reduced mir578 expression in shcirczfrtransduced mcf7 and bt549 cell lines as expected in comparison to the negative control the reduced level of mir578 significantly reversed circzfr knockdowninduced antiviability fig a0 6a anticolony formation fig a0 6b proapoptosis fig a0 6c antimigration fig a0 6d antiinvasion fig a0 6e and antiglycolysis fig a06fhaca aga a was circzfr modulated hif1a expression via a0acting as a0a a0sponge of a0mirusing the software targetscan a predicted mir578binding sequence identified within the ²utr of hif1a fig a0 6a when we cloned the ²utr fragment containing the putative mir578binding sites downstream of a luciferase coding sequence the cotransfection of the luciferase reporter hif1a3²utrwt and mir578 mimic into the two bc cell lines produced lower luciferase activity than in cell lines cotransfected with the mirnc control fig a07b c however the mutation of the target sequence hif1a²utrmut prominently abolished the suppression of mir578 fig a07b c by contrast hif1a mrna and protein levels were significantly reduced by mir578 overexpression in the two bc cell lines fig a07d e these data together pointed that hif1a in bc cell lines was directly targeted and inhibited by mir578 0cchen a0et a0al cancer cell int page of fig circzfr directly interacted with mir578 by binding to mir578 a venn diagrams representing the putative mirnas that bind to circzfr identified by circinteractome and circbank online algorithms b c the levels of mir578 mir944 and mir5323p by qrtpcr in shncinfected or shcirczfrtransduced mcf7 and bt549 cell lines d qrtpcr for mir578 expression in the two bc cell lines transfected with mirnc mimic or mir578 mimic e schematic of the mir578binding sites within circzfr and the mutation of the seed sequence f relative luciferase activity in mcf7 and bt549 cell lines cotransfected with circzfrwt or circzfrmut and mirnc mimic or mir578 mimic g qrtpcr for circzfr level in cell lysates incubated with bionc or biomir578 and streptavidin beads p we then examine whether circzfr influenced hif1a expression in bc cell lines as expected in comparison to their counterparts hif1a expression was remarkably downregulated by circzfr silencing at both mrna and protein levels in the two bc cell lines and this effect was significantly abrogated by antimir578 introduction fig a0 7f g additionally qrtpcr data showed a striking upregulation of hif1a mrna level in bc tissues fig a0 7h furthermore in bc tissues hif1a mrna expression was positively correlated with circzfr expression and negatively correlated with mir578 level fig a07ihif1a was a0a a0functional target of a0mir in a0regulating bc cell viability colony formation migration invasion glycolysis and a0apoptosis in a0vitroto provide further insight into the link between mir and hif1a in bc progression we elevated hif1a expression using a recombinant overexpressing plasmid in mir578 mimictransfected bc cell lines as a result hif1a protein level was prominently increased by the overexpressing plasmid in the two cell lines fig a0 8a functional experiments results revealed that the upregulation of hif1a dramatically abolished mir578 overexpressionmediated antiviability fig a0 8b proapoptosis 0cchen a0et a0al cancer cell int page of fig mir578 overexpression hindered bc progression in vitro a qrtpcr for mir578 expression in pairs of tumor tissues and adjacent normal tissues b correlation between mir578 expression and circzfr level in bc tissues using the spearman test c mir578 expression by qrtpcr in mcf10a mcf7 bt549 mdamb231 and mdamb453 cell lines mcf7 and bt549 cell lines were transfected with mirnc mimic or mir578 mimic followed by the determination of cell viability by cck8 assay d colony formation using a standard colony formation assay e cell apoptosis by flow cytometry f cell migration g and invasion h by transwell assay glucose uptake lactate product and atp level using assay kits ik p fig a08c antimigration fig a08d antiinvasion fig a08e and antiglycolysis fig a08fhdiscussionto date the emerging links between circrnas and bc progression have opened up a novel field for cancer diagnosis and treatment [ ] in the meantime understanding the molecular basis underlying the actions of circrnas has been still challenging in this study we identified the biological role of circzfr in bc progression in a0 vitro and in a0 vivo and investigated the mechanisms governing ithere we firstly demonstrated that circzfr was overexpressed in bc and the elevated expression of circzfr was associated with the poor prognosis of these patients by using lossoffunction in a0vitro and in a0vivo analyses we were first to uncover that the silencing of circzfr performed a suppressive effect in bc progression previous reports had highlighted the potential oncogenic role of circzfr in several other malignancies such as hepatocellular carcinoma papillary thyroid cancer and nonsmall cell lung cancer [ ] conversely circzfr was reported as a tumor suppressor in colorectal cancer and gastric cancer [ ] these contradictory findings may attribute to different type tumor or complex tumor microenvironmentusing the online algorithms we first identified that circzfr sequestered mir578 through sponging mir in bc cell lines ji et a0al showed that the increased mir578 level weakened osteosarcoma progression via directly interacting with circ_001621 farhana et a0al unraveled that in pancreatic cancer mir578 was 0cchen a0et a0al cancer cell int page of fig circzfr knockdown repressed bc malignant progression by mir578 in vitro shncinfected or shcirczfrtransduced mcf7 and bt549 cell lines were transiently transfected with antinc or antimir578 a cck8 assay for cell viability b a standard colony formation assay for cell colony formation c flow cytometry for cell apoptosis d e transwell assay for cell migration and invasion fh glucose uptake lactate product and atp level using assay kits p associated with the tumor cell apoptosis as it has been reported our data validated the downregulation of mir578 expression in bc tissues and cells moreover we first identified that mir578 overexpression restrained bc cell malignant behaviors in a0 vitro more importantly for the first time we substantiated that circzfr knockdown hampered bc progression in a0vitro by mir578using targetscan software we identified that mir in bc cell lines directly targeted and inhibited hif1a hif1a a transcriptional regulator in response to intratumoral hypoxia [ ] contributes to bc metastasis and malignant progression [] we also uncovered that the upregulation of mir578 hampered bc malignant progression via downregulating hif1a in a0 vitro previous studies had reported that several 0cchen a0et a0al cancer cell int page of fig circzfr modulated hif1a expression via sponging mir578 a schematic of the putative mir578binding sequence and mutated the target sequence b c relative luciferase activity in mcf7 and bt549 cell lines cotransfected with hif1a3²utrwt or hif1a3²utrmut and mir578 mimic or mirnc mimic hif1a mrna and protein levels by qrtpcr and western blot in mcf7 and bt549 cell lines transfected with mir578 mimic or mirnc mimic d e shncinfected or shcirczfrtransduced mcf7 and bt549 cell lines transfected with antinc or antimir578 f g h relative hif1a mrna expression by qrtpcr in pairs of tumor tissues and adjacent normal tissues i correlation between hif1a expression with circzfr or mir578 level in bc tissues using spearman test p other mirnas such as mir18a and mir497 exerted an antitumor activity in bc through targeting hif1a [ ] furthermore our data first illuminated the role of circzfr as a sponge of mir578 to mediate hif1a expression in bc cell lines the findings by liang et a0al underscored that circrna circdennd4c contributed to bc cell proliferation under hypoxia via regulating hif1a in our present study demonstrated that circzfr was overexpressed in bc and the silencing of 0cchen a0et a0al cancer cell int page of fig the repression of mir578 upregulation on bc progression in vitro was mediated by hif1a a hif1a protein level by western blot in mcf7 and bt549 cell lines transfected with vector or hif1a mcf7 and bt549 cell lines were transfected with mirnc mimic vector mir578 mimic vector or mir578 mimic hif1a followed by the determination of cell viability by cck8 assay b cell apoptosis by flow cytometry c cell migration and invasion by transwell assay d e glucose uptake lactate product and atp level using assay kits fh vector negative control plasmid hif1a recombinant hif1a overexpressing plasmid p circzfr suppressed bc malignant progression via the regulation of the mir578hif1a axis this is the first report of circzfr in bc pathogenesis providing evidence for the crucial involvement of circzfr in bc progressionsupplementary informationsupplementary information accompanies this paper at https doi101186s1293 additional file a0 supplement material the str authentication of mcf7 a and bt549 b cellsadditional file a0 supplement material the detailed quantification of the western blot analysisabbreviationsbc breast cancer hif1a hypoxiainducible factor 1α cck8 cell counting kit8 atp adenosine triphosphate anova analysis of varianceacknowledgementsnoneauthors contributionszc and xq designed and performed the research fw yx nw and yg analyzed the data zc wrote the manuscript all authors read and approved the final manuscriptfundingno | Colon_Cancer |
" the influence of anastomotic leakage al on local recurrence rates and survival in rectal cancer remainscontroversial the aim of this study was to analyze the effect of asymptomatic anastomotic leakage aal andsymptomatic anastomotic leakage sal on short and longterm outcome after curative rectal cancer resectionmethods all patients who underwent surgical resection of nonmetastatic rectal cancer with curative intent fromjanuary to december were retrospectively analyzed shortterm morbidity longterm functional andoncological outcomes were compared between patients with sal aal and without al walresults overall patients were included and al was observed in patients aal and sal with amedian followup of months rectal cancer location and preoperative neoadjuvant treatment was similar betweenthe three groups postoperative 30day mortality rate was nil the permanent stoma rate was higher in patients withsal or aal compared to wal patients and vs p the mean wexner continence grading scalewas significantly different between aal ± sal ± and wal ± groups p the and year overall and diseasefree survival rates were similar between the groups vs vs and vs vs for patients with sal aal and wal p and p the permanent stoma rate was significant higher in patients with sal or aal compared to wal patients aldid not impair longterm oncological outcomekeywords rectal cancer surgery anastomotic leakage local recurrence low anterior resection score functionaloutcome longterm outcome the management of rectal cancer has substantially improved and standardized in the past twenty years preoperative chemoradiotherapy crt followed by totalmesorectal excision tme is now the gold standard for correspondence mouaissichutoursfr alice artus and nicolas tabchouri contributed equally to this work1department of digestive oncological endocrine hepatobiliary pancreaticand liver transplant surgery trousseau hospital chambray les toursavenue de la rpublique chambray les tours francefull list of author information is available at the end of the locally advanced rectal carcinoma [] indeed tmehas led to decreased local recurrence rates and increasedcancerspecific survival rates more recently technical advances include also an intersphincteric approachfor resection [ ] which combined with a neoadjuvanttreatment aims to reduce the permanent stoma ratein patients undergoing low rectal cancer surgery despitenumerousimprovements anastomotic leakage alremains the most dreaded complication following rectalresection []its occurrence is associated with 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 0cartus bmc cancer page of impaired postoperative functional results anal incontinence low anterior resection syndrome and ultimately an increased rate of permanent stomata reachingnearly [] furthermore al is also associatedwith impaired oncological results with increased localandor distal recurrence rates as well as decreased overall survival rates [] however it is important todiscriminate symptomatic from asymptomatic al sincethese two conditions do not seem to have the sameoncological and functional postoperative outcome although several previous studies did not find a difference between without al and asymptomatic anastomotic leakage aal with regard to early postoperativefunctional and oncological outcome these studies didnot analyze and report longterm data from patientswith aal furthermore these previous studies analyzedheterogeneous patient populations including also patients suffering from distant metastasis [ ] it isobvious that the inclusion of such patients has a negativeimpact on anastomotic healing rates increases the riskfor a permanent stoma and reduced recurrencefree andoverall survival rates respectively therefore theobjective of our study is to report and analyse longtermoncological and functional outcome in a homogeneouspopulation of patients with aal and sal who hadundergone curative surgical resection for nondistantmetastatic rectal cancer in a tertiary referral centremethodsstudy populationretrospective study at a single tertiary referral center ofpatients who underwent curative intended surgery foradenocarcinoma of the rectum upper rectum to cm mid to cmlow to cm from theanal verge between january to december patients suffering from distant metastatic disease orlocal tumor extension requiring resection of adjacentans genitourinary or vascular structures were excluded further exclusion criterion was underlying inflammatory bowel disease familial adenomatous polyposis orpelvic recurrence after primary surgery data were collected from medical records for each patient and includeddemographic parameters primary tumor characteristicsneoadjuvant treatment intraoperative and pathology variables as well as shortterm and longterm oncological andfunctional outcomesneoadjuvant treatmentrectal cancer staging included digital rectal examinationrectosigmoidoscopy total colonoscopy endorectal ultrasound andor pelvic resonance imaging mri all patientshad thoracoabdominal computed tomography ctscanthe upper l5s1 vertebrae and the lower levator anianal verge tumor limit was assessed by mri andorendorectal ultrasound respectively neoadjuvant therapyconsisted of either longcourse treatment or shortcourseradiotherapy in case of combined radiochemotherapycrt patients received gray gy radiotherapy givenin fractions over five weeks with concomitant chemotherapy using capecitabine xeloda® surgery was scheduled eight to weeks after the end of combined radiochemotherapy shortcourse radiotherapy included gy delivered in five fractions over a time span of five toseven days followed by curative surgery one weeklater the inferior border ofsurgical proceduresall patients had preoperative mechanical bowel preparation laparoscopic tumor resection was the standardapproach however patients with t4 tumors underwentprimary open tumor resection a medialtolateral approach was the technique of choice the inferior mesenteric vein was ligated atthepancreas followed by the mobilization of the left colonthe splenic flexure the extend of mobilization was left atdiscretion of the surgeon and ligation of the inferior mesenteric artery if possible the left colic artery was preserved the rectum was transected by the use of linerstapler in double stapling technique the proximal colonwas transected approximately cm above the upperborder of the tumor the specimen was retrieved from theabdominal cavity via a small abdominalincision mechanical colorectal or manual coloanal anastomosessidetoend or endtoend were performed depending ontumor level patients with rectal cancer of the upper rectum underwent partial mesorectal excision with a distal cm margin from the lower border of the tumor all otherpatients underwent standard tme in case of very lowrectal cancer a total or partial intersphincteric resectionwas performed when ever feasible a closed suctiondrainage was placed in the small pelvis for to h postoperatively a protective stoma ileostomy was performedroutinely in selected patients with upper rectal cancerwith no neoadjuvant radiochemotherapy and the intraoperative conditions were assessed by the surgeon asfavorable no deviation ileostomy was placedshortterm outcomesany postoperative clinical sepsis peritonitis emissionof gas pus or feces from the pelvic drain purulent discharge per anus or rectovaginal fistula andor biologicalsuspicion increased crp and white blood cells count ofal led to an earlier ctscan assessment in case ofasymptomatic al antibiotic treatment was given to all patients if required ctscan guided transanal drainageplacement or even redosurgery was performed after hospital discharge all patients underwent structured outpatient followup including physical examination routine 0cartus bmc cancer page of blood tests including crp and a ctscan with watersoluble contrast enema within three months postoperatively anastomotic leakage was defined and gradedaccording to the international study group of rectalcancer patients were divided in three groups as follows symptomatic al sal including grade b and cals asymptomatic radiologic al aal diagnosed onenemacontrast ctscan routinely performed to weeks postoperatively before considering stoma reversaland without al wal ileostomy closure was scheduledsix to eight weeks postoperatively if a control ctscanwith water soluble contrast enema showed no al in patients with aal stoma reversal was delayed until ctscanning results showed no signs for al or after a timeinterval of at least months following rectal resection asdescribed recently shortterm 30day postoperativecomplications were graded according to the dindoclavien classification inhospital stay was measuredfrom the day of surgery including the day of hospitaldischargepathological resultssurgical specimens were analyzed using a standardizedprotocol tumors were staged using the tnm classification according to the 8th edition of american jointcommittee of cancer ajcc circumferential and distalmargins were defined as positive r when mm or lessprimary tumor nodes or tumor deposit within themesorectum and as negative r0 when greater than mm total or partial mesorectal excision colloid component degree differentiation grade as well as presence ofvascular lymphatic or peri nervous emboli were statedin pathology reportlongterm outcomeslongterm functional and oncological outcomes were recorded in all patients patient followup was performedevery months for the first postoperative years every months for the next years and then annually thereafter followup was updated until december andconsisted of clinical examination ctscan and bloodtests with colonoscopy performed one and then every years after surgery local recurrence was defined astumor recurrence at the anastomotic site or in the pelviccavity while distant recurrence was defined as tumor recurrence beyond the locoregional area including liverlung andor other extra pelvic sites followup data wasobtained from medical records outpatient clinic orphone interview longterm functional genitourinaryand digestive outcomes as well as quality of life assessments were recorded using a french translation of thelow anterior resection score the wexner continencegrading scale the iief5 erectile dysfunction score the sf36 health survey a french validatedversion of the european anization for research andtreatment of quality of life questionnaire for colorectal cancer eortc qlqcr30 the euroqolnon diseasespecific instrument for evaluation healthrelated quality of life and the fecal incontinencequalityoflife fiql scale all previously mentioned surveys and questionnaires were collected in outpatient clinic or by phone interview whenever possiblefor all functional scales a greater score represented amore impaired quality of life and for all symptom scalesa greater score was associated with a greater severity ofsymptoms furthermore surgeryrelated longterm complications such as small bowel occlusion anastomosisstenosis and incisional hernias were also recordedpatients who were unable to undergo protective stomareversal and those who required secondary stoma placement colostomy or ileostomy after protective stoma reversal were also identified pathological response toneoadjuvant chemotherapy was considered partial if anytumor downstaging was noted on specimen total if noresidual tumor was found and absent if no downstagingwas notedstatistical analysisbaseline and demographic characteristics of the studiedpopulation intraoperative and pathological characteristicsas well as short and longterm postoperative outcomeswere analyzed patients who presented with sal aal andwithout al wal were compared categorical variableswere compared using the Ï2 test with bonferroni correctionwhenever necessary the kaplanmeier method was usedto estimate recurrencefree survival rfs and overall survival os which were compared using the logrank testall statistical analyses were performed using spss version spss inc chicago il and statistical significance wasaccepted at the level continuous variables were compared using anova or nonparametric anova tests accordingly significant prognostic factors associated withpermanent stoma were identified by univariable logistic regression and included in a multivariable analysis to determine independent risk factors this study was conductedaccording to the ethical standards of the committee onhuman experimentation of our institution and reported according to the strengthening the reporting of observational studies in epidemiology strobe guidelines resultspatients characteristicsduring the study period patients underwent surgeryfor rectal cancer in our department among these patients presented with synchronous metastasisieliver pulmonary peritoneal andor other ansand underwent abdominoperineal resectionandor combined an resection and were therefore 0cartus bmc cancer page of excluded a total of patients who underwentsphincterpreserving resection with curative intent forupper patients mid patients and low patients rectal adenocarcinomawere included patients baseline characteristics are reported in table overall there were menwith a median age of iqr years patients comorbidities were comparable between the three groupsasa score p neoadjuvant therapy was administered in patients of whom underwent longcourse radiation therapy and shortcourse radiation therapy with no differencesfound between the sal aal and wal group p overall patients underwent laparoscopic procedure of whom required conversion with no differences noted between the three groupsp vs p total mesorectal excision wasperformed in patients with no differencesfound between the three groups p in patientssufering from low rectal adenocarcinoma patientsunderwent intersphinteric resection one total and partial and patients had a manual coloanal anastomosis with no differences found between the threegroups p vs p a diverting stoma wasplaced in patients with no differences foundbetween the three groups p detailed intraoperative variables are reported in table shortterm postoperative outcomespostoperative al occurred in patients ofwhom had sal and aal stomadetails in the different analysed groups are summarizedin fig postoperative mortality rate in the total studypopulation was nil in the sal group sepsis was themain symptom in patients additinonally infour other sal patients ileus n and analpus discharge n were the only clinical symptomsobserved symptomatic al was diagnosed after a medianof days postoperatively redo abdominalsurgery was performed after a median of iqr days in patients anastomosis resectionand colostomy placement n and peritoneal lavagetable demographic and perioperative characteristicsn overall population symptomatic al sal age years median ± iqr¥ years n years n sex ratio femalemalebmi kgm2 median ± iqrasa score n arteriopathy n diabetes n tumor diagnosis n screening testsymptoms rectal adenocarcinoma location n upper cmmid cmlow cm neoadjuvant radiation therapy n asymptomatic al aal without al wal plongcourse radiotherapywith chemotherapyshortcourse radiotherapywithout chemotherapynone al anastomotic leak sal symptomatic al aal asymptomatic al and wal without al asa american society of anesthesiologists bmi body mass index iqrinterquartile range 0cartus bmc cancer page of table operative detailsn laparoscopic n conversion to laparotomycomplete tme n pme n rectal anastomosis technique n latero terminaltermino terminal colo anal manual anastomosis n intersphincteric resection isr n total isrpartial isr overall population symptomatic al sal asymptomatic al aal without al al pdiverting stoma n al anastomotic leak sal symptomatic al aal asymptomatic al and wal without al tme total mesorectal excision pme partial mesorectal excision with anastomosis repair and drainage n three patients suffered from one or more an dysfunctions afterredo surgery and had therefore to be admitted to theintensive care unit transanal drainage was performed in patients under general anesthesia overall patients were managed with antibiotics only apermanent stoma was required in patients colostomy n ileostomy n the diagnosis of aal was made by ctscan after amedian of iqr days postoperatively three patients had stoma reversal before aal wasdiagnosed systematic ctscan before reversal did notfig short and longterm postoperative outcome of all patients who underwent sphincterpreserving rectal cancer surgery symptomaticanastomosis leakage sal acute management as well as asymptomatic anastomotic leakage aal management is shown below one patientwas diagnosed with aal although presenting with pelvic collection on ctscan and underwent ctguided drainage 0cartus bmc cancer page of show signs of al seven patients had aal diagnosed before reversal ctscan drainage was required inone patient while the other patients were treated conservatively with antibiotics only overall nine patientsunderwent stoma reversal of which required permanent colostomy placement due to persistent fistula associated with unsatisfactory functional results a total ofthree patients finally required a permanent stomathere were patients who presented withsevere postoperative complications in sal group withnone in the aal group and patients withoutal p severe postoperative complications inpatients without al occurred in patients which had finally to undergo redo surgery for evisceration and smallbowel ileus both on postoperative day sevena total of patients were without al walgroup among wal patients patients required permanent stoma placement colostomy due topelvic local tumor recurrence n colostomy for perforation after dilatation of a anastomotic stenosis n and ileostomy related to severe anastomosis stenosisn postoperative details are summarized in table and in fig a primary diverting stoma reversal was perfomed in patients without al patients withaal and patients with sal respectively p the time intervall from primary surgery to stomaclosure was similar in patients with sal iqr and patients with aal iqr however this time span was significantly longercompared to patients without al iqr days p overall complete and partial pathological responses were recorded in and patients with no differences observed betweenthe three groups p resection margins wereconsidered incomplete in patients with again nodifferences between the three groups p in patients without al three patients underwent colostomyplacement after local pelvic tumor recurrence whereasone patient required colostomy due to a postinterventinal colonic perforation after dilatation of the stenoticanastomosis furthermore another eleven other patientsrequired an ileostomy for severe anastomotic stenosislongterm oncological outcomesduring the postoperative followup after a median timeinterval of months iqr after curative surgery local and distal recurrences occurred in and patients resepectively no differenceswere found between the three groups p oncological results are detailed in table there were no patients in the aal group who presented with local ordistal recurrence diseasefree survival comparison wastherefore performed between the sal and wal groupno significant difference was observed the 3yeardiseasefree survival rate was for patients withsal for those with aal and for those without al p at years the diseasefree survivalrate was similar in the three groups and respectively p overall patients underwent postoperative chemotherapy with no differencebetween the three groups p median followupwas months and longterm mortalityrate was n with no difference between thethree groups p and p respectivelyeight percent of patients died during followup fromcancer progression n and nine patients from other causes the 3year overall survival rate fortable early postoperative outcomen emergency surgery n laparoscopic abdominal drainage n open abdominal drainage n hartmanns procedure n ileostomy repair n transanal drainage n ctguided drainage n antibiotics treatment only n hospital stay days median ± iqrinitial diverting stoma reversal n yesnooverall population symptomatic al sal asymptomatic al aal without al wal p days before stoma reversal days median ± iqral anastomotic leak sal symptomatic al aal asymptomatic al and wal without al iqr interquartile range 0cartus bmc cancer page of overall population symptomatic al sal asymptomatic al aal without al wal p table pathology resultsn t stage n t0t1t2t3t4n stage n nn0pathological response n absentpartialcompletelymph nodes median ± iqrtumor diameter mm median ± iqrresection margins n incomplete rcomplete r0 al anastomotic leak sal symptomatic al aal asymptomatic al and wal without al iqr interquartile range patients in the sal group was in the aalgroup and in the wal group resepectively p similarly at 5years overall survival did not differbetween the three groups and p details about the overall and diseasefree survival ratesare presented in fig 2abstoma placementlongterm functional resultsthere were patients requiring permanentstoma placement n wal group n salgroup and n in aal group p in thewal group three patients underwent colostomy placement after local tumoral recurrence one patient had colostomy for perforation after stenosis dilatation andeleven patients had ileostomies related to severe anastomosis stenosis in patients with sal patients had permanentredosurgery patients required colostomy placement aftersoma reversal for septic complication and patients required secondary colostomy placement after soma reversal for poor functional results in patients with aal onepatient required a permanent stoma due to medical comorbidities cardiac and pulmonary insufficiency andtwo other patients required a secondary colostomyplacement after stoma reversal with poor functional results furthermore analysis over time revealed a significantly different permament stoma rate between thethree groups p as shown in fig longtermafter postoperativefunctional results of patients are shown in table excluding deceased patients n and patients withpermanent stoma placement n this represented aresponse rate of for functional assessment scoresonly the wexner continence grading scale was significantly higher in patients who presented with al p wexner continence grading scale was also significantly different between the aal sal and wal groupp permanent stoma rate was higher in patientswith sal compared to wal patients vs p and similar compared to the aal group p data are summarized in fig overall patients with al n had a permanent stoma rate of compared to in the wal group n p multivariate analysis showed that r1 resectionand sal were the only independent factors which werepredictive for a permanent stoma or p and or p data are presented in table discussionin this retrospective single center study we conducted acomparative analysis of asymptomatic and symptomatical in patients who underwent rectal resection withcurative intent both shortterm and longterm morbidity functional and oncological outcomes in this homogeneous population were analyzed patients suffering fromsal were found not to be at risk for an increased 0cartus bmc cancer page of ano at risksymptomatic leakageasymptomatic leakageno leakagebno at risksymptomaticleakageasymptomaticleakageno leakagewalsalaalwalsalaalp0527p0480fig a symptomatic anastomotic leak sal group solid line and asymptomatic al aal group point line without al wal group dashlines overall survivals in sal aal and without al anastomotic leak al groups symptomatic al sal asymptomatic al aal and without alwal x axis months y axis percentage survival b symptomatic anastomotic leak sal group solid line and asymptomatic al aal grouppoint line without al wal group dash lines disease free survival in sal aal and without al anastomotic leak al groups symptomatic alsal asymptomatic al aal and without al wal x axis month y axis percentage survivalnumber of local andor distant tumor recurrence ratescompared to the group of aal and wal patients however in the group of patients suffering from postoperative anastomotic leakage aal and sal we observed animpaired longterm outcome of postoperative functionalresults furthermore there is an increased risk for a permanent stoma in aal and sal patients which is as highas and compated to patients with no postoperativeanastomotic leakage is significantly higherthe effect of postoperative anastomotic leakage alon the oncological outcome following rectal cancersurgery is still debated controversially and remainsunclear some authors have reported about reducedlocal recurrence rates whereas distant recurrence ratesand overall survival rates were found to be similar incontrast equivalentlocal distant disease recurrencerates and overall survival rates were observed by others[ ] furthermore two metaanalysis reportedthat al was associated with higher local disease recurrence rates and reduced longterm cancer specific survival no impact was found on the incidence of distantdisease recurrence rates [ ] moreover five pooled 0cartus bmc cancer page of fig permanent stoma rate determined as a time to event analysis in sal aal and without al anastomotic leak al groups symptomatic alsal asymptomatic al aal and without al wal x axis month y axis cumulative percentage of patients with permanent stomarandomized trials published in concluded thatthe diseasefree survival was not affected by the postoperative appearance of symptomatic al however symptomatic al was observed to be associated with animpaired overall survival in such patients such discrepancies between the reported results in the aforementioned studies might be be explained by considering twoimportant facts about postoperative al in more detailfirst in most previously published studies any clinicalimpact of postoperative aal was not assessed since nodiscrimination between aal and sal was made so faronly a few studies analyzed the subgroup of aal andobserved that the short as well as the and longtermoutcome differed from the sal and wal group respectively second the diagnosic workup definition of aal andalso the discrimination from patients without al are stillnot clearly defined and are controversially discussedtherefore it is obvious that the rate of patients with diagnosed postoperative al varies considerably betweenthe different studies with a reported prevalence which issomewhere in the range between [] to []in this present study we observed an incidence of alof which is indeed somewhat higher compared to other studies [] however our findingscould be explained by the fact that all patients in ourstudy were subjected to al screening which in accordance with the recommendations of the working group ofpanis included routine blood testing and water soluble enema contrast ctscan six weeks postoperativelyand before stoma reversal []in contrast to the study published by hain we observed no significant difference of the overall anddiseasefree survival between the aal sal and walgroup respectively indeed we are aware that an important limitation of our study besides that it is retrospectiveis the limited number of included patients whichhampers statistical data interpretation nevertheless thehomogeneity of our study population is an importantquality criterion compared to other studies which alsoincluded patients suffering from distant metastatic disease [ ] including such patients suffering fromdistant metastases carries a relevant risk to bias studydata since such patients suffer from impaired postoperative anastomotic healing with allthe correspondingknown negative sequelae finally the managementof rectal cancer has substantially improved in the lastdecade ie rectal surgery neoadjuvant treatment diagnosis and treatment of al therefore data from previous studies to [] are not comparableto present data since they do not reflect current standards for diagnosis and treatment of rectal cancerhence the interpretation of our current data in the context of such previous studies is probably oflimitedrelevancealthough we did not find a negative impact of saland aal on the oncological outcome in our present 0cartus bmc cancer page of table late postoperative outcomen oncological resultsoverall population symptomatic al sal asymptomatic al aal without al wal padjuvant chemotherapy n recurrence n localdistancetotal patients with recurrencetime to recurrence month median ± iqrmedian of follow up months ± iqroverall survival at yearsoverall survival at yearsdisease free survival at yearsdisease free survival at yearsfunctional resultspermanent stoma n ileostomycolostomyother late complications n stenosisherniaocclusionmean of quality of life assessmentslars scorewexner scoreiief5sf36eortc qlqc30euroqolfiqleortc qlqmy20 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± al anastomotic leak sal symptomatic al aal asymptomatic al and wal without al iqr interquartile rangetable multivariate analysis of factors associated withpermanent stomaodds ratioci ageasatmer1fistula occurrenceasymptomatic fistulasymptomatic fistulap study we observed an impaired short and longtermfunctional outcome of aal compared to patients without al wal this is again in contrast to a recentlypublished study by hain which found no differencebetween the lars score in the aal sal and walgroup respectively however the authors of this studydid not discriminate between wal and aal in theirmultivariate analysis of predictive factors of the postoperative functional outcome although it is obviousthat al has a negative impact on the postoperative functional outcome [] it remains still unclear for thesubgroup of patients with aal similar findings as ourswere found by lim who reported that the bowelfunction after ileostomy closure was equally impaired inthe aal and sal group respectively 0cartus bmc cancer page of in our study the postoperative time interval to stomareversal was similar in ssl and aal but significantlylonger compared to wal patients these results areconsistent with findings in other previous studies furthermore we observed thatthe permanentstoma rates were quite similar in the aal andsal group but significantly higher compared tothe wal group some other authors have reported that all aal patients can be managed conservatively with spontaneous healing whereas sal patientsshowed only a percent rate of spontaneous healing an important finding in our study is the fact thateven if aal patients can be managed conservatively thefunctional postoperative outcome can be impairedour multivariate analysis revealed that sal and r1rectal tumor resection were the only independent riskfactors for a permanent stoma for the presence of aalonly a trend towards a higher stoma rate was foundwe are aware about some limitations of our studysmall number retrospective nonetheless our study i | Colon_Cancer |
we report the case of a 24yearold man who presented with chief complaintsof shortness of breath and haemoptysis chest radiography revealed completecollapse of the left lung bronchoscopy revealed an endobronchial tumourwith complete obstruction of the left main bronchus cryosurgical excisionwas performed tissue pathology conï¬rmed the diagnosis of metastatic embryonal carcinoma the patient underwent a right orchiectomy followed by ableomycin etoposide cisplatin bep chemotherapy regimenkeywordscryosurgery embryonal carcinoma endobronchialmetastases endobronchial tumourcorrespondencewenchien cheng division of pulmonary and critical care medicine department of internal medicinechina medical university hospital no yuderoad north dis taichung city taiwanemail wcchengdrgmailcomreceived july revised july accepted july associate editor james horespirology case reports e00644 101002rcr2644introductionlung metastases from extrapulmonary malignancies areobserved frequently in clinical practice by contrastendobronchial metastases ebms from extrapulmonarymalignancies are rare and may have distinct histopathological etiologies [] primary lung cancer is the most common cause of endobronchialtumours extrapulmonarymalignancies that are frequently associated with metastasesto the central airways include tumours of breast colorectalthyroid origin [] although mediastinalrenal orlymphadenopathy is frequently observed in associationwith testicular seminoma ebms from embryonal carcinomas are extremely rare in this report we present acase of ebm from a primary embryonal carcinomacase reporta 24yearold man with no relevant past medical historypresented to our hospital with a chief complaint of shortness of breath lasting for several days upon questioningthecoughexperiencedrevealedpatientthatheanendobronchialrevealedleft main bronchushaemoptysis shortness of breath and occasional chest painfor the past several days he reported no fever chills coldsweats weight loss or decreased appetite a chest radiograph at admission revealed complete collapse of the leftlung fig 1a computed tomography ct was notable forleft pleural masses an endobronchial tumour obstructingthe left main bronchus and complete collapse of the leftlung and a soft tissue mass lesion in the right scrotumbronchoscopytumourobstructing thefig 1b theendobronchialtumour was excised with bronchoscopiccryosurgery a followup chest radiograph revealed someimprovement in the status of the left lung immunohistochemical staining of the tumour tissue revealed that the cellswere thyroid transcription factor1 ttf1negative sallike protein sall4positive and cluster of differentiation cd30positive these ï¬ndings were consistentwith a ï¬nal diagnosis of metastatic embryonal carcinomafig we checked the levels of tumour markers in thepatient including those of alphafetoprotein afp betahuman chorionic gonadotropin bhcg and lactate dehydrogenase ldh each tumour marker was found to be the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirologythis is an open access under the terms of the creative commons attributionnoncommercialnoderivs license which permits use and distribution in any mediumprovided the original work is properly cited the use is noncommercial and no modiï¬cations or adaptations are made vol iss e00644page 0cebm from embryonal carcinomack teng figure chestradiograph and bronchoscopic view of the endobronchial metastasesebm a complete collapse of the left lungon chest radiograph b bronchoscopic viewof the endobronchial tumour within the leftmain bronchusfigure tumour pathology of metastatic embryonal carcinoma a embryonal carcinoma with a complex glandular growth pattern the characteristic large cohesive and highly pleomorphic tumour cells with moderate amounts of amphophilic cytoplasm overlapping nuclei vesicular chromatinand frequent mitoses are shown as indicated by the arrows original magniï¬cation b immunohistochemical staining with antithyroid transcription factor1 ttf1 highlighting cells in the alveolar space original magniï¬cation c immunohistochemical staining with antisallikeprotein sall4 revealed diffuse nuclear staining original magniï¬cation d immunohistochemical staining with anticluster of differentiation cd30 highlighting diffuse membranous staining original magniï¬cation presentin high levels afp ngml bhcg miuml and ldh iul the patientunderwent a right orchiectomy followed by a bepbleomycin etoposide cisplatin chemotherapy regimendiscussionwe report here the case of a young man with an ebmfrom a primary embryonal carcinoma who presentedshortness of breath and haemoptysis chest radiography atpresentation revealed complete collapse of the left lungtumourslikewise manykindslung metastases from extrapulmonary malignancies arereported relatively frequently in clinical practice howeverebms from extrapulmonary malignancies are rare [] primary lung cancer is the most common cause ofendobronchialofextrapulmonary primary tumours have been associatedwith ebm primarily breast colon and renal carcinomas[] ebms from testicular seminomas are also extremelyrare the majority of testicular tumours arise from testicular germ cells and are frequently composed of multiple celltypesaretumours most ofie mixedtypethese the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirology 0cck teng ebm from embryonal carcinomatable reports of previous cases of ebmslocationdiagnostic methodpathology¶zt¼rk moreirameyer case case the oriï¬ce of right upper loberight main bronchusleft main bronchus main carina andright main bronchus fibreoptic bronchoscopy mixed gctfibreoptic bronchoscopy mixed gctvideobronchoscopyembryonal carcinoma¶zsu the oriï¬ce of the right upper lobefibreoptic bronchoscopytesticular seminomaturan varkey our caseand right intermediary loberight intermediate bronchusleft main bronchusleft main bronchusebm endobronchial metastases gct germ cell tumourembryonic carcinomas or seminomas there are only a few published reports of primary testicularembryonic carcinomas resulting in ebms []the mostofcommon symptomsendobronchialtumours are haemoptysis and coughing with shortness ofbreath and wheezing reported less frequently howeversome patients are asymptomatic in our patient symptoms on presentation included haemoptysis and shortnessof breathresults from chest radiography in patients with ebmcan be quite variable and may include mediastinal lymphadenopathy hilar masses atelectasis and multiple pulmonary nodules chest radiographs may also be normal onpresentation our patient presented with complete collapse of the left lung that was revealed initially by chestradiographyhowever the full diagnosis cannot be made based onlyon symptoms and chest radiographs it can be difï¬cult todistinguish between primary lung cancer and tumours ofextrapulmonary origin based on these ï¬ndings alone toconï¬rm the diagnosis we obtained a bronchoscopic biopsyspecimen to examine the tumour tissue the ï¬exible bronchoscopy ï¬breoptic bronchoscopy can be performedunder sedation without general anaesthesia as comparedwith rigid bronchoscopy the former is a simple techniquewhich is well tolerated and most commonly performed asan outpatient day procedure the patient underwentbronchoscopic cryosurgery under sedation in our bronchoscopy room to excise the tumour the ï¬nal pathology reportconï¬rmed the diagnosis of metastatic embryonal carcinomawe had evaluated the presence of afp bhcg andldh tumour markers elevated afp levels can be secretedby germ cell tumours gcts including embryonal carcinoma yolk sac tumour or teratoma in gcts detectablerigid bronchoscopybronchoscopyfibreoptic bronchoscopyand cryosurgerysomatictype gctembryonal carcinomaembryonal carcinomabhcg elevation is observed in both seminomas and nonseminomas the serum level of ldh was directly correlated with tumour burden in nonseminomatous gctswhich is also useful for the surveillance of patients withadvanced seminoma the tumour markers in ourpatient showed elevated levels of afp bhcg and ldhthis was compatible with the diagnosis of embryonal carcinoma moreirameyer also evaluated the patienttumour markers and found elevated levels of afp ngml and bhcg miuml the elevatedlevels of both tumour markers contribute to the diagnosisof metastatic embryonal carcinoma ¶zsu onlyevaluated the patients bhcg level which was found to beelevated miuml and the ï¬nal diagnosis wasmetastatic testicular seminoma on comparison with previous case reports table ours was the ï¬rst case in which the tissue was obtainedusing cryosurgery other reports obtained tissue samplesusing forceps biopsy alone or forceps biopsy combinedwith argon plasma coagulation apc to control bleedingcryobiopsy provided us with enough sample to performmore extensive immunohistochemical staining cryobiopsyhas more successful diagnostic results than forceps biopsiesdue to larger and highquality artefactfree sampleshaemorrhage was observed to be similar during both procedures further study on this topic will be needed toevaluate which of the diagnostic methods result in superioroutcomesin conclusion ebms from primary gcts notably thoseassociated with total or partial collapse are extremely rarewe have presented this case to emphasize the importanceof distinguishing ebm from primary lung carcinoma andto report the ï¬rst case in which metastatic embryonalcarcinoma was diagnosed by bronchoscopic cryosurgery the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirology 0cebm from embryonal carcinomadisclosure statementappropriate written informed consent was obtained forpublication ofand accompanyingimagesreportcasethisreferences ¶zt¼rk a aktas¸ z and yılmaz a endobronchialmetastasis of mixed germ cell tumors two cases tuberktoraks lee sh jung jy kim dh endobronchialmetastases from extrathoracic malignancy yonsei med j ikemura k lin dm martyn cp endobronchialmetastasis from extrapulmonary neoplasms analysis ofclinicopathologic features and cytological evaluation bybronchial brushing lung moreirameyer a bautistaherrera d hern¡ndezembryonal endobronchialgonz¡lez mck teng carcinomaj bronchologyinterv pulmonol ¶zsu s erol mm oztuna f endobronchial metastasis from testicular seminoma med princ pract tumoraltesticular germ cell turan d akif ¶zg¼l m kirkil gendobronchial metastasis ofeurasian j pulmonol et varkey b and heckman mg diagnosis of a case ofembryonal carcinoma by bronchial biopsy chest paradis tj dixon j and tieu bh the role of bronchoscopy in the diagnosis of airway disease j thorac dis aktas z gunay e hoca nt endobronchialcryobiopsy or forceps biopsy for lung cancer diagnosisann thorac med barlow lj badalato gm and mckiernan jm serumtumor markers in the evaluation of male germ cell tumorsnat rev urol the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirology 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 |
the anization of cells into multiple membranous compartments with speciï¬c biochemicalfunctions requires complex intracellular traï¬c and sorting of lipids and proteins to transport themfrom their sites of synthesis to their functional destination intracellular transport involves lipidvesicles or tubules with the capacity to fuse with one another or to be secreted they collectivelyparticipate in the dynamic exchanges necessary for cell homeostasis rothman s¸reng membrane traï¬c is tightly coordinated with protein synthesis signal transduction ofenvironmental stimuli and cytoskeleton anization allowing the implementation of key cellularfunctions such as endocytosis exocytosis or migration mcmahon and gallop habtezion vegacabrera and pardolpez macgillavry and hoogenraad margaria tapia buratta stalder and gershlick several families of molecular components required for orchestrating membrane vesicle exchangeand transport during this process are conserved they include adaptor and coat proteins smallgtpbinding proteins gtpases as well as synaptosome associated protein snap receptoredited byroberto botelhoryerson university canadareviewed bydaniel g s capellutovirginia tech united statesbrian paul ceresauniversity of louisville united statescorrespondencermy charcharcimlunivmrsfrphilippe pierrepierrecimlunivmrsfrspecialty sectionthis was submitted tomembrane trafï¬ca section of the frontiers in cell and developmentalbiologyreceived june accepted july published august citationchar r and pierre p therufys a family of effector proteinsinvolved in intracellular trafï¬ckingand cytoskeleton dynamicsfront cell dev biol 103389fcell202000779frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchar and pierrerufys proteins and trafï¬ckingsnare proteins and snare binding proteins juliano the vast superfamily of gtpases is involved in the establishmentor regulation of virtually every step of intracellular membranetraï¬cking they behave as molecular switches that can alternatebetween active and inactive states through gtp binding andhydrolysis into gdp takai stenmark thelargest group of gtpases involved in intracellular membranetraï¬c is the rab proteins family lamb rab gtpasesspeciï¬cally localize to diï¬erentintracellular compartmentsregulating vesicle formation and sorting as well as transportalong the cytoskeletal network each rab protein can be recruitedto speciï¬c membrane subdomains of a deï¬ned anelle andis associated to multiple eï¬ectors controlling membrane fusionand traï¬cking rab interaction with the membrane fusioncomplexes and cytoskeleton regulators is therefore crucial forcellular functions including endocytosis and autophagy chenand wandingerness bruce geng thomas and fromme yuan and song here we review the literature concerning a lesswell knownfamily of proteins involved in the complex biochemical crosstalkestablished between the cytoskeleton and intracellular vesiclesthis small group of proteins was named rufy for run andfyve domaincontaining rufys share a common structuraldomain anization including an nterminal run domainone or several coiledcoil cc repeats and a cterminal fyvedomain figure 1a the molecular structures of the diï¬erentrufy proteins has been described dunkelberg and gutierrezhartmann mari kukimotoniino kitagishi and matsuda but their function in endocyticregulation and their physiological relevance at the anismallevel are still poorly characterized kitagishi and matsuda terawaki we revisit here how the rufy genefamily was annotated and propose the addition of a novelmember the fyco1 fyve and coiledcoil containing domain gene given its sequence and functional similarities withthe other rufy genes pankiv terawaki we also highlight recent ï¬ndings on the implication ofrufy proteins in the regulation of cytoskeleton and endosomedynamics and their contribution to immunity cancer andneurodegenerative diseasesendocytosis and autophagyendocytosis and autophagy are membrane traï¬c pathwaysrequired for degradation and recycling ofextracellularand intracellular components respectively birgisdottir andjohansen these pathways have a common endpoint at thelysosome where their cargo is degraded these both pathwaysintersect at several stages throughout vesicle formation transportand fusion and share some of the components of their molecularmachineries figure 1bthere are numerous coexisting endocytic pathways whichinitiate by the formation of nascent endocytic vesicles formedfrom plasma membrane invaginations and scissions theseendocytic vesicles undergo homotypic fusion and are rapidlytargeted to sorting endosomes se sorting events initiated inse determine the fate of internalized cargo molecules such asrecycling to plasma membrane degradation in lysosomes orother traï¬cking events naslavsky and caplan figure 1bon their way to degradation sorted cargo accumulate inearly endosomes ee that further mature into late endosomeslethrough multiple events of cargo and lipid sortinglate endosomes adopt a membrane anization termedmultivesicular bodies that are enriched in lysobisphosphatidicacid and contain intraluminal vesicles gruenberg nextle potentiate their hydrolytic competence by fusing withlysosomes pillay resulting in the degradationoftheir contents providing nutrients and key factors tothe cell doherty and mcmahon kaksonen and roux notably endosomes play a role in signal transductionby serving as signaling platforms either for surface activatedreceptors like tolllike receptors and epidermal growth factorreceptor or metabolic sensors such as mechanistic targetof rapamycin complex mtorc1 arg¼ello often they promote the degradation of their targets leadingto signaltermination chung the endocyticpathway has also specialized functions in diï¬erentiated cellssuch as neurotransmitter release and recycling in neurons orantigen processing and presentation in professional antigenpresenting cellsarg¼ello soldom¨nech hinze and boucrot endocytosis events and endosomes positioning ishighly dependent on the dynamic and spatial reanizationofinclude actinintermediate ï¬laments or microtubules fletcher and mullins pegoraro the diï¬erent cytoskeleton networks thatlike b cells or dendritic cellscomplementary to endocytosis autophagy is an intracellularprocess by which cells degrade and recycle their own cytoplasmicmaterials mizushima and komatsu autophagy plays acentral role in many physiological processes including stressmanagement development immunity and aging puleston andsimon zhong f®lfan moretti doherty and baehrecke autophagy ispartially controlled though mtorc1 activity and is responsiblefor degradation and recycling of misfolded proteins as wellas obsolete anelles galluzzi the endpoint ofautophagy is to deliver cytoplasmic material to lysosomes wherelike for endocytosed cargo it is degraded several autophagyprocesses can be distinguished based on the entry mode ofthe cytosolic components destined for degradation figure 1bmacroautophagy involves engulfment of cytoplasmic contentsinto a double membrane vesicle termed the autophagosomethe autophagosome fuses then with lysosomes becomingin which its cargo is degraded galluzzian autolysosome the presence ofspeciï¬c phosphoinositideslipidstogether with rab gtpases at a given membranecompartment is often directly correlated with compartmentfunction one of the common mechanism regulating endocytosisand autophagy is an accumulation of phosphatidylinositol phosphate ptdins3p at surface of ee and on intraluminalvesicles of multivesicular endosomes and on autophagosomesnascimbeni figure 1b ptdins3p is also observedat sites of lc3associated phagocytosis another pathway ofinternalization used by the cells to ingest large particulatematerial or microbes ptdins3p is therefore a beacon used by thefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchar and pierrerufys proteins and trafï¬ckingfigure run and fyve domain containingproteins in the endolysosomal pathway a schematic representation of the rufy proteins family b description ofthe endolysosomal and autophagy pathways and presumed functional locations of rufy proteins extracellular material is ingested by endocytosis orphagocytosis the action of different endosomes allows cargo to be sorted recycled or degraded in a complex and regulated process involving fusion maturationand transport along the cytoskeleton alternatively during autophagy obsolete components present in cytosol are captured in autophagosomes prior fusion withlysosomes and degradation macroautophagy or directly internalized through endosomal invagination microautophagy se sorting endosome ee earlyendosome tgn trans golgi network le late endosome mvbs multi vesicular bodies re recycling endosome mt microtubule ct centrioles er endoplasmicreticulum the location of pi3p and rufy proteins known activity is shown created with biorendercomfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchar and pierrerufys proteins and trafï¬ckingcellular machinery to regulate endosomal sorting and autophagybirgisdottir and johansen run domainsthe presence of a single copy of a run and a fyve domainat their extremities is the key characteristic deï¬ning the rufyfamily members run domains were named after three proteinsbearing similar peptide motifs rpip8 unc14 and nescanew molecule containing sh3 at the carboxyterminus ogura matsuda run domains are present inmultiple proteins run proteins in a large panel of anismsfigure and principally allow direct interactions with smallgtpases of the rap and rab families callebaut yoshida run domains adopt a hydrophobicglobular structure bearing six conserved blocks named a to ffigure 3a these blocks correspond to eight αhelices andsome 310helices the ï¬rst helix is crucial to limit hydrophobicexposure and maintain protein solubility of runcontainingproteins callebaut kukimotoniino in spite of strong conservation among the domains presentin runcontaining proteins the proteins they interact withtheir eï¬ectors are highly variable mari and thestructural features of the run domain alone are not suï¬cientto deï¬ne binding speciï¬city for one or several members of thegtpase superfamily fukuda most run domainbearing proteins bind small gtpases but interactions with othermolecules like kinesin have also been described boucrot a direct physical link between run proteins withactin ï¬laments and microtubules has been also demonstratedtorti reinforcing the idea that these molecules arealso critical for cellular functions requiring actin remodelingsuch as migration or phagocytosis price and bos bos miertzschke xu figure 4aadditional functions for run domains have been describedfor example for the run domain present in nesca whichblocks traf6mediated polyubiquitination of the nfkappabessential modulator and consequently induces nfkb activationthis is just one of the ways in which run proteins canact in signal transduction and the coordination of membranetraï¬c with actin dynamics upon external stimulation yoshida as well as promoting endosomal fusion throughtheir binding to rab or rap gtpases callebaut figure evolution of run and fyve domain or rufy genes among living anisms diagram illustrating the evolution of the rufy genes species representative ofvarious taxonomic groups are listed data were extracted from the differential expression atlas genes database emblebi next to each species studied thenumber corresponds to the number of genes having in its sequence a fyve green run blue or both red domain the x corresponds to the appearance of acommon rufy ancestor genefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchar and pierrerufys proteins and trafï¬ckingfigure molecular anization of run and fyve domains from the rufy proteins family alignment of the protein sequences of the run a and fyve bdomains of the rufy proteins family in human and mouse a run consensus blocks are represented by segments af rpip8 sequence is used as run domainreference b fyve conserved motives and zinc ï¬ngers are represented by segments in the alignment x is any amino acid and represents positively chargedamino acid eea1 sequence is used as fyve domain reference for all alignment amino acids are colored according to their properties cyan for hydrophobicpositions a v i l m turquoise for aromatic positions f y w h red for basic residues k r purple for acidic residues d e green for polar uncharged n q st salmon for cysteine c orange for glycine g and yellow for proline p gray numbers below alignment means the amino acids position after alignment blacknumbers surrounding the alignments represent the start left and end right positions of the domains in the peptide sequence of each protein alignment wererealized with seaviewer analyzer software gouy accession numbers for protein are following human rpip8 np_0011382971 mouse rpip8np_0580391 human eea1 np_0035573 mouse eea1 np_0010019321 human rufy1 np_0794343 mouse rufy1 np_7661451 human rufy2np_0604574 mouse rufy2 np_0817012 human rufy3 np_0557761 mouse rufy3 np_0818061 human rufy3xl np_0010325191 mouserufy3xl np_0012767031 human rufy4 np_9408852 mouse rufy4 np_0011641121 human fyco1 np_0787892 mouse fyco1np_0011037232yoshida their interaction with motor proteins likekinesin or myosin suggests a role for run domains in regulatingvesicular and anelle transport callebaut yoshida via these diï¬erent mechanisms run proteins havebeen implicated in neuronal development honda 2017bsignaling sun migration yoshida and regulation of various cellular function like endocytosis orexocytosis kitagishi and matsuda fyve domainsfyvedomainbearing proteins for fab1 yotbzk63212vac1 and eea1 are speciï¬cally found in association withmembranous anelles enriched in ptdins3p and highlyconserved among eukaryotes including yeast hayakawa figure fyve domains adopt a zinc ï¬nger conformationmisra and hurley kutateladze and overduin inaddition to fyve ten types of zinc ï¬nger folds have beenfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchar and pierrerufys proteins and trafï¬ckingfigure rufy proteins are important for intracellular trafï¬cking signaling and cytoskeleton dynamics a schematic representation of the run and fyvedomains activity of rufy proteins run domains act on signaling endosomal protein trafï¬cking and cytoskeletal network dynamics via small gtpase proteins fyvedomains bind ptdins3p and regulates autophagy and endosome trafï¬cking b function of rufy proteins in homeostatic conditions c consequences ofalterations in rufy proteins functions at the cellular and anismal levelcharacterizedincluding conventional gal4 gata1 tfiismetrs lim ring domain pkc crd and phd domainszinc ï¬ngers are structural conformations adopted by peptidechains upon coordination of two zn2 cations within a cysteinerich region schwabe and klug stenmark unlike most molecules bearing zinc ï¬ngers fyve proteinsdisplay only one copy of the domain located at any positionalong the peptide chain highlighting its autonomy as a structuralunit fyve zinc ï¬ngers can stabilize proteinprotein or proteindnarna interactions dunkelberg and gutierrezhartmann a classical fyve domain has eight potential zinccoordinating tandem cysteine positions and is characterized byhaving basic amino acids around the cysteines many membersof this family also include two histidine residues in a sequencemotif including wxxd cxxc rhhcxcg and rvc wherex means any amino acid and a positively charged aminofrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchar and pierrerufys proteins and trafï¬ckingacid figure 3b most deviations from this sequence canreduce the domain aï¬nity for zinc and destabilize it stenmark misra and hurley stenmark and aasland kutateladze and overduin within this structuralframework speciï¬c modiï¬cations in the nonconserved residuesof the domain can radically aï¬ect fyve protein subcellularlocalization and function by forming a turret loop and adimerization interface hayakawa with regard to their aï¬nity for ptdins3p fyve domaincontaining proteins are mostly found associated to ee orphagosomes stenmark gaullier stenmark and aasland figure 4a the presence offyve domains is therefore correlated to the regulation ofmembrane traï¬c through speciï¬c recognition of ptdins3pdomains by rhhcxcg motifs gaullier and modulation by associated phosphatidylinositol kinasesptdins3p is generated from phosphatidylinositol by class iiiptdins 3kinases pi3klike vps34 on target membranessuch as nascent autophagosome omegasomes melia or ee di paolo and de camilli raib scott figure 1b in turn accumulation ofptdins3p recruits and activates eï¬ector proteins containingfyve domains favoring transport or fusion of target anellesstenmark and gillooly axe burman andktistakis schink aï¬nity for ptdins3p isdetermined by the pair of histidine residues present in therhhcxcg motif of the fyve domain stahelin diraviyam lee he this aï¬nity can also be harnessed by fyve proteins to linkendosomes with mrna ribonucleoprotein ps mrnpand associated ribosomes playing a role in their longdistancetransport in the cell pohlmann importantlymany fyve proteins homodimerize dimerization multiplies theconserved residues displayed by the diï¬erent signature motifspresent in the fyve domain and contributes to a network ofhydrogen bonding and electrostatic interactions that providespositive selection for binding several ptdins3p head groupsphdependent insertion of fyve domain into cell membraneshe pankiv is reinforced by additionalhydrophobic membrane interactions with the turret loop andortandem lysine residues these nonspeciï¬c interactions promotefyve domain access to phosphate head groupsthat arehindered by the close packing of lipid molecules this bivalentmechanism increases therefore greatly fyve domains speciï¬cityfor ptdins3penriched domains and discrimination againstother mono or polyphosphorylated ptdins species misra andhurley stenmark and aasland dumas kutateladze and overduin fyve proteins are therefore key players in endocytosis andautophagy and mutations in fyve domains can alter profoundlythese functions as well as cellular homeostasis kamentseva for example eea1 protein early endosome antigen isknown to be crucial for endosome dynamics and any mutation inits conserved residues or the oligomerization site can drasticallyreduce the aï¬nity between its fyve domain and ptdins3pstenmark gaullier in this contextrufys proteins by bearing a nterminal run domain oneor several copies of a coiledcoil domain next to a cterminalfyve domain figure 1a have all the features required tocarryout speciï¬c adaptor functions to regulate endocytosis orautophagy by impacting on anelle fusion and mobility alongthe cytoskeletonthe rufy proteins familythe rufy family encompass four genes named rufy1 to sharing homologies and displaying speciï¬c tissue expressionand alternative splicing rufy genes are relatively conservedgenes absent from prokaryotes and fungi upon evolution theemergence of the common ancestor appeared in vertebrates andarthropods which possess one ortholog cg31064 figure no rufy protein could be detected in caenorhabditis elegansand only a fyvebearing protein t10g35 considered as anortholog of human eea1 shows some sequence similaritieswith the rufy family t10g35 exhibits ptdins3p bindingactivity and is involved in endocytosis being mostly expressedin epidermis and intestine of c elegans hayakawa in chordates rubicon run domain and cysteinerichdomain containing beclin 1interacting protein and fyve andcoiledcoil domain autophagy adaptor fyco1 displaystructural and functional features potentially categorizing themas rufy proteins rubicon was identiï¬ed as a componentthe class iii pi3k complex and a negative regulatorofof autophagy and endosomaltraï¬cking matsunaga zhong like rufys rubicon containsmultiple functional domains that interact with other proteinsincluding a run a cc and a fyvelike domains wong however despite these similaritiesthe poordegree of sequence homology and the lack of conservationof its fyvelike domain which was found not to bind topi3p burman and ktistakis prevented rubiconsintegration within the rufy proteins family conversely tofyco1 which we propose here to name rufy5 and detail thecharacteristics belowrufy1rufy1 previously named rabip4 is an kda proteinmainly expressed in the brain kidneylung placenta andtestis there are two rufy1 isoforms rabip4 and rabip4that has an additional amino acid upstream ofthenterminal run domain figure 1a they were both shownto interact with the small endosomal gtpases rab4 rab5and rab14 fouraux vukmirica table rufy1 inactivation inhibits eï¬cient recycling ofendocytosed transferrin implicating rufy1 in the regulationof ee functions through cooperative interactions with rab4and rab14 cormont yamamoto nag this was further demonstrated by the alteration ofepidermal growth factor receptor endocytic traï¬cking kineticsin cells depleted of rufy1 gosney and thehijacking of rufy1 by the bacteria p gingivalis to escapelysosomal degradation takeuchi in melanocytesrufy1 was found to form a complex with rabenosyn5frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchar and pierrerufys proteins and trafï¬ckingtable summary of rufy proteins functional interactionsprotein aliasesbinding partnerfunctionsrufy1rabip4 rabip4zfyve12rufy2lzfyve rabip4rkiaa1537 fyve13rufy3singar1 ripxzfyve30 kiaa087rufy4zfyve31fyco1rufy5zfyve7 rufy3ctrct18 catc2rab4etkrab14ap3rabenosyn5kif3abrab4aap3 complexpodxl1rab33arab4arab6aretrap2fascinrab5rab33agpm6arap2stefyial2complexpak1foxk1hoxd9rab7recycling endosomal trafï¬ckingregulation of endocytosis through its interaction with rufy1rufy1s recruitment endosome tethering and fusionregulates spatial distribution of lysosomesorting endosome pathway in endosomal membrane inmelanocytes and segregates tyrosinaserelated protein1increases cell proliferation migration and invasionendosome dynamic golgi complexassociated rab33 andautophagosome formation on omegasomeslead to a fusion of the ret tyrosine kinase domain to a rundomain and a coiledcoil domain appear to be critical fortumorigenesiscontrol neuronal polaritycontrol the growth of axons and neuronal growth coneacts on endosomal trafï¬ckingfacilitates cell polarityinduce cell migration and invasion in gastric cancerincreases cells migration rufy3mediated with metastasis invasionin colorectal cancerhoxd9 transactivate rufy3 and it overexpression induce gastriccancer progression proliferation and lung metastasisautophagosome formation and lysosome clusteringmap1lc3abautophagosome formation and elongationrab7kinesin1endosomal transport by acting with microtubule plus enddirectiontransportallows translocation from the late endosome lysosome andautophagosome to the plasma membrane through plusendmicrotubule transportstudycormont yang yamamoto ivan nag zhi fukuda kitagishiand matsuda staubitz janoueixlerosey wei yoshida fukuda honda 2017awang xie 2017azhu terawaki cheng olsvik wang krau and haucke raib kif3ab rab4a and adaptor protein3 ap3 to diï¬erentiallyregulate tyrosinaserelated protein1 and tyrosinase sorting inendosomes contributing to melanosome maturation nag table moreover silencing the rabip4isoform ofrufy1 was shown to promote outgrowth of plasma membraneprotrusions and to regulate the spatial distribution of lysosomesat their tips through an interaction with ap3 ivan figures 1b 4b rufy1 is also capable of controllingcell migration by regulating integrin traï¬cking vukmirica presumably via endocytosis in full agreementwith a role of rufy1 in regulating endosomal dynamics asingle nucleotide polymorphism s705a in the rufy1 gene wasassociated with high blood glucose levels and type diabetesmellitus susceptibility in an exomewide association studyewas yamada this result is consistent with theearly ï¬nding that rabip4 expression leads to glucose transporter glut1intracellular retention cormont interestingly rufy1 display a sh3binding motif pxxpxpembedded in the fyve domain and is able to interactingwith the epithelial and endothelialtyrosine kinase etkand possibly regulates endocytosis through this interactionyang another ewas aiming to ï¬nd earlyonsetalzheimers disease ad susceptibility genes identiï¬ed rufy1among genes involved in endolysosomal transport and knownto be important for the development of ad kunkle figure 4crufy2lungrufy2 or leucine zipper fyveï¬nger protein lzfyve is a kda protein originally identiï¬ed as an activating transcriptionfactor2 interactor embryogenesis dunkelberg and gutierrezhartmann preferentially located in the nucleus andexpressed during after development rufy2 expression remainshigh in the brainliver and the gastrointestinal tractyang rufy2 displays two nterminalleucinezipper domains as well as a cterminal fyveï¬nger domainalthough it is likely to have a nuclear function at early stagesof embryonic development the presence of a fyve domainsuggests a cytoplasmic role for rufy2 in regulating membranetraï¬c in fully diï¬erentiated cells importantly the run domainof rufy2 was shown to associate speciï¬cally with the golgicomplexassociated rab33a fukuda table givenfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchar and pierrerufys proteins and trafï¬ckingthe reported interaction of rab33a and rab33b with atg16land its putative role in regulating autophagy fukuda and itoh rufy2 could contribute to autophagosome formationthrough a dual interaction with rab33a and ptdins3p onomegasomes figures 1b 4b irrespective of its function rufy2expression is subject to modulation by the micro rna mir155boï¬llde ros which is an important regulator ofimmune cells development and inï¬ammatory responses ceppi the rufy2 gene is also frequently found mutatedin cancer cells with the most frequent mutations convertingit into a strong target for nonsense mediated mrna decaythereby decreasing considerably its expression shin figure 4crufy3rufy3 also known as rap2interacting protein x ripxkukimotoniino or single axonrelated singar1mori is the best characterized member of therufy family rufy3 the smallest of the rufy proteins witha molecular weight of kda figure 1a is mostly expressedin neurons kitagishi and matsuda neuronal rufy3 isatypical since it lacks a fyve domain and is considered as partof the rufy family based on strong sequence similarities withthe other members notably in the run and coiledcoil domainsfigure 2a rufy3 is distributed between the cytosol and at theplasma membrane but not in intracellular vesicles presumablybecause it lacks a fyve domain in artiï¬cial conditions likefollowing expression of the dominant gain of function mutantform of rab5 q79l in u937 cells rufy3 was found associatedin large vesicle structures and to coimmunoprecipitate withrab5 via an interaction with its carboxyl terminal domain andsurprisingly not its run domain yoshida likerufy2 rufy3 was also shown in a 2hybrid screen and bycoimmunoprecipitation to bind rab33 through its coiledcoildomain cc1 fukuda in 293t and 3y1 celllines however rufy3 was shown not to interact with severalsmall gtpasesincluding rab2 rab5 rab7 rho and rasthis suggests that either rufy3 requires cell speciï¬c partnerproteins or posttranslation modiï¬cations to be able to bind tosmall gtpases rufy3 was ï¬rst described as interacting withrap2 a small raslike gtpase via a residue fragment located in the run domain janoueixlerosey kukimotoniino table together with rap1 rap2interacts with ras eï¬ectors such as raf pi3k and ral guaninenucleotide dissociation stimulator inhibiting activation of theirdownstream targets and thus suppressing ras oncogenic activitykukimotoniino nussinov in the adultnervous system rap1 and rap2 also regulate the maturationand plasticity of dendritic spine and synapses by forminga complex together with rap2 and fascin rufy3 interactswith the ï¬lamentous actin network and controls the growth ofaxons and neuronal growth cone wei table recent mechanistic studies indicate that rufy3 accumulates inlipid rafts by forming a glycoprotein m6a gpm6arufy3rap2stefyial2 complex honda 2017a table this complex activates the rac guanine nucleotide exchangefactor honda 2017b impacting actin anization andpromoting neuronal polarity and growth figure 4b rufy3seems therefore to have diï¬erent axogenic functions in brainmori honda 2017b and not surprisingly rolesfor rufy3 in amyotrophic lateral sclerosis arosio major depressive disorder aberg and ad zelaya have been reported olfactory dysfunction occurs in of ad cases and is correlated with elevated rufy3 expressionin glomerular and mitral layers of the olfactory bulb zelaya rufy3 is cleaved by caspase and critically required forcaspasemediated degeneration of tropomyosin receptor kinasea positive sensory axons in vitro and in vivo hertz figure 4c removal of neuronally enriched rufy3 is able toblock caspase 3dependent apoptosis while dephosphorylation ofrufy3 at residue s34 appears required for its degradation hertz analysis of rufy3deï¬cient mice supports a seconddistinct function for rufy3 in neuronal growth and polaritysince mutant embryos show defects in axonal projection patternsthese occur in addition to the prevention of casp3dependentapoptosis in dorsal root ganglions rufy3 appears therefore to bekey for nervous system development remodeling and functionexplaining the embryonic lethality displayed upon rufy3 geneticinactivation in mouse hertz with the current advance in genomics and single cell rnasequencing speciï¬c gene expression patterns can be revised andmore accurately deï¬ned analysis of several genomic databasesbiogps ncbi human atlas protein immgen ensembl revealthat in addition to neurons rufy3 expression can be detectedin other tissues and cell types moreover the rufy3 gene appearsto have many transcriptional variants leading to the expressionof diï¬erent protein isoforms two of these isoforms display acterminal region extended by amino acids compared to thepreviously identiï¬ed neuronal isoform of rufy3 importantlythese previously uncharacterized longer isoforms rufy3xlpossess the same run domain and a putative fyve domainin their cterminus figure 1a indicating that rufy3 is alegitimate member of the rufy family in contrast to classicfyve zinc ï¬ngers genomic databases reveal this putative fyvedomain appears to lack the tandem histidine residue clusterthat deï¬nes aï¬nity for ptdins3p figure 3b interestingly thesh3 binding site embedded in the rufy1 and rufy2 fyvedomains is also present in rufy3xl suggesting a potentialsignal transduction activity for this uncharacterized isoformthe translation of rufy3xl mrna into a functional protein andits capacity to bind ptdins3p remain to be demonstratedif true a role for rufy3 in the coordination of endosomedynamics or anelle transport could be hypothesized thisidea is supported by the observation that rufy3 is present instaufen2containing messenger ribonucleoprotein ps thatare used to transport mrnas along neuronal dendrites to theirsite of translation maherlaporte fyve proteinshave already been implicated in endosomemediated transport ofmrnp pohlmann and rufy3xl could thereforealso perform this function the existence of fyve domainbearing isoforms might extend and diversify its function in otherspecialized cellsfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchar and pierrerufy4rufy4 is a kda that is atypical among the rufy familymembers since it bears several nonconserved residues inits run domain and it lacks the tandem histidine clusterand t | Colon_Cancer |
objectives paired box protein8 pax8 immunohistochemical expression can be used as a diagnostic marker for epithelial cells tumors this study aimed at investigating the immunohistochemical expression of pax8 among sudanese females diagnosed with cervical endometrial and ovarian cancers between december and may by studying their formalinfixed paraffin embedded blocksresults sixty patients diagnosed with female reproductive tract cancers were included who aged ± years range cervix was the most common cancer site in patients regarding cancer stage there was and of the study population had stage 3b and 2b respectively the histopathological diagnosis included and poorly moderately and well differentiated cervical squamous cell carcinoma scc as well as and endometrial adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma respectively pax8 was positively expressed in endometrial adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma poorly and moderately differentiated scc all patients diagnosed with well differentiated scc and metastatic adenocarcinoma showed no expression of pax8 a statistically significant was seen for pax8 expression and the different histopathological diagnosis p value keywords female reproductive cancer paired box protein8 immunohistochemical expressionintroductionpaired box protein8 pax8 is a member of the family paired box proteins paxs [ ] pax8 consists of amino acids with a molecular weight of approximately kilo dalton and its molecular properties are located on chromosome 2q13 [] pax8 is a transcription factor that regulates ans development during the embryonic period as well as to maintain normal cellular functions in some cells after birth [ ] during the embryonic period pax8 also plays a significant role correspondence nouh_saadoutlookcom alfarrabi college for science and technology khartoum sudanfull list of author information is available at the end of the in the development of genital ans derived from the mesonephric and the m¼llerian ducts [] in a previous experiment the deletion of the pax8 gene resulted in dysfunctional uterus absence of the endometrium and the vaginal opening also resulted in poor development of the myometrial tissue several studies have described the immunohistochemical utility of pax8 as a diagnostic marker for epithelial cells neoplasms of many glands and ans such as thyroid thymus and kidney as well as some female reproductive tract tumors [ ]in a healthy female reproductive tract pax8 shown to be overexpressed in the epithelial cells of the endocervix and the endometrium [] pax8 was found to be expressed among endometrioid carcinomas transitional the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cali a0et a0al bmc res notes page of undifferentiated cell carcinomas and the metastatic carcinomas at a range of and [ ] whereas for the ovarian carcinomas pax8 was under expressed considering that few studies have investigated the immunohistochemical expression of pax8 in carcinomas of the endometrium and uterine cervix in the different parts of the world but none from sudan yet [ ] this study aimed at investigating the immunohistochemical expression of pax8 among sudanese females diagnosed with cervical endometrial and ovarian carcinomasmain textmaterials and a0methodsstudy design and a0population characteristicsthis is a descriptive retrospective hospital based study conducted at different histopathology laboratories during the period from december till may in khartoum state sudan we retrieved archived formalin fixed paraffin embedded blocks previously collected from female patients with cervical endometrial or ovarian carcinomas the retrieved formalin fixed paraffin blocks represent all the female population admitted at the hospitals for reproductive malignancies diagnosis the participants demographic data was collected including age place of residence the clinical data including site of cancer cancer grade and the histopathological diagnosis were also collectedsections preparation for a0immunohistochemistry stainingtwo sections were cut using rotary microtome leica germany from each histopathological block then one slide was stained by hematoxylin and eosin staining technique the other slide was mounted onto 3aminopropyltriethoxysilane coated slides for immunohistochemistry to retrieve pax8 tissues antigen we treated the sections with citrate buffer at ° a0c for a0min in a waterbath then the tissue sections were rinsed first in distilled water and later with tris buffer saline tbs this was followed by treatment with peroxidase block hydrogen peroxide in methyl alcohol for a0min to quench endogenous peroxidase activity the slides were then placed in a humid chamber then the slides were drained and rinsed in two successive changes of tris buffer wash buffer for a0 min each nonspecific proteinprotein interactions were blocked by incubating and treating the tissue sections in a humid chamber with the power block casein in phosphate buffered saline for a0 min then the remaining solution was drained from the slides the sections were then incubated in the primary antibody pax8 antipax8 rabbit antihuman monoclonal antibody ab189249 abcam united kingdom at room temperature in the humid chamber according to the manufacture instructionsobserving the yellowishbrown or brown appearance of the nucleus was considered a positive result for the pax8 for the negative control we omitted the incubation with the primary antibody step instead we incubated the section in the phosphate buffer saline pbsresults interpretationsfor the interpretation of the results we depended on the intensity as well as the number of the cells that expressed the marker and the expression was graded into categories negative no staining less than of the cells were expressing the marker of the cells were expressing the marker more than of the cells were expressing the marker more than of the cells were expressing the marker the slides were interpreted and validated by two expert pathologists blindly of each other results photomicrographs were taken using olympus sp350 camera olympus imaging america inc usastatistical analysisthe statistical analysis of the results was done using ibm spss statistics vs the chisquared test was performed to compare the frequencies of categorical variables statistical significance level was defined as p value at confidence intervalresultscharacteristics of a0the a0study participantsthe study included patients diagnosed with female genital tract cancer patients aged ± a0years range a0years patients were grouped into age groups those aged a0 years constituted half of the study participants the remaining were and patients distributed across the remaining age groups of a0 years a0 years and a0 years respectively according to patients place of residence patients were originating from the four regions of sudan most of the patients were from western part of sudan followed by from the central part of sudanregarding the site of cancer the cervix was the most commonly involved patients there were and endometrial and ovarian cancer respectively based on the international federation of gynecology and obstetrics figo cancer grading the majority of the study population was diagnosed with stage 3b and 2b cancer and of the patients respectively the were and stage 4b 3a 2a 1b and 4a respectively 0cali a0et a0al bmc res notes page of no statistically significant association between figo staging and age group was found p value histologically there were squamous cell carcinoma scc all of which were cervical cancers and adenocarcinoma scc and adenocarcinoma were further classified into poorly differentiated scc moderately differentiated scc and well differentiated scc endometrium adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinomabased on age groups age group showed no statistically significant relationship with either patients place of residence cancer site cancer histological type figo staging and cancer histopathological type table a0immunohistochemical expression of a0paxthe immunohistochemical expression of pax8 was shown as a yellowishbrown or brown staining of the nucleus fig a0 based on site of cancer all endometrium carcinoma showed positive expression of pax8 with p value there were only patients who had positive expression of pax8 including adenocarcinoma and scc a statistically significant difference was noted for the pax8 staining and cancer type with p value the analysis of pax8 staining results based on the histopathological diagnosis showed that all patients who were diagnosed with well differentiated scc and metastatic adenocarcinoma had negative results for the pax8 expression while of the endometrium adenocarcinoma were found positive for the pax8 expression a statistically significan was t seen for pax8 expression and the different histopathological diagnosis p value table a0table classification of a0participants demographic and a0clinical diagnosis based on a0age groupage group no total no p value a0years a0years a0years a0yearsresidence of patient central sudan east sudan west sudan north sudansite of cancer cervix endometrium ovarycancer histological type scc adenocarcinomafigo staging stage stage 2a stage 2b stage 3a stage 3b stage 4a stage 4bhistopathological cancer grades well differentiated scc poorly differentiated scc moderately differentiated scc endometrium adenocarcinoma endocervical adenocarcinoma metastatic adenocarcinoma ovarian mucinous cyst adenocarcinomascc squamous cell carcinoma 0cali a0et a0al bmc res notes page of fig immunohistochemical expression of pax8 among the different histopathological cancer types and grades the immunohistochemical expression of pax8 is shown as a yellowishbrown or brown staining of the nucleus a well differentiated scc negative b metastatic adenocarcinoma negative c poorly differentiated scc positive d moderately differentiated scc positive e endometrium adenocarcinoma positive f ovarian mucinous cystadenocarcinoma positive g endocervical adenocarcinoma positive and h endometroid adenocarcinoma positive 0cali a0et a0al bmc res notes page of table association of a0clinical diagnosis and a0the a0immunohistochemical expression of a0pax8pax results no total no p valuepositivenegativecancer histological type scc adenocarcinomacancer site cervix endometrium ovaryfigo staging stage stage 2a stage 2b stage 3a stage 3b stage 4a stage 4bcancer histopathological grading well differentiated scc poorly differentiated scc moderately differentiated scc endometrium adenocarcinoma endocervical adenocarcinoma metastatic adenocarcinoma ovarian mucinous cyst adenocarcinomascc squamous cell carcinoma discussionprevious studies on the immunohistochemical expression of pax8 in the normal female reproductive tract showed that pax8 was expressed in the endometrial endocervical and ovarian epithelial cells as well as in nonciliated epithelial cells of the fallopian tubes [ ] this study investigated the immunohistochemical expression of pax8 in sudanese patients who were diagnosed with female reproductive tract cancers patients on the 5th decade of life were constituting half of the study participants with no statistically significant association between age group and the type of cancer however previous studies had suggested other risk factors which could contribute in the development of certain gynecological cancer [ ]regarding the place of residence the majority of patients coming from western sudan this result is in contrary with a previous study in sudan conducted by saeed et a0al in which they showed that the percentage of patients suffering from different types of cancers residing in central and northern sudan were higher compared to the other regions in sudan nevertheless these findings could suggest the involvement of environmental risk factors however the limited study samples size is insufficient to support this suggestion therefore further research with a larger samples size investigating the potential environmental risk factors is essential for strategic prevention and protection measuresthe reported number of female patients with cervical cancer was high compared to ovarian and endometrium cancer similar results were seen previously among sudanese females also the high frequency of stages 3b and 2b compared to the other stages were comparable to previous study conducted in sudan this similarity underscores a delayed response among sudanese females in seeking healthcare and urge the need for health promotion and education to encourage young sudanese females for the early signs detection and seeking healthcare as early as possible for a better treatmentregarding the classification based on the histopathological diagnosis most of the female diagnosed with scc this result was also similar to previous study investigated the prevalence of the different gynecologic cancer in sudan however the expression of pax8 among the studied samples was relatively low compared to previous studies [ ] this could be attributed 0cali a0et a0al bmc res notes page of to the site of cancer development while agrees with another study where pax8 was expressed only in patient interestingly a high frequency of pax8 expression was noted among females diagnosed with endometrium cancer compared to scc this finding is in contrary with a previous report where pax8 was expressed among only of the studied samples also the result was strongly in accordance with other studies [ ] besides that the lack of pax8 expression among those who were diagnosed with well differentiated scc and metastatic adenocarcinoma could play a significant role in either gynecologic cancer differentiation or in detection of endometrium adenocarcinoma progression to metastatic adenocarcinoma [ ]conclusionalthough pax8 showed a significant expression among adenocarcinomas lesions and negative expression was noted among those with well differentiated scc and metastatic adenocarcinoma pax8 might not be beneficial when used alone as a diagnostic marker for the tumors that occur in the female reproductive tractlimitations¢ the small sample size investigated in this study reduced the ability of using the expression of pax8 as a diagnostic marker therefore a largescale study is needed and it should include other types of malignant tumors encountered in the female reproductive systemacknowledgementsthe authors would like to acknowledge the medical staff for their interest and cooperation during the study and thanks to all who participated in completing this studyauthors contributionseta nsm and ees provided conceptual framework for the study guidance for interpretation of the data and performed data analysis eta ees irs lah and amm performed laboratory work nsm ees msm aay and aa performed the statistical analysis nsm msm ees and aa participated in the manuscript preparation revision and coordination all authors read and approved the final manuscriptfundingnot applicableavailability of data and materialsthe datasets used andor analyzed during the current study are available from the corresponding author on reasonable requestethics approval and consent to participateethical approval was obtained from the research ethics committee of the faculty of medical laboratory sciences university of khartoum sudan ethical approval no fmlsrec002042 all participant approved to participate by signing an informed consentconsent for publicationnot applicablecompeting interestsno competing interests to discloseauthor details department of histopathology and cytology faculty of medical laboratory sciences university of khartoum khartoum sudan department of histopathology and cytology faculty of medical laboratory sciences national university khartoum sudan alfarrabi college for science and technology khartoum sudan faculty of medicine sinnar university sennar sudan molecular biology department faculty of medical laboratory sciences nile university khartoum sudan faculty of dentistry ibn sina university khartoum sudan department of neurology mayo clinic jacksonville fl usa department of radiology mayo clinic jacksonville fl usa institute of endemic diseases university of khartoum khartoum sudan mycetoma research center university of khartoum khartoum sudan faculty of medicine nile university khartoum sudan received july accepted august references gruss p walther c pax in development cell mansouri a hallonet m gruss p pax genes and their roles in cell differentiation and development curr opin cell biol macchia pe lapi p krude h pirro mt missero c chiovato l souabni a baserga m tassi v pinchera a pax8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis nat genet vilain c rydlewski c duprez l heinrichs c abramowicz m malvaux p renneboog bt parma j costagliola s vassart g autosomal dominant transmission of congenital thyroid hypoplasia due to lossoffunction mutation of pax8 j clin endocrinol metab park s vk c genetics of congenital hypothyroidism j med genet dahl e koseki h balling r pax genes and anogenesis bioessays lang d powell sk plummer rs young kp ruggeri ba pax genes roles in development pathophysiology and cancer biochem pharmacol stoykova a gruss p roles of paxgenes in developing and adult brain as suggested by expression patterns j neurosci mittag j winterhager e bauer k grummer r congenital hypothyroid female pax8deficient mice are infertile despite thyroid hormone replacement therapy endocrinology bouchard m de caprona d busslinger m xu p fritzsch b pax2 and pax8 cooperate in mouse inner ear morphogenesis and innervation bmc dev biol mittag j winterhager e bauer k grummer rje congenital hypothyroid female pax8deficient mice are infertile despite thyroid hormone replacement therapy endocrinolog laury ar perets r piao h krane jf barletta ja french c chirieac lr lis r loda m hornick jl a comprehensive analysis of pax8 expression in human epithelial tumors am j surg pathol wong s hong w hui p buza n comprehensive analysis of pax8 expression in epithelial malignancies of the uterine cervix int j gynecol pathol ozcan a shen ss hamilton c anjana k coffey d krishnan b truong ld pax expression in nonneoplastic tissues primary tumors and metastatic tumors a comprehensive immunohistochemical study mod pathol bowen nj logani s dickerson eb kapa lb akhtar m benigno bb mcdonald jf emerging roles for pax8 in ovarian cancer and endosalpingeal development gynecol oncol 0cali a0et a0al bmc res notes page of ozcan a liles n coffey d shen ss truong ld pax2 and pax8 expression in primary and metastatic m¼llerian epithelial tumors a comprehensive comparison am j surg pathol distinguishing ovarian mucinous neoplasms from colonic and appendiceal mucinous neoplasm bmc res notes nesrin r kilic d risk factors for cervical cancer results from a hospital ozcan a liles n coffey d shen ss truong ldjtajosp pax2 and pax8 based casecontrol study int j hematol oncol expression in primary and metastatic m¼llerian epithelial tumors a comprehensive comparison am j surg pathol nonaka d tang y chiriboga l rivera m ghossein r diagnostic utility of thyroid transcription factors pax8 and ttf2 foxe1 in thyroid epithelial neoplasms mod pathol tacha d zhou d cheng l expression of pax8 in normal and neoplastic tissues a comprehensive immunohistochemical study appl immunohistochem mol morphol bowen nj logani s dickerson eb kapa lb akhtar m benigno bb mcdonald jfjgo emerging roles for pax8 in ovarian cancer and endosalpingeal development gynecol oncol k¶bel m kalloger se boyd n mckinney s mehl e palmer c leung s bowen nj ionescu dn rajput a ovarian carcinoma subtypes are different diseases implications for biomarker studies plos medicine 2008512e232 nonaka d chiriboga l soslow ra expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas am j surg pathol tong gx devaraj k hamelebena d yu wm turk a chen x wright jd greenebaum e pax8 a marker for carcinoma of m¼llerian origin in serous effusions diagn cytopathol laury ar perets r piao h krane jf barletta ja french c chirieac lr lis r loda m hornick jljtajosp a comprehensive analysis of pax8 expression in human epithelial tumors am j surg pathol tong gx devaraj k hamelebena d yu wm turk a chen x wright jd greenebaum ejdc pax8 a marker for carcinoma of m¼llerian origin in serous effusions diagn cytopathol chu pg chung l weiss lm lau sk determining the site of origin of mucinous adenocarcinoma an immunohistochemical study of cases am j surg pathol brunner ah riss p heinze g meltzow e brustmann h immunoexpression of pax in endometrial cancer relation to highgrade carcinoma and p53 int j gynecol pathol ozcan a shen ss hamilton c anjana k coffey d krishnan b truong ldjmp pax expression in nonneoplastic tissues primary tumors and metastatic tumors a comprehensive immunohistochemical study mod pathol aldaoud n erashdi m alkhatib s abdo n almohtaseb a graboskibauer a the utility of pax8 and satb2 immunohistochemical stains in saeed me cao j fadul b kadioglu o khalid he yassin z mustafa sm saeed e efferth t a fiveyear survey of cancer prevalence in sudan anticancer res saeed me cao j fadul b kadioglu o khalid he yassin z mustafa sm saeed e efferth tjar a fiveyear survey of cancer prevalence in sudan anticancer res mohamed keh ashmeig aaa cervical cancer our experience in sudan philadelphia aacr elhasan lme bansal d osman of enan k abd farag eab prevalence of human papillomavirus type in sudanese women diagnosed with cervical carcinoma j cancer res ther tacha d zhou d cheng ljai morphology m expression of pax8 in normal and neoplastic tissues a comprehensive immunohistochemical study appl immunohistochem mol morphol ord³±ez ng value of pax immunostaining in tumor diagnosis a review and update adv anat pathol gailey mp bellizzi am immunohistochemistry for the novel markers glypican pax8 and p40 δnp63 in squamous cell and urothelial carcinoma am j clin pathol yemelyanova a gown am holmes bj ronnett bm vang r pax8 expression in uterine adenocarcinomas and mesonephric proliferations int j gynecol pathol liang l zheng w liu j liang sx assessment of the utility of pax8 immunohistochemical stain in diagnosing endocervical glandular lesions arch pathol lab med wong s hong w hui p buza njijogp comprehensive analysis of pax8 expression in epithelial malignancies of the uterine cervix int j gynecol pathol de andrade dap da silva vd de macedo mg de lima ma de andrade vm andrade cemc schmidt rl reis rm dos reis r squamous differentiation portends poor prognosis in low and intermediaterisk endometrioid endometrial cancer plos one 20191410e0220086publishers notespringer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations ¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your ï¬eld¢ rapid publication on acceptance¢ support 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" lymph node staging of ductal adenocarcinoma of the pancreatic head pdac by crosssectionalimaging is limited the aim of this study was to determine the diagnostic accuracy of expanded criteria in nodalstaging in pdac patientsmethods sixtysix patients with histologically confirmed pdac that underwent primary surgery were included inthis retrospective irbapproved study crosssectional imaging studies ct andor mri were evaluated by aradiologist blinded to histopathology number and size of lymph nodes were measured shortaxis diameter andcharacterized in terms of expanded morphological criteria of border contour spiculated lobulated and indistinctand texture homogeneous or inhomogeneous sensitivities and specificities were calculated with histopathologyas a reference standardresults fortyeight of patients had histologically confirmed lymph node metastases pn sensitivityspecificity and youdens index for the criterion size were and for inhomogeneous signal intensity and and for border contour and respectively there was a significant associationbetween the number of visible lymph nodes on preoperative ct and lymph node involvement pn p lymph node staging in pdac is mainly limited due to low sensitivity for detection of metastatic diseaseusing expanded morphological criteria instead of size did not improve regional nodal staging due to sensitivityremaining low combining specific criteria yields improved sensitivity with specificity and ppv remaining highkeywords ductal adenocarcinoma of the pancreatic head staging lymph nodes computed tomography magneticresonance imaging crosssectional imaging neoadjuvant therapy correspondence florianlochcharitede1charit universittsmedizin berlin corporate member of freie universittberlin humboldtuniversitt zu berlin and berlin institute of healthdepartment of surgery campus benjamin franklin hindenburgdamm berlin germanyfull 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 0cloch world of surgical oncology page of pancreatic cancer remains one of the most lethal malignancies being the fourth leading cause of cancerdeath in the usa and predicted to be the secondleading cause of cancer death by the overall5year survival after diagnosis is and at thetime of diagnosis the main proportion of patients hasadvancedstage disease leaving only qualifiedfor resective surgery pancreatic cancer is locatedin the head of the pancreas in of the cases even after successful resective surgery in patients withcancer of the pancreatic head the 5year survival remains as low as these data underline theimportance of establishing multimodaltherapeuticconcepts for patients with pancreatic cancer as perother entities of abdominal cancerapart from the potential to increase the resectabilityrate of pancreatic cancer by neoadjuvant therapy [ ]there is evidence that patients which are successfullydownstaged from nodepositive disease cn1 to nodenegative disease ypn0 prior to surgery benefit in termsof higher 5year survival rate this would qualifynodalinvolvement as a sufficient basis for indicatingneoadjuvant therapy yet even given advanced imagingtechnologies identifying lymph node metastasis remainschallenging consequently the indication of a potentiallyeffective neoadjuvant therapy cn with side effects inlymph nodepositive patients cn is mainly based onunreliable clinical stagingthe established criterion for lymph node involvement in pancreatic cancer is size using the size underlies the assumption that tumor spread to regionallymph nodes leads to an enlargement of the respective lymph node the usual cutoff value is a shortaxis diameter of mm [ ] it has been shownthough that lymph nodes of ¥ mm are not seenmore frequently in patients with histopathologicallymph node involvement pn in various othertumor entities expanded morphological criteria suchas texture and border contour oflymph nodes areused for the assessment oflymph node malignancyon both computed tomography ct and magneticresonance mr imaging this is utilized in order toimprove the accuracy of lymph node staging []by applying morphological criteria instead of brown size criterion alone the sensitivity was improvedfrom to and the specificity from to inlymph node staging of rectal cancer thus the aim of this study was to determine thelymph node staging in patients withaccuracy ofductal adenocarcinoma ofthe pancreatic head byboth computed tomography and magnetic resonanceimaging using sizeand expanded morphologicalcriteriamaterial and methodspatientsin this retrospective singlecenter study approved by thelocal ethics committee consecutive patients with histologically proven ductal adenocarcinoma of the pancreatichead that underwent primary surgery between february and november at the department of surgerycampus benjamin franklin charituniversity medicine berlin germany were included patients were retrieved from the database of our pancreatic cancercenter certified by the german cancer society n inclusion criteria were primary oncologic tumor resection and the presence of preoperative crosssectional imaging of sufficient quality see below exclusion criteriawere neoadjuvant therapy presence of a potential simultaneous cause of lymphadenopathy of the upper abdominal region eg abdominal lymphoma neuroendocrinetumor and main tumor mass located outside the pancreatic head on histopathology the process of patientselection with the respective reasons for inclusion andexclusion is shown in fig crosssectional imagingall images were retrospectively analyzed for the purposeof this study by a single abdominal radiologist with morethan years of experience in staging of tumors of the visceral ans blinded to the results of histopathologyall crosssectional imaging studies were assessed forsufficient image quality by the radiologist prior to commencement for ct imaging the minimum quality wasdefined as either thinsection ct mm reconstructedslice thickness or contrastenhanced ct with a slicethickness of mm for mri minimum quality was defined as availability of an axial t2weighted sequencewith fat suppression slice thickness mm in combination with a venous phase postcontrast 3d gradientecho sequence slice thickness mmfor lymph node assessment all visible regional lymphnodes in the field of view were recorded on a score chartand the total number of visible lymph nodes per patientwas calculated then for each patient all lymph nodeswere characterized in terms of size long and shortaxisdiameter in millimeters and the expanded morphological criteria border contour lobulated spiculated indistinct or unaltered and texture homogeneous orinhomogeneous fig based on kim regional lymph nodes of the pancreas are defined asthe following lymph node station numbers 8a8p 11p 11d 12a 12b 12p13a 13b14p14d 17a17b and in all cases in which a lymph node wasnot definitively regional correlation with postoperativecrosssectional imaging was performed to assess whetherthe lymph node was resected or not only resectedlymph nodes were analyzed in this study 0cloch world of surgical oncology page of fig flowchart of patient recruitment the process of patient selection with the respective reasons for inclusion and exclusion is showna second radiologist with more than years of experience in staging of tumors of the visceral ans alsoblinded to the results of histopathology evaluated thect examinations of a representative subgroup of patients for evaluation of interobserver agreementsurgeryall patients underwent primary oncologic pyloruspreserving pancreaticoduodenectomy or whipple procedure with complete lymphadenectomy of the regionallymph nodes mentioned aboveformalinembedded surgicalhistopathologythe original histopathological reportsfor the studyspecimens wereusingreviewed cancer of the pancreatic head was defined as amalignant tumor located within the pancreas to the rightof the superior mesenteric vein and portal vein each patient with histologically proven lymph node metastaseswas classified as nodepositive pn regardless of thenumber of metastatic lymph nodes patients without anymetastatic lymph nodes were classified as nodenegativepn the ratio of metastatic lymph nodes vs the totalnumber of retrieved lymph nodes was documented infig morphological characterization of lymph nodes based on kim the morphological criteria for lymph node assessment used inthis study are shown smooth and homogeneous lymph nodes were considered normal 0cloch world of surgical oncology page of the histopathological report eg or tumorswere classified according to their respective tnm stageusing the 8th edition of tnm classification of malignant tumors table demographic data of patients with ductal adenocarcinomaof the pancreatic head undergoing primary tumor resectionpatientsagen comparison of crosssectional imaging andhistopathologysensitivity specificity and positive predictive value ofthe nodal status using ct and mri with histopathologyas a reference standard were calculated for lymph nodeinvolvement using size and morphological criteria specifically nodal involvement criteria were based on eithersize shortaxis diameter altered border contour lobulated spiculated or indistinct and inhomogeneous signal intensity fig ct and mri examinations wereconsidered nodepositive cnif at least one lymphnode met one of the respective criteria used for involvement if no lymph node with the respective criteria wasseen on ct or mri then the examination was considered nodenegative cnstatistical analysissensitivities specificities and positive predictive valueppv for the size criterion and all morphological criteria were calculated for their respective cutoff valuesan index summarizing the sensitivity and specificity foryoudens index was calculated sensitivity specificity the number of lymph nodes visible on ctand mr images in the group with pn and withoutpn nodal metastases was compared using the mannwhitney u test when calculating the association between ct and mri criteria and lymph node positivitythe Ï2test was used interobserver agreement was calculated using cohens kappa statistic a p value of was considered to indicate a statistically significantdifferenceresultspatientssixtysix patients were included in the study fig with the characteristics of the patients presented intable sixty of these patients were staged by preoperative ct twelve of which had additional stagingby mri and six patients were staged by only mri intwo patientsthe mri examinations were excludeddue to insufficient imaging quality both patients hadsufficient staging by ct and were therefore includedin the study of the patients patients receivedpreoperative biliary drainage eight ofthem werestaged by ct only and two by mri onlycomputed tomography ctlymph nodes were detected by ct in ofthe patients the median number of visible lymph nodesmedian age yearsage range yearssexfemalemalecrosssectional imagingct onlyct and mrimri onlyhistopathological stagingpnpnpt1pt2pt3 was range the smallest visible lymph node was mm of size whereas the largest measured mmshortaxis diameter the mean time between ct andsurgery was days with a median of days range the slice thickness in of the ct examinations was mm or less in five ct examinations slice thickness was mmsize criterion for lymph node involvement onpreoperative ctfigure shows the percentage of patients with pnand without pn lymph node metastases in which alymph node of the respective size was visible mmin table sensitivity specificity and youdens indexare presented for the respective cutoff values lymphnodes of small and medium size mm were visiblein patients with pn and withoutlymph node metastases pnineven frequency large lymph nodes mm wereseen more frequently in the lymph nodepositive group pnthan in the lymph nodenegative group pn the maximumvalue of youdens index for the size criterion was j ci when a cutoff value of mmwas applied yielding a sensitivity of and specificityof additionallylymph nodesgreater than mm on preoperative ct and the histopathological confirmation of a lymph node metastasispn showed a trend towards significance p the presence of 0cloch world of surgical oncology page of fig graph showing lymph node size and morphological criteria of lymph nodepositive and negative patients frequency of regional lymphnodes of the pancreatic head in percent xaxis with different shortaxis diameters and morphological features yaxis in patients with pn redbars or without histologically proven lymph node metastases pn blue bars on preoperative ct imagingexpanded morphological criteria for lymph node involvementon preoperative ctfigure shows the percentage of patients with pnand pn without lymph node metastases in which alymph node of the respective morphological criterionwas visible and table shows the sensitivity specificityand youdens index of the respective criterionlymph nodes of lobulated border contour were visiblewith a similar frequency in patients with pn and without lymph node metastases pn lymph nodes of spiculated or indistinct bordercontour were only occasionally detected in both groups vs in the lymph nodepositivegroup pn and vs in the lymphnodenegative group pnlymph nodes of inhomogeneous signal intensity were detected in only one patient of the lymph nodenegative group pn and more frequently in patients of the lymphnodepositive group pn resulting in the maximum value of youdens index for the morphological criteriaj ci consisting of a sensitivity of and a specificity of the ppv was comparison of size with expanded morphological criteriathe maximum value of the youdens index of the sizecriterion was j ci cutoff mmwhich is not inferior to the maximum value of the morphological criteria j ci inhomogeneoussignal intensity figure displays respective ct images ofpatients with and without lymph node metastasesfig ct images of patients with and without lymph node metastases a patient with enlarged suspicious lymph node adjacent to the portalvein and hepatic artery who had no lymph node metastases on pathology b patient with enlarged suspicious lymph node adjacent to thehepatic artery who had lymph node metastases on pathology c patient with multiple suspicious lymph nodes based on size and inhomogeneitywho had lymph node metastases on pathology 0cloch world of surgical oncology page of table sensitivity specificity ppv and youdens index for cutoff values and morphological criteria by ct and mrisensitivity specificity ppvyoudens indexctsize cutoff value mm mm mm mm mm mm mm mmmorphological criterionlobulatedspiculatedindistinctinhomogeneous mrisize cutoff value mm mm mm mm mm mm mm mm mmmorphological criterionlobulatedspiculatedindistinctnot visibleinhomogeneousnot visible number of visible lymph nodesthere was a significant association between number of visible lymph nodes seen on preoperativect and histopathologicallymph node involvementpn p seven or more lymph nodes were seen on preoperative ct in of patients with lymph nodemetastases pn and in of patients without lymph node metastasis pn p this resulted in a specificity of youdens index of and ppv of combination of number size and expanded morphologiccriteria on preoperative ctcombining the size criterion and the morphological criterion with the respective highest youdens index cutoff mm and inhomogeneous signalintensity andthe criterion visible lymph nodes n ¥ was significantly associated with nodal metastases pn p for this combined criterion specificity was sensitivity ppv and youdens index ci interobserver agreementinterobserver agreement was calculated for patientspn pn vs pnfor the criteria sizemorphology and number of visible lymph nodes withthe respective highest youdens indexinterobserveragreement was substantial for size mm cutoff κ p moderate for the presence of seven ormore lymph nodes κ p and fair forthe morphological criterion inhomogeneous signal intensity κ p magnetic resonance imaging mrilymph nodes were detected in of patientson preoperative mri the median number of visiblelymph nodes was range there was no significantassociation between number of visible lymph nodes seenon preoperative mri and histopathological lymph nodeinvolvement pn p the smallest visible lymph node was mm of sizewhereas the largest measured mm shortaxis diameter the mean time between mri and surgery was days with a median of days range the cutoff values of the highest diagnostic value were mm or mm for the size criterion sensitivity specificity youdens index the presence ofa lymph node of these sizes was not associated with lymphnode metastases pn p lobulated and spiculated lymph nodes were only seen in a few patients n and n and indistinct and inhomogeneous lymphnodes were not seen at all table discussionin this retrospective singlecenter study on lymph nodestaging by ct in ductal adenocarcinoma of the pancreatic head we could show that the morphologic criteriainhomogeneous signal intensity and size are specificfor regional nodal metastatic disease replacing the sizecriterion by morphologic criteria however did not improve diagnostic accuracy due to sensitivity remaininglow combining specific criteria yields improved sensitivity with specificity remaining highby ct lymph nodes of mm in shortaxis diameterwere seen just as often in patients with and without 0cloch world of surgical oncology page of lymph node metastases resulting in poor discriminationlarger lymph nodes mm had a higher prevalence inthe lymph nodepositive group leading to high specificity however these lymph nodes mm or mmwere seen infrequently resulting in a rather low sensitivity the maximum value of the youdens index for thesize criterion of was achieved when a cutoff valueof mm was applied consisting of a specificity of and sensitivity of yielding a ppv of as for morphologic criteria lymph nodes of lobulatedborder contour were seen in about half of the patients ofboth groups pn and pn and therefore isa criterion that is not suitable to differentiate betweenthe groups lymph nodes of spiculated and indistinctborder contour were seen in few cases in both patientgroups only pn and versus pn and making them poor diagnostic criteria however lymphnodes of inhomogeneous signal intensity were visible in of patients with lymph node metastases pn andonly in of the patients without lymph node metastases pn resulting in a youdens index of whichwas the maximum value for the morphological criteriaand a ppv of ideally a good discriminator for nodal metastases isnegative in patients without nodal involvement and positive for tumors with lymph node metastases in ourstudy each criterion ie size as well as different morphological features only met one of these prerequisitesthe size criterion mm as well as the presence of alymph node of inhomogeneous signal intensity as morphological criterion turned out to be negative in patientswithout nodal involvement pn and therefore highlyspecific yet lymph nodes of the respective characteristicwere not positive in a sufficiently high number of tumorswith lymph node metastases pn to reach high levelsof sensitivity and consequently did not have a significantdiagnostic valuethe maximum value of the youdens index for the sizecriterion was when a cutoff value of mm wasapplied and for the morphological criteria whenthe criterion inhomogeneous signal intensity was usedshowing that morphologic criteria do not yield in higherdiagnostic value than lymph node size in adenocarcinoma of the pancreatic head pdac patients this iscontrary to the findings of brown in rectal cancer one reason might be that brown used mri toassess morphologic criteria which has a higher soft tissuecontrast compared to ct which was used in most patients in our studyinterestingly we could show that with preoperativect the presence of seven or more lymph nodes wasseen more often in patients with lymph node metastasispn than in those without metastasis pn p when applying this as a sole criterion cn for lymphnode metastasis pn this led to a sensitivity of aspecificity of ppv of and youdens indexof in diagnostic test analysis criteria can be combinedin mainly two ways sensitive criteria can be taken together to improve specificity or specific criteria canbe accumulated to improve sensitivity when combining the highly specific criteria size cutoff value mm inhomogeneous signal intensity and number ofvisible lymph nodes n ¥ a highly significant association with nodal metastases pn p wasfound consequently the ct examination was considered nodepositive cn when at least one of thesecriteria was met the application of this criterion improved the sensitivity to with a remaining specificity of and ppv resulting in an alsoimproved youdens index of the results of the mri examinations must be viewedin a rather descriptive manner since the sample size waslimited n lymph nodes were detected in the majority of examinations generally allowing theevaluation of lymph nodes by mri as well upper abdominal mri generally has a lower spatial resolutionbut a higher soft tissue contrast compared to ct forthe size criterion a cutoff value of mm or mm ledto the best diagnostic results sensitivity specificity youdens index lymph nodes of abnormal morphological criteria were seen in only veryfew patients table the main limitation of this study is the retrospectivestudy design in which a nodebynode comparison ofcrosssectionalimaging with histopathology was notpossible this was of minor importance though sincelow sensitivity was the main factor that led to compromised diagnostic performance in our study we werealso able to correlate with postoperative crosssectionalimaging in all cases in which it was unclear whether alymph node had been resected during surgery or notalso in our cohort only patients who had subsequentsurgery were included presuming lower tumor stage ascompared to the average patient who undergoes imagingfor presurgical workupthe strength of our singlecenter study is reinforcedby a defined number of surgeons a high standardizationof the ct technique and an experienced radiologist whoperformed the analysisthe results of our study are consistent with recent andinitial data demonstrating that clinical staging by lowsensitivity underestimates histopathological lymph nodeinvolvement pn [ ] however by adding thecriterion inhomogeneous signal intensity and numberof visible lymph nodes to the size criterion we wereable to increase the sensitivity to in comparison toprevious findings roche nanashima 0cloch world of surgical oncology page of and cao with specificity remainingsufficientan additional imaging modality that has shown the potential to improve the sensitivity of detecting metastaticdisease is positron emission tomographycomputed tomography petct however a beneficial role ofpetct in locoregional nodal staging could not be established to date the majority of initial as well as recentstudies show very limited sensitivities for nodal status between and [] the petpanc study evaluated the incremental diagnostic accuracy and impact ofpetct in addition to multidetector ct in patients withsuspected pancreatic cancer in a prospective multicenterstudy that included patients in this study significantlymore patients with stage iib disease pn were correctlystaged by petct than by multidetector ct p but this only led to a moderate sensitivity of for petct versus for multidetector ct endoscopic ultrasonography eus is a wellestablisheddiagnostic procedure in pancreatic cancer with the benefitof a dynamic diagnostic examination that allows fineneedle aspiration for cytologic diagnosis two metaanalyses evaluating diagnostic accuracy of eus for locoregional nodal staging the pooled sensitivities and specificities were and li studies n patients and and nawaz studiesn patients advanced techniques such ascontrastenhanced eus cheus and eus elastographyare currently in evaluation to date ct remains the standard staging imaging modality recommended by nccn guidelines for locoregional staging of pancreatic cancer neither petctnor eus yields reliable diagnostic accuracy for nodalstagingan advantageous effect on resectability and overallsurvival os in unresectable cases including both borderline resectable and unresectable of pdac by multimodality therapy including neoadjuvanttherapy hasalready been described in several studies the benefit of neoadjuvant therapy in cases of primarily resectable disease at diagnosis is yet less revealedseveral phase ii trials showed that patients who completed neoadjuvant chemoradiation without progressivedisease at restaging had a higher chance of achieving r0resection and consequently higher median and oswhen compared to historical data as seen in othertumor entities a potential benefit of neoadjuvant therapyon the basis of positive nodal status cn is stronglyimplied cao found that the of patients thatwere successfully downstaged from nodepositive diseasecn1 to nodenegative disease ypn0 by neoadjuvanttherapy benefit in terms of higher rates of 5year survivalypn0 vs ypn1 p this is consistent with the findings of portuondo 5yearsurvival ypn0 vs ypn1 p the nccn guidelines for pancreatic adenocarcinomaappreciates these results by stating that considerationcan be given to neoadjuvant therapy for selected patientswith resectable tumor but poor prognostic features suchas large regional lymph nodes markedly elevated ca large primary tumors extreme pain and excessiveweight loss further clarification on this matter isexpected to come from the ongoing neonax trialnct02047513 a phase ii study comparing neoadjuvant plus adjuvant with only adjuvant nabpaclitaxelplus gemcitabine therapy forresectable pancreaticcancer theiii neopa trialphasenct01900327 compares neoadjuvant chemoradiotherapy with upfront surgery of resectable pancreatichead cancer a subgroup analysis in terms of nodalstatus would present reliable dataongoinggiven the suggested benefit of neoadjuvant therapybased on lymph node staging there is an urgent need tofind criteria and modalities to further improve the diagnostic value of lymph node staging by pretherapeuticcrosssectional imaging in patients with ductal adenocarcinoma of the pancreatic head to date none of theexisting modalities and criteria accomplishes reliablenodal staging larger prospective studies are ongoingand necessary to get a more precise idea of the prognostic advantage of neoadjuvant therapy in patients with regionalcn of pdac inpretherapeutic staginglymph node metastasisslymph node staging in pdac patients when using ctmorphological criteria such as border contour or homogeneity compared to diameter cutoff values does notlead to reliable diagnostic value diagnostic accuracy islimited due to low sensitivity for detection of metastaticdisease combining specific criteria yields improved sensitivity with specificity and ppv remaining high theseresults suggest an attentive interpretation of the resultsof pretherapeutic lymph node staging particularly incases in which lymph node metastases are absentabbreviationspdac adenocarcinoma of the pancreatic head ct computed tomographymri magnetic resonance imaging pn histopathologically involved lymphnodes pn histopathologically no involved lymph nodes cn clinicallyinvolved lymph nodes cn clinically no involved lymph nodes ppv positivepredictive value petct positron emission tomographycomputed tomography eus endoscopic ultrasonography cheus contrastenhancedendoscopic ultrasonography os overall survivalacknowledgementswe acknowledge support from the german research foundation dfg andthe open access publication funds of charit universittsmedizin berlinauthors contributionsall authors were involved in data acquisition and manuscript revision theconception of the design of the study and drafting of the manuscript was 0cloch world of surgical oncology page of done by fn loch c kamphues and p asbach image analysis was performedby p asbach and assisted by fn loch image analysis of the subgroup foranalysis of interobserver agreement was performed by m haas all authorshave approved the submitted version of the manuscript and account fortheir own contribution and the accuracy as well as the integrity of the workpresentedfundingthe study was not fundedavailability of data and materialsthe datasets used during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatethe study was approved by the local ethics committee of the charituniversity medicine berlin informed consent of participation was waived bythe irb given the retrospective study design irb no ea411418consent for publicationconsent for publication is not applicable for this studycompeting intereststhe authors declare that they have no competing interestsauthor details1charit universittsmedizin berlin corporate member of freie universittberlin humboldtuniversitt zu berlin and berlin institute of healthdepartment of surgery campus benjamin franklin hindenburgdamm berlin germany 2charit universittsmedizin berlin corporatemember of freie universitt berlin humboldtuniversitt zu berlin and berlininstitute of health department of radiology campus benjamin franklinhindenburgdamm berlin germany 3the johns hopkins universityschool of medicine department of surgery n wolfe street blalock baltimore md usareceived december accepted july zeman rk cooper c zeiberg as kladakis a silverman pm marshall jl tnm staging of pancreatic carcinoma using helical ct am jroentgenol m¼ller mf meyenberger c bertschinger p schaer r marincek b pancreatictumors evaluation with endoscopic us ct and mr imaging radiology midwinter mj beveridge cj wilsdon jb bennett mk baudouin cj charnleyrm correlation between spiral computed tomography endoscopicultrasonography and findings at operation in pancreatic and ampullarytumours br j surg r¶sch t braig c gain t feuerbach s siewert jr schusdziarra v staging of pancreatic and ampullary carcinoma by endoscopicultrasonography comparison with conventional sonography computedtomography and angiography gastroenterology prenzel kl h¶lscher ah vallb¶hmer d drebber u gutschow ca m¶nig sp lymph node size and metastatic infiltration in adenocarcinoma of thepancreatic head eur j surg oncol rollvn e blomqvist l ¶istm¶ e hjern f csanaky g abrahamnordling mmorphological predictors for lymph node metastases on computedtomography in colon cancer abdom radiol brown g richards cj bourne mw newcombe rg radcliffe ag dallimorens morphologic predictors of lymph node status in rectal cancer withuse of highspatialresolution mr imaging with histopathologic comparisonradiology kim jh beets gl kim mj kessels agh beetstan rgh highresolution mrimaging for nodal staging in rectal cancer are there any criteria in additionto the size eur j radiol isaji s murata y kishiwada m new japanese classification of pancreaticcancer in neoptolemos j urrutia r abbruzzese j b¼chler mw editorspancreatic cancer new york springer p brierley jd gospo | Colon_Cancer |
thyroid carcinoma is presently the malignancy with the most rapidly increasing incidence in the worldand is the most widely recognized endocrine carcinoma in the western world thyroid cancers derivedfrom follicular thyroid cells can be sorted into papillary thyroid carcinoma ptc follicular thyroid carcinoma ftc and anaplastic thyroid carcinoma atc according to the histological subtype clinicalresults vary across these subtypesthe annual rate of thyroid cancer has more than doubled within the past two decades with the vast majority of this increase being ascribed to ptc which accounts for of all thyroid carcinomas inaddition patients with ptc suffer from cervical lymph nodes metastasis or remote metastasis which leadsto unfavorable results and approximately of cases may progress to a potentially fatal recurrentailment due to these reasons uncovering the causes of ptc and its fundamental mechanisms andfinding molecular biomarkers for early diagnosis and customized treatment are significant and important the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555taskswith the advancement and continuous improvement of gene sequencing and geneediting technology it is nowconvenient to recognize the hub biomarkers related to neoplasm metastasis and survival status using a large amountof information available by applying bioinformatics currently there are no effective sensitive biomarkers for earlydiagnosis treatment and prevention of lymph node metastasis of ptc an examination of differentially expressedgenes degs between tumor and paracarcinoma tissue may help identify critical biomarkers of papillary thyroidcarcinoma as a form of molecular marker mrna containing the most abundant genetic information is necessaryfor protein translation and it is separate from the pathological process of cancer at various stages some studiesutilized public databases such as the cancer genome atlas tcga and the gene expression omnibus geo toidentify significant biomarkers of papillary thyroid carcinoma however these investigations were only founded onsingle datasets with constrained sample sizes or just based on online databases used to screen out the degsin the present study we analyzed the degs in ptc tissues versus the matched adjacent tissues by rnaseq andbioinformatics methods to obtain the degs then we screened out the key modules and extracted the key genes inthose modules by constructing degs interaction network then the possible role of differentially expressed geneswas analyzed using go annotation and kegg pathway enrichment analysis the expression validation survivalanalysis and functional enrichment analysis of key genes were conducted by using relevant databases finally wefound that the three genes adora1 apoe and lpar5 were highly expressed in ptc and were associated withptc methylation tnm staging and immune infiltrationmethodstissue samplesthirty pairs of ptc and adjacent tissues were collected from january to july at the first affiliated hospitalof hebei north university this experiment was approved by the ethical committee of the first affiliated hospitaland all patients provided informed consent all tissues were frozen in liquid nitrogen after surgical resectionrna library construction and sequencingtotal rna was isolated from four adjacent normal and cancerous thyroid samples utilizing trizol reagent qiagenvalencia ca usa as indicated by the manufacturers guidelines rnas of ptc tissues and paracancerous tissuessample numbers 1c 1p 2c 2p 3c 3p 4c 4p the number represents different samples the c indicates a cancersample and the p represents a matched paracancerous tissue sample were used six libraries were built utilizingan illumina standard kit as indicated by the manufacturers protocol all sequencing was carried out on an illuminahiseq lc bio chinadifferentially expressed genes screeningthe level of expression of mrnas was evaluated using stringtie by calculating fpkm the degs between ptcand paracancerous tissue were screened with log2 fold change1 and p005 was regarded as statistically significant the analyses were conducted using the r package ballgown functional enrichment analysis and pathway analysisto reveal the functional roles of the degs the annotation visualization and integrated discovery function annotation tool david httpdavidabccncifcrfgovhomejsp was used to perform gene ontology go enrichmentanalysis and kyoto encyclopedia of genes and genomes kegg pathway enrichment analysis p values less than were considered as cutoff criteriappi network construction and identiï¬cation of hub genesppi networks were constructed successively using string database tringdb the interactions ofdegs with a combined score were set as significant and cytoscape software version was utilized tovisualize and analyze the results of the string database to find key hub genes in this ppi network the significantmodule was analyzed by using the plugin mcode of cytoscape software the criteria for selection were set to thedefault the key genes were chosen with degrees ¥ subsequently genes in that module were used to analyse theirfunctional roles with funrich software the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555table pcr primersgene symboladora1actinapoelpar5bp base pair f forward primer r reverse primerprimer sequencef5cid3ccacagacctacttccacacc3cid3r5cid3taccggagagggatcttgacc3cid3f5cid3cactcttccagccttccttcc3cid3r5cid3aggtctttgcggatgtccac3cid3f5cid3 gttgctggtcacattcctgg 3cid3r5cid3 gcaggtaatcccaaaagcgaccid3f5cid3 cacttggtggtctacagcttg3cid3r5cid3 gcgtagtaggagagacgaacg3cid3data validation and analysisto verify the accuracy of our rnaseq results we used the gene expression profiling interactive analysis databaseto verify the expression of key genes in ptc and adjacent tissues the overall survival and diseasefree survivalanalyses were performed by kaplanmeier plots for these ptcrelated hub genes genetic alterations of hub genesin ptc and their correlations with other genes were analyzed utilizing the cbioportal for cancer genomics hub genesrelated to clinicopathological features were analyzed using the online database ualcan httpualcanpathuabedu the correlation of adora1 apoe and lpar5 expression with the immune infiltration level in ptc and theexpression of these three genes in different kinds of cancers was performed using the tumor immune estimationresource database for qrtpcr analysis total rna was isolated from normal and cancerous papillary thyroid samples utilizingtrizol reagent qiagen valencia ca usa cdna was synthesized with rna reverse transcription kit tiangenbiotech beijing china qrtpcr was performed with an abi realtime pcr system applied biosystems life technologies usa the expression of the genes of interest was normalized to actin the primers foradora1 apoe lpar5 and actin are shown in table for western blot ripa buffer was used to extract protein from four pairs of tissue from ptc patients and theprotein concentrations were measured via bca methods briefly the sdspage gel was used for electrophoresis andpdvf membrane was used for transmembrane transfer pdvf membrane was blocked and then incubated with primary antiadora1 antibodies dilution bioss bs6649r apoe dilution bioss bs4892r lpar5antibodies dilution bioss bs15366r and actin dilution bioss bs0061r at ¦c overnight followed by incubation with secondary antibodies zhongshanjinqiao dilution at ¦c for h the signal wasdetected using ecl methodstatistical analysisall the data were analyzed by r and spss spss inc usa kaplanmeier method was used to estimate thesignificant difference in survival between the overexpression group and the lowexpression group of key genes inpapillary thyroid carcinoma the statistical difference was set at p resultsdifferentially expressed genes screening based on rnaseqto screen out the genes or modules that may play a role in promoting cancer in papillary thyroid carcinoma weperformed rnaseq experiments on four pairs of thyroid cancer tissues and their matched paracancerous tissues toobtain differentially expressed genes after rnaseq we acquire million reads for each sample the fold changesbetween ptc cancer tissues and matched paracancerous samples were calculated setting the cutoff criterion as pvalue and a fold change there were upregulated and downregulated genes these degswere considered to be candidate genes for subsequent study figure 1a showed the expression of the top genes inptc versus matched paracancerous tissuesfunctional enrichment analysis and pathway analysisconsidering that there were many falsepositive genes among these degs we verified our results one by onethrough the tcga database we found that only genes in our data were consistent with the gene expression of the the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure identiï¬cation of degs by rnaseqthe heat map a and ppi network of the degs b c the volcano plots of the degs d the most significant module was selectedby mcode in cytoscape red represents the upregulated genes and blue represents the downregulated genesfigure go and kegg pathway enrichment analysis of degs through rnaseqa bubble plot of gene ontology enrichment analysis of degs b bubble plot of kyoto encyclopaedia of genes and genomespathway enrichment analysis of degstcga database to investigate the potential function of these degs in ptc genes functional enrichment was conducted by using go and kegg pathway analyses for the biological process category the degs were significantly involved in the regulation of axonogenesis regulation of cell morphogenesis extracellular structure anization extracellular matrix anization synapse anization cellsubstrate adhesion and urogenital system development thecellular component category results showed ptcrelated degs were enriched in collagencontaining extracellularmatrix synaptic membrane cellcell junction glutamatergic synapse neurontoneuron synapse postsynaptic membrane basolateral plasma membrane degs in molecular function were mainly involved in cell adhesion moleculebinding passive transmembrane transporter activity extracellular matrix structural constituent glycosaminoglycanbinding growth factor binding transmembrane receptor protein kinase activity and transmembrane receptor proteintyrosine kinase activity figure 2aas figure 2b showed the kegg pathway results showed degs were enriched in cytokinecytokine receptorinteraction mapk signaling pathway proteoglycans in cancer rap1 signaling pathway axon guidance cushing the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure go enrichment analysis and kegg analysis for the key genesa top elements involved in biological processes b top elements involved in molecular function c top elementsinvolved in cellular components d top pathways related to the key genes through kegg analysissyndrome parathyroid hormone synthesis secretion and action agerage signaling pathway in diabetic complications growth hormone synthesis secretion and action salivary secretion circadian entrainment cholinergic synapse p53 signaling pathway ecmreceptor interaction arrhythmogenic right ventricular cardiomyopathyarvc endocrine resistance renin secretion type ii diabetes mellitus bladder cancer nicotinate and nicotinamidemetabolism and apoptosismultiple speciesppi network construction and module analysisppi networks were constructed successively by the database by loading the ptc related dges into the stringdatabase figure 1bc using cytoscape we analyzed the most significant module in the ppi network figure 1dthe ppi network consisted of nodes and edges following the use of mcode in cytoscape the significantmodule was selected the top hub genes adcy8 adora1 adra2c apoe c5ar1 ccl13 ccl20 cdh2chgb cxcl12 eva1a fam20a fn1 gnai1 gpc3 grm4 lpar5 meltf or mfi2 mfge8 nmu oprm1serpina1 sstr3 timp1 and tnc were evaluated by degree in the ppi network figure 1d the resultsshowed that the functions of the key genes were mainly concentrated in signal transduction cell communicationgprotein coupled receptor activity cell adhesion molecule activity and gpcr ligand binding figure data analysis and validationafter the key genes were selected the expression of key genes in ptc and its adjacent tissues were verified by thegepia database figure adora1 apoe eva1a lpar5 mfge8 oprm1 serpina1 sstr3 and timp1were positively related to the overall survival analysis of ptc patients while c5ar1 and gnai1 were negativelyrelated figure adcy8 adora1 chgb fn1 lpar5 nmu and tnc showed positive associations withdiseasefree survival analysis of ptc patients but not apoe figure next we analyzed the alterations of the key genes by using the cbioportal database figure the key geneswere changed in of queried samples figure 7b figure 7a showed the frequency of alterations of eachptc related key gene sstr3 fn1 and adora1 were altered the most and respectively figure 7dshowed the network of the genes and their altered neighbouring genes in ptc patients out of a total of among these genes only adora1 apoe and lpar5 genes simultaneously showed statistical significance foroverall survival analysis and diseasefree survival analysis of ptc patients the qpcr experiments and western blotdata verified that these three survivalrelated genes were all overexpressed in ptc figure then based on ual the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure validation of the key degs in the gepia databaseadora1 apoe ccl13 cdh2 cxcl12 eva1a fam20a fn1 gnai1 lpar5 mfge8 nmu serpina1 timp1 and tnc areoverexpressed in ptc tissues compared with paracancerous tissue while gnai and gpc3 are downregulated the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure overall survival analysis of key genes in ptc using kaplanmeier plotsexpression levels of adora1 apoe c5ar1 eva1a fam20a gnai1 lpar5 mfge8 oprm1 serpina1 sstr3 and timp1are related to the overall survival of patients with ptccan the clinical features and degree of methylation of these three genes were analyzed the transcription levels ofadora1 apoe and lpar5 were significantly higher in ptc patients than normal tissues according to subgroupsof sample types individual stages and nodal metastasis status figure in addition ador1 and lpar5 exhibiteda hypomethylation state in the cancer group but apoe showed a hypermethylation state in ptc samples figure10ato further clarify the role of these genes we conducted an analysis of immune infiltration the ador1 expression level was positively corelated with infiltrating levels of b cells r0111 p151e2 cd8 t cells r0246p396e neutrophils r0162 p331e and dcs r0232 p232e the expression of apoe was the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure diseasefree survival analysis of key genes in ptc using kaplanmeier plotsexpression levels of adcy8 adora1 apoe chgb fn1 lpar5 nmu and tnc are significantly related to the diseasefreesurvival of patients with ptcpositively associated with b cells r0228 p439e cd8 t cells partialcor p930e neutrophils r0197 p114e and dcs partialcor p358e lpar5 expression level was positively related to b cells r0259 p815e cd4 t cells r0238 p103e macrophages r0175p105e neutrophils r027 p142e and dcs r0256 p104e and negatively related to purity r p294e and cd8 t cells r p618e figure 10b these findings stronglysuggested that lpar5 ador1 and apoe may play specific roles in immune infiltration in ptc especially those ofdcs finally we examined the expression of these three genes in common cancer tissues and adjacent tissues and wefound that these three genes were highly expressed in most cancer tissues figure the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure the key genes expression and mutation analysis in ptc by the cbioportal for cancer genomicsa the genetic alterations of key genes of ptc samples queried genes are altered in of queried patientssamplesb the expression heatmap of key genes c the alteration frequency of key genes in ptc d network of key genesmutations and their frequently altered neighboring genes in ptc the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure the mrna and protein expressions of adora1 apoe and lpar5 in ptc tissuesac validation of expression levels of adora1 apoe and lpar5 by rtqpcr in cases of ptc and matched adjacent tissuesd adora1 apoe and lpar5 protein levels are increased in four cases of ptc and matched adjacent tissues as measured bywestern blot p0001discussionptc is a common cancer with great heterogeneity in morphological features and prognosis although most papillary thyroid carcinomas exhibit low biological activity there are still a few patients with higher invasive and metastaticclinical features activation of oncogene expression and loss of function of tumor suppressor genes may lead tothe development or progression of ptc to better clarify the molecular mechanism of ptc occurrence development and metastasis we identified key genes related to ptc progression through comprehensive bioinformaticsmethods and we screened three of the ptc prognosisrelated genes for a comprehensive analysisin the present study we identified differentially expressed genes by rnaseq with go enrichment analysis showing that the degs were enriched in the regulation of the axonogenesis regulation of cell morphogenesis extracellular structure anization extracellular matrix anization synapse anization cellsubstrate adhesion urogenital system development collagencontaining extracellular matrix synaptic membrane cellcell junction glutamatergic synapse neuron to neuron synapse postsynaptic membrane basolateral plasma membranecell adhesion molecule binding passive transmembrane transporter activity extracellular matrix structural constituent glycosaminoglycan binding growth factor binding transmembrane receptor protein kinase activity andtransmembrane receptor protein tyrosine kinase activity and kegg pathway results showed degs were enrichedin cytokinecytokine receptor interaction mapk signaling pathway proteoglycans in cancer rap1 signaling pathway axon guidance cushing syndrome parathyroid hormone synthesis secretion and action agerage signalingpathway in diabetic complications growth hormone synthesis secretion and action salivary secretion circadian entrainment cholinergic synapse p53 signaling pathway ecmreceptor interaction arrhythmogenic right ventricularcardiomyopathy arvc endocrine resistance renin secretion type ii diabetes mellitus bladder cancer nicotinateand nicotinamide metabolism and apoptosismultiple speciesto further explore the interrelationship of differentially expressed genes in papillary thyroid carcinoma we constructed a ppi regulatory network a total of degs with nodes greater than were screened out in the networkthe key genes were adcy8 adora1 adra2c apoe c5ar1 ccl13 ccl20 cdh2 chgb cxcl12 eva1afam20a fn1 gnai1 gpc3 grm4 lpar5 meltf mfge8 nmu oprm1 serpina1 sstr3 timp1 andtnc biological process and molecular function analyses of these key degs indicated that they were significantly the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure relative expression of adora1 apoe and lpar5 in normal thyroid tissues and ptc tissues individual cancerstages and nodal metastasis status respectively ualcanp0001involved in cancer regulation processes such as adjustment of cell growth or maintenance cell immune response celladhesion molecular activity and extracellular matrix structural constituentto verify the credibility of the experiments and data the degs screened were verified by the gepia databaseamong the selected genes genes showed expression differences consistent with our rnaseq data among the the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure methylation level and immune inï¬ltration level of adora1 apoe and lpar5a relative methylation level of adora1 apoe and lpar5 based on normal thyroid tissues and ptc tissues individual cancerstages and nodal metastasis status respectively ualcan b the correlation between the three genes and tiics timer tiicstumor infiltrating immune cells genes adora1 apoe ccl13 cdh2 eva1a fam20a fn1 lpar5 mfge8 nmu serpina1 timp1and tnc levels were overexpressed in ptc tissues while gpc3 and gnai1 were downregulatedadora1 belongs to the gprotein coupled receptor family and protects human tissues and cells under physiological conditions lin et al suggested that adora1 may promote the proliferation of breast cancer cellsby positively regulating oestrogen receptoralpha in breast cancer cells similarly jayakar indicated that knockdown of apoe expression can reduce the level of mmps by regulating the ap1 signaling pathway and thus reducethe invasion and metastasis of oral squamous cell carcinoma bioinformatics predictions were that apoe mrnashows a significant increase in ptc ccl13 is a coding gene involved in immune regulation and inflammatoryresponses and it has been reported that ccl13 has a role in promoting the proliferation of tumorforming volumein nude mice cdh2 is overexpressed in various cancers some research results indicate that overexpression ofcdh2 can increase the invasive ability and induce emt in lung cancer cells qiu et al confirmed cdh2 actsas an oncogene in papillary thyroid carcinoma which is consistent with our findings eva1a acts as a regulatorof programmed cell death and shen et al indicates that eva1a can inhibit the proliferation of gbm cells by inducing autophagy and apoptosis via inactivating the mtorrps6kb1 signaling pathway fam20a may play a keyrole in haematopoiesis there are few reports on the relationship between fam20a and cancer and our experimentfound that fam20a is more highly expressed in papillary thyroid carcinoma than in other cancers fn1 is involvedin regulating cell adhesion cell movement wound healing and maintaining cell morphology some researchersindicated that fn1 participates in regulating many types of cancer progression such as gastric cancer skin squamous cell carcinoma and papillary thyroid carcinoma it has been shown that lpar5 is related tothe pathogenesis of pancreatic cancer consistent with our study zhang et al believes that lpar5 may be involved in the development of papillary thyroid carcinoma according to previous reports mfge8 is involvedin the progression of various malignancies such as breast cancer melanoma bladder tumors and ovarian cancer the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure the expression of adora1 apoe and lpar5 in thyroid cancer tissues and normal thyroid tissuesthe three genes expression were analyzed in different kind of cancer tissues and normal tissues via the timer database p005p001 p001 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555[] mfge8 is considered to be a potential therapeutic target for ovarian cancer owing to its carcinogenic effect consistent with our data zhang et al indicate that nmu is one of the degs of papillary thyroid carcinoma recently a researcher has shown that abnormal expression of nmu is associated with a variety of cancers for serpina1 there are currently six s pointing out that serpina1 may be a key gene for ptc consistentwith our results [] clinical studies have shown that high expression of timp1 is positively correlated witha poor prognosis of colon brain prostate breast lung and several other cancers tnc is a component of theextracellular matrix ecm and is closely related to the malignant biological behavior of cancer in particular tncoverexpression is positively associated with liver cancer oral squamous cell carcinoma and lymph node metastasisof breast cancer gpc3 belongs to the glypicans family it has been reported that overexpression of gpc3can promote the metastasis of hepatocellular carcinoma but we found that it is expressed at low levels in ptcsimilar to gpc3 some scholars believe that gnai1 is a tumorpromoting gene and reported upregulated gnai1mrna in human glioma which is inconsistent with our data only the adora1 apoe and lpar5 genes simultaneously showed statistical significance for overall survivaland diseasefree survival of ptc patients considering that the occurrence and metastasis of cancer is a complexand multiregulated process we further analyzed the regulatory mechanisms of these three genes we found thatthe mrna and methylation levels of these three genes were significantly correlated with tnm staging in additionadora1 apoe and lpar5 were all related to immune infiltration especially to dendritic cells finally we foundthat these three genes were more highly expressed in cancer tissues than matched adjacent tissueshowever our research has certain limitations first only four pairs of cancer and adjacent tissues were analyzedusing rnaseq in this experiment so further research requires a larger sample size second further experiments areneeded to validate the specific mechanisms of these key genesdata availabilitythe data used to support the findings of this study are available from the corresponding author upon requestcompeting intereststhe authors declare that there are no competing interests associated with the manuscriptfundingthis study was supported by grants from the hebei provincial department of finance specialist capacity building and specialistleadership program [grant number ] the hebei provincial natural science foundation project [grant number h201840505]and the hebei north university basic research business expenses project [grant number jyt2019015]author contributionxu lin conducted the bioinformatics analysis xu lin and gang xue contributed as first authors xu lin wrote the manuscriptjingfang wu and gang xue critically revised the gang xue and da pei obtained clinical specimens and the others contributed to verification of the rnaseq resultsabbreviationsatc anaplastic thyroid carcinoma ecm extracellular matrix ftc follicular thyroid carcinoma ptc papillary thyroid carcinomareferences kitahara cm and sosa ja the changing incidence of thyroid cancer nat rev endocrinol 101038nrendo2016110 aschebrookkilfoy b ward mh sabra mm and devesa ss thyroid cancer incidence patterns in the united states by histologic type thyroid 101089thy20100021 pourseiraï¬ s shishehgar m ashraf mj and faramarzi m papillary carcinoma of thyroid with nasal cavity metastases a case report iranj med sci ullmann tm gray kd moore md zarnegar r and fahey iii tj current controversies and future directions in the diagnosis andmanagement of differentiated thyroid cancers gland surg 1021037gs20170908 jin x deng b ye k et al comprehensive expression proï¬les and bioinformatics analysis reveal special circular rna expression and potentialpredictability in the peripheral blood of humans with idiopathic membranous nephropathy mol med rep 103892mmr201910671 rapisuwon s vietsch ee and wellstein a circulating biomarkers to monitor cancer progression and treatment comput struct biotechnolj 101016jcsbj201605004 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc by 0cbioscience reports bsr20201555101042bsr20201555 pertea m pertea gm antonescu cm et al stringtie enables improved reconstruction of a transcriptome from rnaseq reads natbiotechnol 101038nbt3122 frazee ac pertea g jaffe ae et al ballgown bridges the gap between transcriptome assembly and expression analysis nat biotechnol 101038nbt3172 von mering c huynen m jaeggi d et al string a database of predicted functional associations between proteins nucleic acids res 101093nargkg034 chandrashekar ds bashel b balasubramanya sah et al ualcan a portal for facilitating tumor subgroup gene expression and survivalanalyses neoplasia 101016jneo201705002 li t fan j wang b et al timer a web server for comprehensive analysis of tumorinï¬ltrating immune cells cancer res e108e11010115800085472can170307 nikiforov ye and nikiforova mn molecular genetics and diagnosis of thyroid cancer nat rev endocrinol 101038nrendo2011142 pusztaszeri m and auger m update on the cytologic features of papillary thyroid carcinoma variants diagn cytopathol 101002dc23703 borea pa gessi s merighi s and varani k adenosine as a multisignalling guardian angel in human diseases when where and howdoes it exert its protective effects trends pharmacol sci 101016jtips201602006 lin z yin p reierstad s et al adenosine a1 receptor a target and regulator of estrogen receptoralpha action mediates the proliferativeeffects of estradiol in breast cancer oncogene 101038onc2009409 jayakar sk loudig o brandweingensler m et al apolipoprotein e promotes invasion in oral squamous cell carcinoma am j pathol 101016jajpath201706016 tan j qian x song b et al integrated bioinformatics analysis reveals that the expression of cathepsin s is associated with lymph nodemetastasis and poor prognosis in papillary thyroid cancer oncol rep kuo cy wang jc hsu sl and hwang gy functional characterization of hepatitis b virus x protein based on the inhibition oftumorigenesis in nude mice injected with ccl13hbx cells intervirology 101159000158522 yamauchi m yoshino i yamaguchi r et al ncadherin expression is a potential survival mechanism of geï¬tinibresistant lung cancer cellsam j cancer res qiu j zhang w zang c et al identiï¬cation of key genes and mirnas markers of papillary thyroid cancer biol res 101186s4065901801881 shen x kan s liu z et al eva1a inhibits gbm cell proliferation by inducing autophagy and apoptosis exp cell res 101016jyexcr201702003 liao yx zhang zp zhao j and liu jp effects of fibronectin on cell proliferation senescence and apoptosis of human glioma cellsthrough the pi3kakt signaling pathway cell physiol biochem 10115 | Colon_Cancer |
tenascinc tnc is an extracellular matrix ecm glycoprotein that plays an important rolein cell proliferation migration and tumour invasion in various cancers tnc is one of themain protein overexpressed in breast cancer indicating a role for this ecm molecule in cancer pathology in this study we have evaluated the tnc lossofffunction in breast cancercells in our approach we used dsrna sharing sequence homology with tnc mrna calledatnrna we present the data showing the effects of atnrna in mdamb231 cells bothin monolayer and threedimensional culture cells treated with atnrna were analyzed forphenotypic alterations in proliferation migration adhesion cell cycle multicaspase activation and the involvement in epithelial to mesenchymal transition emt processes as complementary analysis the oncogenomic portals were used to assess the clinical implication oftnc expression on breast cancer patients survival showing the tnc overexpression associated with a poor survival outcome our approach applied first in brain tumors and then inbreast cancer cell lines reveals that atnrna significantly diminishes the cell proliferationmigration and additionally reverses the mesenchymal cells phenotype to the epithelial onethus tnc could be considered as the universal target in different types of tumors wheretnc overexpression is associated with poor prognosisa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation wawrzyniak d grabowska m gÅodowiczp kuczyÅski k kuczyÅska b fedorukwyszomirska a downregulation oftenascinc inhibits breast cancer cells developmentby cell growth migration and adhesionimpairment one e0237889 101371 pone0237889editor lucia r languino thomas jeffersonuniversity united statesreceived may accepted august published august copyright wawrzyniak this is anopen access distributed under the terms ofthe creative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportingintroductioninformation filesfunding this work was supported by the ministryof science and higher education of the republic ofpoland by know program kk was supported byncbr program powr03020000i03216competing interests the authors have declaredthat no competing interests existthe tumor microenvironment is composed of the surrounding stromal cells such as endothelialcells in blood vessels immune cells fibroblasts and the extracellular matrix ecm [ ] during carcinogenesis is often perturbed and deregulated while during embryonic development isstrictly controlled to maintain homeostasis in tumors the composition of the ecm differsfrom that of normal tissue and enables new interactions that affect the function of cancer cellsand are critical in modulating invasion associated with cell migration and growth the tumorassociated ecm presents several tumorassociated antigens that are generally more abundant one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentand possibly more stable than those of the cell surface [] consequently these proteins represent possible valuable targets for tumor imaging and therapy [ ] ecm proteins such as fibronectin fn and tenascin have isoforms that are expressed in a tissue specific manner generatedby alternative splicing of their primary transcripts one of the most consistent isoform changesin the ecm of many tumors is the upregulation of the glycoprotein tenascinc tnc tncalongside tenascinx tnx tenascinr tnr and tenascinw tnn are members of wellconserved among vertebrates tenascin family tn [] numerous isoforms of tnc can beproduced through alternative splicing of nine fibronectin type iii regions between repeats and at the premrna level there is a considerable amount of literature on the contribution of different splicingdependent tnc domains in specific biological functions changes in thetnc isoforms expression pattern have been then described in a number of malignancies andtheir nature appears to be tumortype specific recent studies have demonstrated that somesplice variants are specific to diseased tissues [] in breast tissues expression of two tncvariants one containing domain d and the other both b and d was found to be associated withinvasive phenotype tnc promotes cell migration angiogenesis inhibit focal contact formation and also act as a cell survival factor [] its importance was found in the development and progression of different types of neoplasm including colon and breast cancerfibrosarcoma lung cancer melanoma squamous cell carcinoma bladder cancer and prostaticadenocarcinoma [ ] tnc is also highly expressed in highgrade gliomas which correlatesas well with the invasiveness of glioma cells [] in the brain it is important for the development of neural stem cells [ ] and moreover is suspected to be a potential marker for glioblastoma multiforme gbm stem cells gsc previously we have shown that tnc is overexpressed in gbm and can be a good target inrnai approach with 164nt long dsrna complementary to the mrna of tnc which wecalled atnrna we conducted the experimental therapy for gbm patients the discoverythat tnc presents a dominant epitope in glioblastoma prompted us to investigate the potentialof atnrna to block the tnc expression and its effect on the growth of human breast cancers where tnc overexpression was also established and linked with the highest malignancyinvasion capability and metastasis ability this view is supported by mock who showedthat gbm patients with antibodies against the egflike repeats of tnc antibody targetvcedgftgpdcae have a significantly better prognosis than other patients thus weassumed that in the light of the satisfactory results of brain tumors experimental therapy breastcancer could be the next possible object of interest to establish the atnrna approachhere we demonstrate that atnrna approach can be successfully used in breast cancercells impairing the basic hallmarks of tumor cells with the performed analysis of proliferation migration rate multicaspases induction pathway cell cycle analysis spheroids viabilityand the involvement of tnc in emt induction we have then interrogated the impact of tncon breast cancer growth showing its potency to be also the promising therapeutic target inbreast cancer treatmentresultsoncogenomic in silico analysis reveals the tnc correlation with poorsurvival of breastcancer patientsto look deeper into the tnc function we performed the analysis of genomewide breast cancerdata with available oncogenomic portals such as gepia the human protein atlas cbioportaland ppisurv based on the status of three important receptors conventionally used for breastcancer subtyping ie estrogen receptor er progesterone receptor pr and human epithelialreceptor her2 breast cancer is classified as luminal a luminal b her2 positive and triple one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentnegative basallike triplenegative and her2overexpressing breast cancer yields a poorpatient prognosis because of a high incidence of metastases disease progression and resistanceto current chemotherapy regimens we first compared the expression level of tenascincin subtypes of breast cancer using gepia program fig a in s1 file mrna level of tncwere higher in triplenegative and her2 subtypes compared to the luminal a and luminal bsubtypes which have a better prognosis for patient survival therefore we chose mdamb231cells as a model for in vitro experiments because it is the most invasive cell line from breast cancer models mdamb231 cell genome clusters with the basal subtype of breast cancer sincethe cells also lack the growth factor receptor her2 they represent a good model of triplenegative breast cancer what is important adams showed that only invasive cell linessuch as mdamb231 or mdamb468 express tenascinc whereas the tumor cell lines witha low invasive capacity mcf7 and t47d do notas a next step we compared the expression levels of tenascin genes tnc tnn tnrtnxb in invasive breast cancer using gepia program fig 1a mrna level of tnc washighly expressed in breast cancer tissue brca interestingly expression levels of tnn andtnxb were significantly lower in breast cancer tissues fig 1a there was no significant difference in tnr gene expression between breast cancer and nontumor tissueswe also examined the expression of tenascin proteins in normal and malignant tissues byquerying data from the human protein atlas tnc in most cases and partly tnn wereexpressed at medium levels whereas tnr was not detected fig 1b and 1c tnc and tnrlevels were undetectable in samples from normal breast adipocytes glandular and myoepithelial cells taken together our results demonstrate that mrna and protein levels of tnc is relatively higher in invasive breast cancer tissues than those in normal tissuesthe cbioportal analysis enabled to look for the mutations in the tnc gene it appeared thattnc gene mutations measured for breast cancer patients are present as somatic mutation only in cases since these mutations seem to be irrelevant for breast cancer wedid not perform any further analysiswith ppisurv portal we looked through the transcriptomic data to correlate the tncexpression with the different clinical parameters such as survival or prognosis of the canceras the initial step of analysis we performed the alignment of tnc and other proteins from thetenascin family such as tenascinx tnx looking for the homology between these two proteinsat the top of that we made the analysis of the homology between the tnc and tnx with the relation to atnrna sequence the alignment of these two ecm proteins shows that they shareonly limited number of nucleotides query cover and for short and long transcriptionalvariants in the protein nterminus region respectively the sequence alignment analysis clearlyshow the atnrna matching exclusively to the tnc sequence identity fig 2appisurv analysis based on the kaplanmeier statistics showed very clearly the strong correlation with tnc expression and patients survival the high expression level has a greatimpact on the shorter survival for the patients thus suggesting also that tnc can be also considered as the prognostic factor for breast cancers p fig 2b at the same time weanalyzed also the available data for the tnx the results showed an inverse correlation oftnxb mrna expression and survival p fig 2c analysis was carried out on thegroup of patient n for tnc and n for tnxbatnrna mediates the downregulation of tnc mrna and proteinexpression in breast cancer cellsto achieve downregulation of tnc expression in breast cancer cell line transfection withvarious concentration of atnrna and nm was performed h after one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig tenascin is highly expressed in breast invasive carcinoma tcgabrca a messenger rna levels of tnc tenascinctnn tenascinw tnr tenascinr tnxb tenascinx genes in specimens from patients with invasive carcinoma of the breast one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentvs nontumor samples rna sequencing data were retrieved from the database of tcga and analysed using the gepia geneexpression profiling interactive analysis online web server httpgepiacancerpkucn the red boxes represent cancer specimensgrey boxes represent healthy breast specimens significance value � p b summary of tenascin expression patterns in breastcancer tissues and healthy breast determined by immunohistochemical staining data were retrieved from the human protein atlasdatabase case numbers of invasive breast cancer are shown na not available nd not detectable in healthy breast column means that tnc and tnr are not detected in nontumor samples results in normal breast are based on immunohistochemical stainingof a single sample c representative images of immunohistochemical staining for tnc tnn and tnr in breast healthy tissue andinvasive breast carcinoma specimens the images shown here are of the tissue sections from tissue microarray arrays tmas stainedwith appropriate antibodies tnccab004592 tnncab010907 tnrcab022343 all the images were taken at à magnification101371 pone0237889g001transfection the expression level of tnc was examined by qrtpcr analyses significantdownregulation of tnc mrna expression was observed compared to control treated withscrambled rna the level of tnc was decreased from at a concentration of nmatnrna up to for cells treated with nm atnrna in comparison to the controlp fig 3afig the oncogenomic analysis of the survival association with tenascinc and tenascinx in breast cancer asequence alignment of tenascinc versus tenascinx with their relation to atnrna the sequence alignment analysisclearly show the atnrna matching exclusively to the tnc sequence identity relation of tnc b and tnxbc gene expression to survival of breast cancer patients survival analysis performed with the use of a dataset breastcancer geo gse7390 and gse3494 deposited in and tools available from the ppisurv web portal tnxbl longtranscription variant tnxbs short transcription variant101371 pone0237889g002 one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig expression level of tnc and immune response genes after atnrna treatment in mdamb231 cell line a relative expression level of theexpression of tnc oas1 oas3 rig1 ifi16 and tlr3 established by qrtpcr relative expression was calculated using theδδcp method statisticalevaluation of atnrna versus scrambled sirnas ccontrol cells was performed using oneway anova followed by tukeys posthoc test effect of poly ic μgml on immune response genes oas1 oas3 rig1 ifi16 tlr3 in the figure presented as purple bars the results for hprtnormalized expressionof mrna are expressed as fold change of target gene expression relative to the control without poly ic treatment which is defined as b the proteinexpression levels of tnc and hprt c western blot analysis reveals efficient tnc silencing in mdamb231 cells with atnrna compared to cells treatedwith sirnas ccontrol the data represents the means ± sd from independent experiments significance value � p �� p ��� p 101371 pone0237889g003the qrtpcr analysis was also supported by direct analysis of the protein expression levelwe have observed already a decrease in tnc protein expression upon 50nm atnrnatreatment the highest concentration 100nm used led to the dramatic drop of the protein one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentexpression measured as the of the decrease fig 3b and 3c these observations were fullyconsistent with relative tnc expression level measured by qrtpcrinterferon response to atnrnato establish interferon induction in breast cancer mdamb231 cultured cells we looked forinterferon stimulated genes isg including oas1 oas3 rig1 tlr3 and ifi16 genes theanalysis was carried out with the qrtpcr fig 3a changes after atnrna measured byqrtpcr were not significant as shown basically for all of the genes in the concentrationrange of nm in parallel a synthetic form of dsrna polyipolyc poly ic wastransfected as a positive control poly ic has been used extensively as a tlr3 ligand to induceantiviral response [] we showed that transfection with poly ic μgml efficientlyinduced the expression of immune response genes oas1 oas3 rig1 ifi16 tlr3 inmdamb231 cells this enhancement in mrna expression was 7fold higher in poly ictreated cells than in untreated cells fig 3a purple barstnc knockdown inhibits cells proliferation and leads to the changes inmigration rate and adhesion potential of breast cancer cellsin order to investigate the involvement of tnc on breast cancer cells proliferation mdamb cell line was treated with atnrna and the realtime cell proliferation assay was performed the cells ability to proliferate was measured for h we have noticed time and concentrationdependent decrease in proliferation rate the most effective concentration ofatnrna was nm with decrease from after and h respectively fig 4anoteworthy nm and nm of atnrna was already sufficient concentration for the efficient inhibition of breast cancer cells proliferation the dosedependent effect of atnrna inmdamb231 proliferation potential resulted in standard sigmoidal doseresponses with ic50of ± nm after h ± nm after h and ± nm after h of treatmentfig 4bto get more insight into the downregulation of tnc expression by atnrna on themobility of breast cancer cells realtime measurements of migration was carried out wefound that downregulation of tnc expression by atnrna significantly impaired the cellmigration in breast cancer cell lines fig 4c the results were quantitatively assessed during h of experiment and showed that mdamb231 cells transfected with atnrna had thelowest motility beginning from h post transfection it was established that atnrnadelayed the migration of mdamb231 cells by ± h ± h ± h and ± h with and 100nm concentration respectively notably the most effective concentration which affected the migration potential of the cells was 10nm when compared to the control the observed delay was ± hsince tnc is implicated also in cellmatrix attachment we further looked at the adhesionability of atnrna treated cells the cells were conducted to realtime adhesion assay withxcelligence system compared with the controls tnc knockdown resulted in increased cellsadhesion on average fig 4dtnc promotes apoptosis and is involved in cell cycle regulation in breastcancer cellsto determine additionally the effect of atnrna on the cell cycle progression themdamb231 cells were treated with different concentrations of atnrna for h andthe cell cycle analysis was assessed by muse1 cell analyzer cells transfected with atnrna one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig activity of atnrna in proliferation migration and adhesion proliferation of breast cancer in culture was monitored in realtime using xcelligencesystem a impedance was recorded every min but to improve the clarity of the graphs only every fourth readout was plotted data show the mean ± sd ofthree independent measurements b dosedependent effects of atnrna on proliferation was evaluated using nonlinear regression by fitting experimentalvalues to sigmoidal bellshaped equation c migration of mdamb231 cancer cells was studied using xcelligence system serumdepleted cells weretransfected with increasing concentrations of atnrna from to nm or scrambled sirnas ccontrol impedance ci values of each experimentalcondition was recorded over time plotted against time fitted to fourparameter logistic nonlinear regression model and et50 was calculated for each atnrnaconcentration to generate doseresponse curves et50 value was normalized to the data obtained for untreated cells and plotted as normalized half maximaleffective time et50 of cell migration against atnrna concentrations d adhesion of mdamb231 cell line was observed in realtime using xcelligencesystem graph shows the final impendence values minus the initial values for the respective samples differences between ci values for atnrna treated andcontrol cells were statistically evaluated using oneway anova followed by tukeys posthoc test symbols above the bars significance value ��� p compared to cells treated with scrambled sirnas ccontrol101371 pone0237889g004showed the cell cycle distribution with the concentrationdependent decrease in cell numberin g0g1 phase increased in s phase and unchanged in g2m phase compared to the cellstransfected with unspecific control rna fig 5a atnrna impacted the cell cycle by almostdoubling the cells sphase fraction from to for the highest atnrna concentrationthus resulted with the cells arrest in s phase the cell cycle analysis proved the nontoxic effectof atnrna since we did not observe the increase in g1 population s phase arrest persistsfollowing up to h of atnrna treatment fig 5bthus to examine whether atnrnainduced apoptosis would be associated with the caspases activation the expression levels and activity of caspases such as caspase1 and in the atnrnatreated mdamb231 cells were assessed using muse1 cell one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effect of atnrna on breast cancer cell cycle phase analysis mdamb231 cells were transiently transfected with increasing concentrations ofatnrna from to nm or scrambled sirna ccontrol for a and h b the cells were then fixed and added with propidium iodide pirnase a staining solution and analyzed in muse1 cell analyzer using modfit lttm software a percentage of cell distribution in each cell cycle phase wassummarized and shown the cell cycle distribution profile image is shown as a representative result of three independent experiments101371 pone0237889g005analyzer as shown in fig 6a breast cancer cells treated with atnrna exhibited enhancedmulticaspase activity in a concentrationdependent manner the multicaspase activity was ± ± ± and ± respectively at and nm atnrna concentration compared with the control fig 6b thus we observed almost5fold increase of the population of the apoptotic cells with the lowest atnrna concentration whereas almost 12fold with the highest oneatnrna has an impact on spheroids integrityto visualize the involvement of tnc in tumor formation the 3d culture model was appliedsince 3d cell culture models mimic better the in vivo behavior of cells in tumour tissues andare excellent surrogates to predict tumorigenic potential in vivo the ability to spheroid maintenance of breast cancer cells was assessed after atnrna treatment we have observed that one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effect of atnrna on multiple caspase activation caspase1 and in mdamb231 cell linebreast cancer cells were transiently transfected with increasing concentrations of atnrna from to nm orscrambled sirnas ccontrol for h the transfected cells were then incubated with muse1 multicaspase reagent followedby analysis of the percentage of cell population in live caspase caspasedead and dead in muse1 cell analyzer a thepercentage of live caspase caspasedead and dead cells profile image is shown as a representative result from one of threeindependent experiments b the graphical representation of percentage of live and exhibiting caspase activity cellpopulation transfected with atnrna and scrambled sirnas statistical evaluation of atnrna versus scrambled cells one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmenttreated with scrambled sirnas was performed using oneway anova followed by tukeys posthoc test significance value��� p compared to scrambled control ccontrol error bars represent sd101371 pone0237889g006downregulation of tnc led to the disintegration of the spontaneously forming spheroids ofmdamb231 the clearly visible effect on the spheroid viability was observed even with thelowest atnrna concentration the increased concentrations of dsrna had a great impacton spheroids integrity resulting in structure disintegration at the highest concentration of nm fig 7a the atnrna application influenced the spheroid volume and shape displaying the total shrinking of the compact structure into the small fragments with the highestatnrna concentration fig 7ato have a better insight into the mdamb231 spheroids structure and the atnrnaimpact on their viability the confocal microscopy imaging was assessed the analysis of fluorescent labelling with greenfluorescent calceinam of living and dead cells within the spheroid with the livedead viabilitycytotoxicity kit was carried out as revealed by the image ofthe untreated mdamb231 cells compact multicellular spheroids were obtained fluorescence images revealed the overall morphology of the mdamb231 spheroids fig 7b thecell density in the core of the untreated spheroid was high and no dead cells were identifiedthe similar pictures were obtained for the control furthermore all conditions with differentatnrna concentrations resulted with losing the spheroid density increased dimensionsand appearing a higher population of dead cells it is worthy of note that the spheroids treatedwith increasing atnrna concentrations did not display a smooth contour following h oftreatment and subsequently their round shape was markedly altered by the treatments afterthe treatment with 50nm concentrations the spheroids showed the strong overrepresentationof dead cells fig 7b thus the 100nm concentration of atnrna was most likely too highfor the cells viability and we were not able to keep the spheroids in shape that would allow forthe imagingtnc is involved in emt processesas the consequence of these finding we analyzed the expression level of proteins involved inemt processes we took into account two main emt markers ecadherin and vimentinwestern blot analysis shows the significant increase of ecadherin level followed by the dropof the expression of vimentin protein fig 8a these observations were concentrationdependent showing the highest efficacy for atnrna at the concentration of 100nm for ecadherin expression similarly for vimentin we have observed the highest decrease of expressionupon atnrna transfection at 100nm concentration fig 8bdiscussiontnc is the main ecm protein of various tumors and its overexpression is repeatedlyobserved in breast cancer cells both in vitro and in vivo indicating a role for this extracellularmatrix glycoprotein in neoplastic pathology moreover its high expression correlates withworsened patient survival prognosis in several cancer types in breast cancers severalstudies demonstrate that high expression of tnc is not only an indicator of poor prognosisbut also correlates with metastasis to distinct ans such as lymph nodes liver and lung [] tnc plays a substantial role in emt that is believed to be a key mechanism in cancer progression whereby cancer cells acquire more aggressive behavior [ ] in human breast cancer specimens tnc is coexpressed with the mesenchymal marker vimentin themechanistic role of tnc in the process of emt remains poorly defined however studies one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effects of atnrna on viability and spheroid structure in mdamb231 cells a monolayer cultures weretransfected with indicated amounts of atnrna oligonucleotides and after hrs cellular spheroids of mdamb cells were generated from cells in perfecta3d1 96well hanging drop plate and cultured for up to hoursscale bars μm scrambled sirnas cscr b the viability of the atnrna transfected cells within spheroidsusing livedead cell imaging kit left and middle panels present live cells green and dead cells red respectively one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentthe right panels show merge of two fluorescent images scrambled sirnas cscr concentrations ofatnrna used for transfection fluorescence images were taken using leica tcs sp5 confocal laser scanningmicroscope and plan apo à na oilimmersion objective scale bars μm101371 pone0237889g007suggest that tnc can induce an emt like phenotype in mcf7 breast cancer cells via theαvβ6 and αvβ1 integrins [ ] many studies on various cancer tissues have demonstrateddownregulation of epithelial markers including ecadherin plakoglobin and cytokeratin aswell as the upregulation of mesenchymal markers such as ncadherin and vimentin andexpression of emt transcription factors snai and twist since these changes towardsmesenchymal phenotype could correlate with invasiveness metastatic potential and poorpatients outcome we have investigated the effect of tnc knockdown on the expression levelsof emt markers our results show that down regulation of tnc reverses the malignant phenotype of the cancer cells as the experimental result we observed the downregulation of mesenchymal markervimentin followed by the upregulation of epithelial markerecadherinthis indicates atnrna as a potential therapeutic agent which could switch the mesenchymal phenotype of breast cancer cells to the epithelial one inhibiting the ability to metastasisand invasion additionally it has been also shown that tnc as the ecm component plays alsofig the effect of tnc downregulation on emt process of mdamb231 cells a the protein expression levelsof ecadherin vimentin and gapdh b western blot analysis of atnrna effects on the emt process revealed asignificant increase in ecadherin level followed by the drop of the expression of vimentin protein the data representsthe means ± sd from independent experiments101371 pone0237889g008 one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmenta role in cell to cell or cellmatrix attachment most probably inhibiting the cells migration inapproach with atnrna it seems that tnc in breast cancer cell line plays an antiadhesiverole which would affect the cell migration and invasion ability in addition to emt processesthus tnc downregulation seems to enhances the adhesiveness of cancer cells showing thedirect involvement of tnc in cell adhesive properties targeting the tnc in potential therapymight be also highly beneficial since it has been already established that tnc maintain a stemcell niche in the brain tumors thus could promote the tumor cell invasion therefore its overexpression largely contributes to radiochemotherapy resistance and tumor recurrence infact it has been shown that targeting gbm invasion increases tumor sensitivity to temozolomide tnc also promotes stromal events such as the angiogenic switch and the formationof more but leaky blood vessels involving wnt signaling and inhibition of dickkopf1 dkk1in a neuroendocrine tumor model we observed significant atnrnamediated downregulation of tnc was in concordance with the observed changes in proliferation and migration rates the results show thatthe atnrna transfected cells lose their ability to migrate thus showing the involvement oftnc in breast cancer invasiveness our data indicate also that the downregulation of tncexpression in mdamb231 cancer cell lines inhibits proliferation along with induction of celldeath we were able to determine the tnc impact on the apoptosis by measuring level of bothcaspases initiating intracellular events caspase2 and effector caspases3 and orend demonstrated that tnc causes cdk2 inactivation and blocks cell cycle progression from g1 phase to s phase of anchoragedependent fibroblasts by interfering withfibronectinsyndecan4 interactions for the proliferation of anchoragedependent cellsattachment to the ecm is required detachment of fibroblasts by a pure tenascinc substratum results in g1phase arrest by inactivation of the cdk2 complex in a ckidependent manner in contrast to fibroblasts proliferation of most tumor cells is stimulated by tnc this shows that the effect of tnc on proliferation is cell typespecific and suggests that in cancer cells the cdk2 complex is not repressed in the presence of tnc in leukemia breast carcinoma and glioma were found subpopulations of stemlike cells that support tumor growth in such cells adhesion on the tnc substratum may override the g0g1 and gls cellcycle checkpoints which may explain the increased proliferation rate the g1s transitionis enforced by cyclin ecdk2 activation via syndecan4 related signalling it has been shownthat tnc in tumor cells binds to the syndecan4 binding site in fibronectin thereby blockingsyndecan4 ligation releasing the tumor cells from the suppressive effect of fibronectin ontheir proliferation this would a | Colon_Cancer |
" excessive perioperative fluid administration may result in iatrogenic endothelial dysfunction andtissue edema transducing inflammatory markers into the bloodstream colloids remain longer in the circulationrequiring less volume to reach similar hemodynamic endpoints compared to crystalloids thus we tested thehypothesis that a goaldirected colloid regimen attenuates the inflammatory response compared to a goaldirectedcrystalloid regimemethods patients undergoing moderate to highrisk open abdominal surgery were randomly assigned to goaldirected lactated ringers solution n or a hydroxyethyl starch n fluid regimen our primaryoutcome was perioperative levels of pro and antiinflammatory cytokines secondary outcome was perioperativelevels of white blood cell count wbc creactive protein crp procalcitonin pct and lipopolysaccharidebindingprotein lbp measurements were performed preoperatively immediate postoperatively on postoperative day onetwo and fourresults the areas under the curve of interleukin il p il p il p and tumor necrosisfactor α p levels did not differ significantly between the groups wbc crp and pct values were alsocomparable lbp although significantly higher in the crystalloid group remained in the normal range patientsassigned to crystalloids received a median iqr amount of ml of crystalloid patients assigned tocolloids received ml of crystalloid and ml of colloid cytokine and inflammatory marker levels did not differ between goaldirected crystalloid and colloidadministration after moderate to highrisk abdominal surgerytrial registration clinicaltrialsgov nct00517127 registered 16th august correspondence barbarakabonmeduniwienacat1department of anaesthesia general intensive care medicine and painmedicine medical university of vienna spitalgasse vienna austriafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cobradovic bmc anesthesiology page of is crucialintroductionvolume replacementin the perioperativeperiod and has great impact on postoperative outcome fluid restriction may cause hypotension and hypoperfusion leading to an dysfunction on the otherhand excessive fluid administration leads to destructionof the endothelial surface layer and consequently to tissue edema with harmful side effects [ ]fluidgdtbasedtherapygoaldirectedonoptimization of flowrelated hemodynamic parametersimproves clinical outcome in low to highrisk surgicalpatients compared to fixed fluid protocols [ ] specifically gdt enhances cardiac performance and gutmicrocirculation while avoiding iatrogenic hyperhydration [ ] in addition to hypervolemia the inflammatoryaggravatesdegradation of the endothelial barrier the socalled glycocalyx inflammation leads to cytokine release andmay thus worsen outcome for example high postoperative interleukin il levels are independently associatedwith postoperative complications response dueto surgicaltraumaso far in most previously performed gdt studieshemodynamic algorithms were based on colloid bolusadministration to improve hemodynamic variables colloids better maintain the intravascular oncotic pressure and provide a higher volume effect when used incase of hypovolemia goaldirected colloid administration reduces intraoperative fluid requirement and improves cardiac performance compared to crystalloids[ ] whether this translates into better outcomespecifically in a decreased postoperative inflammatoryresponse is still a matter of research the comparisonbetween colloid versus crystalloid based fluid regimenswas still lacking therefore we tested the primary hypothesis that perioperative levels of pro and antiinflammatory cytokines il il il and tumor necrosis factor alpha tnf α are reduced by goaldirectedcolloid versus crystalloid administration during the firstfour postoperative days in patients undergoing moderateto highrisk open abdominal surgery in addition wemeasured white blood cell wbc count creactive protein crp procalcitonin pct and lipopolysaccharidebinding protein lbp levelsmaterials and methodsthis prospective randomized controlled trial was conducted at the department of anesthesia intensive caremedicine and pain medicine medical university ofvienna vienna austria the institutional review boardof the medical university of vienna approved it as partof a large multicenter outcome trial evaluating the effectof goaldirected crystalloid and colloid on postoperativecombined morbidity and complications the ethicalcommittee of medical university of vienna viennaaustria provided ethical approval for this trial the trialwas conducted in accordance with the declaration ofhelsinki and good clinical practice and registered atclinicaltrialsgov nct00517127 and eudract a written informed consent was obtainedfrom all patients the authors have followed the applicable consort guidelinesfor this single center substudy consecutive eligiblepatients were included patients aged to yearsundergoing elective moderate to highrisk open abdominal surgery with american society of anesthesiologistsasa physical status iiii were included we excludedpatients with severe obesity body mass index bmi kgm cardiac insufficiency ejection fraction ef coronary artery disease with angina severe chronicobstructive pulmonary disease autoimmune diseases coagulopathies renal insufficiency creatinine clearance mlmin or renal replacement therapy symptoms of infection or sepsis and preoperative crp higher than mgdl allpatientsreceivedprotocolpreoperativelyantimicrobialprophylaxis using a single dose of a 2nd generationcephalosporine according to our clinicalstandardsanesthetic management wasstandardized standardmonitoring included electrocardiography ecg invasiveblood pressure surveillance pulse oximetry and esophageal core temperature monitoring a central venouscatheter was inserted when deemed clinically necessarywe used balanced anesthesia with sevoflurane none ofour patients received locoregional anesthesia accordingto patients requirements additional fentanyl and nondepolarizing neuromuscular blocking were administeredventilatory rate was adjusted to maintain endtidal carbon dioxide partial pressure etco2 of mmhgnormothermia was maintained with forced air warmingpatients were randomized to crystalloid lactatedringers solution or colloid hydroxyethyl starch voluven fresenius kabi germany grouprandomization was based on computergenerated codesto conceal allocation sealed opaque envelopes wereopened only shortly before induction of anesthesiaall patients were given mlkg oflactatedringers solution during induction of anesthesia followedby mlkg per hour for maintenance normalized toideal body weight ibw throughout surgery we calculated ibw according to the robinson formula thereafter the randomized fluid crystalloid or colloidwas esophageal dopplerguided cardiac q deltex medical group plc chichester uk according to a standardalgorithm this method is based on corrected aorticflow time ftc as well as stroke volume sv and allowsdistinguishing whether a patient is a fluid responder or 0cobradovic bmc anesthesiology page of not if mean arterial pressure map was below mmhg and no signs of hypovolemia were detected vasopressors were administratedpatients were transferred to postanesthetic care unitpacu or intensive care unit icu at the discretion ofthe attending anesthesiologist fluid management wasstandardized for the first postoperative hours in whichpatients received mlkg ibw crystalloid per hourmeasurementsdemographic and morphometric data were recorded aswell as asa score medical history type of surgery andpreoperative laboratory values duration of anesthesiaand surgery were recorded we also recorded intraoperative fluid requirements estimated blood loss transfusion requirements and urinary output for evaluation ofanesthetic management the total amount of fentanylendtidal sevoflurane concentration core temperatureandnotedhemodynamic parameters such as map heart ratehr ftc sv and cardiac output co were recorded at10min intervals the application of phenylephrine usewas notedicu admission werepostoperativethe primary outcomes were the areas under the curveaucs of postoperative levels of pro and antiinflammatory cytokines il il il and tnf α andtheir differences between the crystalloid and the colloidgroup secondary outcomes were aucs of wbc crppct and lpb and their differences between the groupsall blood samples for parameteranalyses were obtainedbefore surgery as baseline values t0 immediately postoperatively t1 as well as on postoperative days onetwo and four t2 t3 and t4 respectively for analysisof il il il and tnf α blood samples were centrifuged within h at g for min and plasma wasimmediately stored at °c for later enzymelinkedimmunosorbent assay elisa analyses the serum concentrations of il il il and tnf α were determined according to the manufacturersinstructionshuman sil6 instant elisa human il8nap1 instant elisa and human sil10 instant elisaebioscience vienna austria wwwebiosciencecom human tnf α duoset rd systems minneapolis minnesota wwwrndsystemscom for that purpose opticaldensity was measured with a victor microplate readerat a wavelength of nm multiple testing of sampleson different plates revealed an intraassay variability of for il for il for il for tnf α andan interassay variability of for il for il for il and for tnf αfor investigation of wbc crp pct and lpb separateblood samples were obtained their analysis took placeimmediately after blood sampling as routine laboratoryanalysessample size calculation and statistical analysissample size calculations for our trial were based on thestudy of steppan and colleagues they observed amean standard deviation sd of pgml in il h after surgery in abdominal surgery patients assuming a similar coefficient of variation sdmean for each of the four cytokines primarily plannedfor evaluation in our study we calculated a total of patients in order to obtain a power to detect a reduction in any of the cytokines at an overall significance level with powergroups were primarily compared for balance in patients demographic data intraoperative characteristicsand postoperative variables absolute standardized differences asd were calculated for patients baseline covariates subsequent measurements ofintraoperativeparameters were first averaged within each patient andthen averaged among the patients in each treatmentgroup for descriptive analysis normal distribution wasassessed with qq plots and kolmogorowsmirnow testsnormally distributed variables were with unpaired twotailed ttests otherwise the in case of normally distributed values wilcoxon ranksum test was used for notnormally distributed continuous data paired comparisons between baseline data and postoperative data wereperformed with paired sample ttest or wilcoxonsignedrank test as applicable nominal data were analyzed with chisquare or fishers exact test for low expected cell counts data were presented as means ± sdmedians iqr or as numbers percentage as applicableadjustment for multiple testing was performed with thebonferroni method a p value was considered statistically significantanalysis was conducted with spss software version armonk ny ibm corp r for macintosh version321 r core team r a language and environmentfor statistical computing r foundation forstatistical computing vienna austria was used to calculate asdresultsa total of patients were included between november and october in the colloid group and in the crystalloid group fig at t0 all values weremeasured overall in the crystalloid group and inthe colloid group of the preplanned blood sampleswere collected and analyzedpatients baseline characteristics did not differ exceptfor height bmi with a slightly higher bmi in the colloidgroup type of surgery and crp also higher in the colloid group table duration of anesthesia and surgerywere comparable between both groups patients assignedto crystalloid administration received a median of ml crystalloids whereas patients assigned 0cobradovic bmc anesthesiology page of fig consort patient flow chartto the colloid group received ml ofcrystalloid solution and ml of colloidsblood loss transfusion requirements and urinary outputdid not differ between the groups anesthetic management map and hr did not differ between the groupsftc sv and co were significantly higher in the colloidgroup compared to the crystalloid group ftc ms versus ms p sv ml versus ml to p and co ± lmin versus ± lmin p thenumber of patients requiring vasopressor support wascomparable between groups the incidence of postoperative icu admissions did not differ in the crystalloid versus in the colloid group p table baseline values of il il il and tnf α in thecrystalloid group were comparable to values in the colloid group il pgml versus pgml p il pgml versus pgml p il pgml versus pgml p tnf α pgml versus pgml p immediatepostoperative values of il and were significantlyhigher compared to baseline values in both groupsp for all measurements while tnf α did notshow any significant increase in the crystalloid p and the colloid group p fig aucs of il il il and tnf α did not differ significantly between the groups table wbc values at baseline were gl in thecrystalloid versus gl in the colloidgroup p crp pct and lbp baseline valueswere also comparable in both groupscrp mgdl versus mgdl p pct ngml versus ngml p lbp mcgl versus mcgl p immediate postoperative values of wbc and pctwere significantly higher compared to the baseline valuesin both groups p for all measurements fig 0cobradovic bmc anesthesiology page of table baseline characteristicsage yrsweight kgheight cmbmi kgm gender no menwomenasa score no iiiiiimedical history no pulmonary diseasecardiovascular diseasediabetes type idiabetes type iitype of surgery no colorectalliverpancreaticcrystalloidsn ± ± ± ± colloidsn ± ± ± ± asd preoperative laboratory valuescrp mgdl ± ± patient characteristics data are presented as means ± sd or as counts for thecategorical outcomesabbreviations asd absolute standardized differences absolute difference inmeans or proportions divided by the pooled sd asd values of and represent small median and large differencesbmi body mass index m male f female asa american society ofanesthesiologists crp creactive protein sd standard deviationthe aucs for wbc crp pct and lbp for the timeperiods from t1 to t4 did not differ significantly between the groups fig table however lbpshowed significantly higher levels in the crystalloidgroup in the immediate postoperative period comparedto the colloid group mcgl versus mcgl p at all other postoperativetime points there were no significant differences betweenthe groups fig discussionthis trial is a substudy of a large multicenter randomized trial evaluating the effect of goaldirected crystalloidversus goaldirected colloid fluid administration on acomposite of serious complications after moderate tohighrisk open abdominal surgery the overall trial concluded that colloids did not decrease the composite ofmajorincomplicationsare ourresultsconcordance as they did not show any differences inperioperative pro and antiinflammatory markers between a crystalloid and a colloid fluid regimendespite multimodal care and enhanced recovery programs it still remains challenging to blunt the inflammatory response to surgery systemic inflammationafter abdominal surgery impairs outcome and thereforemany attempts have been made to alter the inflammatory response several factors influence the perioperative inflammatoryresponse such as the underlying disease type and invasiveness of surgery as well as type of anesthesia [] themost important factor is the magnitude of surgical traumaand tissue damage which induce proliferation and activation of immune competent cells in turn triggering cytokine and inflammatory marker release so far veryfew trials have specifically investigated the influence offluid therapy and differences in terms of the type of fluidon the extent of inflammatory marker releaseto investigate the potential influence of goaldirected hydroxyethyl starch versus a lactated ringers solution fluid regimen on inflammatory response pro il il and tnf α and antiinflammatory cytokine il serum levels were measured during the perioperativeperiod additionally we measured wbc crp pct andlbpgenerally the most commonly measured biomarkersare crp and wbc if levels of crp are above mgdl after postoperative day four a postoperative infection can be suspected crp levels in our studygroups increased on the first postoperative day droppingon the fourth postoperative day to nearly mgdl inboth study groupswbcs are an imprecise marker to detect postoperativecomplications after major abdominal surgery amore sensitive parameter in predicting postoperativecomplications after major abdominal surgery is il surgical trauma and hypoperfusion of the colon aremain sources of il release in colorectal surgery noblett demonstrated that gdt during elective colorectal surgery significantly reduced il levels in comparison to a control group yates showed no differencesof il and il levels between goaldirected colloidand crystalloid fluid therapy during the first h in asubgroup of patients undergoing colorectal surgery although patients in the crystalloid group received significant more volume amount as compared to the colloid groupthere was no significant difference inhemodynamic variables our patients showed similar courses of il and il levels in the immediatepostoperative period in contrast to the trial of yateswho measured cytokine levels up to the first h aftersurgery we extended our measurement period to fourpostoperative days we showed comparable circulating 0cobradovic bmc anesthesiology page of table intraoperative dataduration of anesthesia minduration of surgery minfluid managementtotal fluid intake mlacrystalloid mlcolloid mlestimated blood loss mltransfusion yesno urinary output mlanesthesia managementfentanyl mcgtwa et sevoflurane core temperature °cicu admission yesno hemodynamictwa map mmhgtwa hr beatsmintwa ftc mstwa sv mltwa co lmin phenylephrine yesno crystalloidsn ± ± [ ] ± ± ± ± colloidsn ± ± [ ] ± ± ± ± p value ° ° °° °°intraoperative data are presented as means ± sd medians iqr or as counts for the categorical outcomes means were compared with an unpaired twosided ttests ormannwhitneyu tests as appropriate medians with wilcoxon ranksum tests and counts with chisquare or fishers exact tests ° represents statisticalsignificance p abbreviations et end tidal icu intensive care unit twa time weighted average map mean arterial pressure hr heart rate ftc corrected flow time sv strokevolume co cardiac output sd standard deviationa total fluid intake includes baseline fluid boluses antibiotics analgesics and additional fluid administered at the discretion of attending anesthesiologistfig ad pro and antiinflammatory cytokines il il il and tnf α over time a il b il c il and d tnf α data arepresented as medians iqr abbreviations il interleukin tnf α tumor necrosis factor alpha pod postoperative day 0cobradovic bmc anesthesiology page of table areas under the curve of inflammatory markerspcrystalloidsn colloidsn auc il pgml 1dauc il pgml 1dauc il pgml 1dauc tnf α pgml 1d auc wbc gl1dauc crp mgdl 1dauc pct ngml 1dauc lbp mcgl 1dtable areas under the curve of il il il and tnf α as well as wbccrp pct and lbp are presented as medians iqr medians were comparedwith wilcoxon ranksum testsabbreviations il interleukin tnf α tumor necrosis factor alpha wbc whiteblood cells pct procalcitonin lbp lipipopolysaccharidebinding protein il and il levels between a gdt crystalloid and colloid administration these surrogates of inflammatoryresponse imply that gut perfusion during surgery waswell preserved with both types of fluid and suggest thatthe type of fluid might be of minor importance as longas the fluid is administered in a goaldirected fashionthe fact that tnf α levels in both groups remainedstable over the entire measured period further supportsour theory the course of tnf α levels during the perioperative period was in accordance with the study ofin which fluid therapy was guided withszakmanypicco versusin majorvenous pressurecentralabdominal surgery in patients at risk for postoperativecomplications as tnf α per se triggers glycocalyxdegradation we anticipate that tnf α did not influence glycocalyx shedding and thus possible fluid shifts inour study populationpct is an early predictive marker for systemic inflammation after abdominal surgery values above ngml are associated with postoperative complicationssuch as pneumonia or anastomotic leakage in ourstudy median pct levels did not exceed ngml atany measured time point these results are in concordance with our main study where infectious complications rate were held low and did not differ between thegroups moreover as pct production can also beinduced by tissue hypoperfusion we might assume thatgoaldirected fluid administration contributed to lowpct values by optimizing cardiac performance furthermore we measured lbp a prognostic markerfor bacterial infections patients in the crystalloidgroup showed significantly higher levels immediatelyafter surgery however the measured values remainedwithin the normal range therefore this difference ismost likely not to of clinical importancethe vascular endothelium is one of the earliest sitesinvolved in the inflammatory response syndrome an adequate perioperative fluid management has a major impact on the integrity of the glycocalyx with goaldirected fluid management individualized and time appropriate fluid resuscitation can be achieved enablingfig ad inflammatory markers wbc crp pct and lbp over time a wbc b crp c pct and d lbp data are presented as medians iqrabbreviations wbc white blood cells crp creactive protein pct procalcitonin lbp lipopolysaccharidebinding protein pod postoperative day
represents significant difference in lbp in the immediate postoperative period between the crystalloid and the colloidgroup p 0cobradovic bmc anesthesiology page of preservation of endothelial surface layer and sufficientan perfusion thus improving postoperative outcomesafter major surgery patients in the colloid group received significantly lessfluid ml confirming the previously publishedfluid sparing effect of colloids however clinical significance of this difference may be questionable during aperioperative period of nearly five hours further ourhemodynamic data showed significantly higher values ofsv and co with colloid administration though the absolute difference of ml in sv most likely has onlylimited clinical relevance it might very well be that thesignificant differences in sv and co are the result ofour number of patients included in this substudyassurrogatesfirst limitation of our study is that we measured inflammatory markers that reflect the inflammatory responseand not direct markers ofglycocalyx degradation like syndecan1 therefore wecannot draw any s about the preservation ofthe endothelial surface layer in our patients secondlywe did not control postoperative fluid management during the postoperative followup period a further limitation isthe time between patient enrolment andsubmission of our current results due to the fact thatthe main trial has been published recently a delay of oursubmission occurred nevertheless our results canstill be extrapolated to current clinical practicein summary goaldirected hydroxyethyl starch administration did not attenuate the inflammatory responseexpressed by cytokine levels of il il il and tnfα in patients undergoing moderate to highrisk open abdominal surgery wbc crp and ptc values did not differ between the different fluid regimes as wellabbreviationsasa american society of anesthesiologists asd absolute standardizeddifference auc area under the curve bmi body mass index co cardiacoutput crp creactive protein ecg electrocardiography ef ejectionfraction elisa enzymelinked immunosorbent assay etco2 endtidalcarbon dioxide ftc corrected flow time gdt goaldirected therapyhr heart rate ibw ideal body weight icu intensive care unitil interleukin iqr interquartile range lbp lipopolysaccharidebindingprotein map mean arterial pressure pacu postanesthetic care unitpct procalcitonin picco pulse contour cardiac output spss statisticalpackage for the social sciences sv stroke volume tnf α tumor necrosisfactor alpha twa time weighted average wbc white blood cell countacknowledgementsassistance with this we thank bianca tudor md department ofanesthesia intensive care medicine and pain medicine medical university ofvienna spitalgasse vienna austria for her support in laboratoryskills performing elisasauthors contributionsall authors have read and approved the manuscript mo patientrecruitment data acquistion and prepratation of the manuscript ak studyprotocol writing and preparation of the manuscript bk study protocolwriting and preparation of the manuscript statistical analyisis gr dataanalysis revision of the manuscript ok data analysis preparation of themanuscript oz patient recruitment data acquisition data managementab patient recruitment data acquisition revsion the manuscript cr dataacquistion revision of the manuscript as patient recruitment revision ofthe manuscript ef study protocol writing and preparation of the manuscriptfundingmedical university of viennapartially funded by fresenius kabi deltex medical provided oesophagealdoppler monitors and disposablesthe sponsors were not involved in protocol development data acquisitionor data analysisavailability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestbarbarakabonmeduniwienacatethics approval and consent to participatethe main trial was approved by the local ethics committee of the medicaluniversity of vienna in ek and was registered atclinicaltrialsgov nct00517127 and eudract a writteninformed consent was obtained from all patients prior to participationconsent for publicationnot applicablecompeting intereststhe authors declare no competing interestsauthor details1department of anaesthesia general intensive care medicine and painmedicine medical university of vienna spitalgasse vienna austria2department of outcomes research and general anesthesiologyanesthesiology institute euclid avenue cleveland clinic cleveland ohusa 3department of anesthesiology and general intensive care franziskushospital nikolsdorfergasse vienna austria 4department ofgynecology klinik ottakring montleartstrasse vienna austria5department of surgery medical university of vienna spitalgasse vienna austriareceived july accepted august referencesbrandstrup b tonnesen h beierholgersen r effects of intravenousfluid restriction on postoperative complications comparison of twoperioperative fluid regimens a randomized assessorblinded multicentertrial ann surg myles ps bellomo r corcoran t restrictive versus liberal fluidtherapy for major abdominal surgery new engl j med chappell d jacob m hofmannkiefer k conzen p rehm m a rationalapproach to perioperative fluid management anesthesiology lowell ja schifferdecker c driscoll df benotti pn bistrian brpostoperative fluid overload not a benign problem crit care med sun y chai f pan c romeiser jl gan tj effect of perioperative goaldirected hemodynamic therapy on postoperative recovery following majorabdominal surgerya systematic review and metaanalysis of randomizedcontrolled trials critical care calvovecino jm ripollesmelchor j mythen mg effect of goaldirected haemodynamic therapy on postoperative complications in lowmoderate risk surgical patients a multicentre randomised controlled trialfedora trial br j anaesth noblett se snowden cp shenton bk han af randomized clinical trialassessing the effect of doppleroptimized fluid management on outcomeafter elective colorectal resection br j surg 0cobradovic bmc anesthesiology page of alphonsus cs rodseth rn the endothelial glycocalyx a review of thevascular barrier anaesthesia pearse rm harrison da macdonald n effect of a perioperativecardiac outputguided hemodynamic therapy algorithm on outcomesfollowing major gastrointestinal surgery a randomized clinical trial andsystematic review jama publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationskimberger o arnberger m brandt s goaldirected colloidadministration improves the microcirculation of healthy andperianastomotic colon anesthesiology kolarova h ambruzova b svihalkova sindlerova l klinke a kubala lmodulation of endothelial glycocalyx structure under inflammatoryconditions mediat inflamm rettig tc verwijmeren l dijkstra im boerma d van de garde emnoordzij pg postoperative interleukin6 level and early detection ofcomplications after elective major abdominal surgery ann surg gan tj soppitt a maroof m goaldirected intraoperative fluidadministration reduces length of hospital stay after major surgeryanesthesiology jacob m chappell d rehm m clinical update perioperative fluidmanagement lancet feldheiser a pavlova v bonomo t balanced crystalloid comparedwith balanced colloid solution using a goaldirected haemodynamicalgorithm br j anaesth orbegozo cortes d gamarano barros t njimi h vincent jl crystalloidsversus colloids exploring differences in fluid requirements by systematicreview and metaregression anesth analg kabon b sessler di kurz a effect of intraoperative goaldirectedbalanced crystalloid versus colloid administration on major postoperativemorbidity a randomized trial anesthesiology robinson jd lupkiewicz sm palenik l lopez lm ariet m determination ofideal body weight for drug dosage calculations am j hosp pharm steppan j hofer s funke b sepsis and major abdominal surgery leadto flaking of the endothelial glycocalix j surg res wilmore dw from cuthbertson to fasttrack surgery years of progress inreducing stress in surgical patients ann surg desborough jp the stress response to trauma and surgery br j anaesth veenhof aa sietses c von blomberg bm the surgical stress responseand postoperative immune function after laparoscopic or conventional totalmesorectal excision in rectal cancer a randomized trial int j color dis gilliland he armstrong ma carabine u mcmurray tj the choice ofanesthetic maintenance technique influences the antiinflammatory cytokineresponse to abdominal surgery anesth analg wichmann mw huttl tp winter h immunological effects oflaparoscopic vs open colorectal surgery a prospective clinical study archsurg watt dg han pg mcmillan dc routine clinical markers of themagnitude of the systemic inflammatory respon | Colon_Cancer |
" national economies are increasingly facing the challenge of having to finance the prevention andtreatment of human diseases and of having to compensate for the resulting loss of economic production physicalinactivity is demonstrably closely related to the risk of developing certain disease group physical inactivity results indirect and indirect burdens that the present study intends to quantify in hungary for the period between and methods based on the data of the hungarian public finances this study determines the direct and indirect costsincurred by hungary due to illnesses and through the par method it quantifies the financial burden of physicalinactivity incurred by the hungarian treasuryresults the total financial burden of illnesses in hungary showed a decreasing tendency from to eventhough the year saw an increase in costs compared to similarly while total public expenditure on illnessesassociated with physical inactivity increased by when compared to the total amount attributable to medicalconditions stemming from physical inactivity still showed a decrease of billion huf in the overall period the biggesteconomic burden is posed by cardiovascular diseases hypertension and type diabetess the increase in the economic burden associated with physical inactivity can be attributed to thecombined effect of two factors changes in total expenditure on specific disease groups which showed an increase inthe period under review and changes in the physical activity levels of the hungarian population which showed animprovement over the period under review initiatives in hungary aimed at encouraging an active lifestyle fromchildhood onwards should be continued since beyond the initial impact that has already been felt to some extent inrecent years these initiatives will come to their full fruition in the coming decadeskeywords physical inactivity economic burden parmethod direct costs indirect burden population attributable risk the fundamental change towards a more sedentary lifestyle has a serious impact on peoples health physical inactivity is one of the most important global issues of thetwentyfirst centuryleading to an increased risk ofchronic diseases such as type diabetes cardiovascular correspondence davidpaaretkptehu1university of pecs faculty of health sciences pecs hungaryfull list of author information is available at the end of the disease certain types of cancer rectal colon breastobesity and osteoporosis these diseases may even become the cause of death the world health anizationwho has also identified these medical conditions as themost burdensome noncommunicable diseases of todaysdeveloped world regular moderate physical activity reduces the risk of the most common of these diseases andcontributes to an increased sense of wellbeing [ ] acinactivity ranks as thecording to the who physical the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0c¡cs bmc public health 20suppl page of fourth most significant mortality factor in the world with million deaths a year worldwide [ ]another study suggests that there is a lower likelihoodof health problems among people engaging in regularphysical activity than among those leading a sedentarylifestyle furthermore there is convincing evidence thatregular physical activity increases life expectancy and reduces the likelihood of developing coronary and cardiovascular problems of suffering a stroke or developingcolon cancer inactive and sedentary lifestyles directly affect metabolism bone mineral composition and magnify the healtheffects of cardiovascular disease furthermore thereis epidemiological evidence to suggest that a sedentarylifestyle increases the risk of cancer obesity metabolicand psychosocial problems according to oecd data the average life expectancyof hungarians at birth in was years which is years below the oecd average actually one of the lowest on the list for men this value is years forwomen years both showing an increasing trend in recent years the hungarian government has made anumber of efforts to bring about significant changes in theinactive lifestyle of the hungarian population these include measures to increase the number of physical education lessons and to improve the conditions in pe lessonsat school also the development and construction of sportsfacilitiesincreased funding for sports associations andeven the use of corporate tax incentives for sporting purposes while improving the conditions alone does notresult in a change in the attitudes of the population towards sport it is certainly a prerequisite procedures that quantify the burden on the hungarianeconomy resulting from physicalinactivity are one ofthe ways of measuring the effectiveness of state intervention [ ] this study aims to contribute to this bodyof research and proposes to analyse a longer timespectrummethodsto analyze the economic burden of physical inactivity weneed to start with the burden of diseases on the nationaleconomy as physical inactivity plays a vital role in the onset of several diseases and leads to various causes of deathat a national level diseases have direct and indirect costsdirect costs of diseases include treatments medications sickpay allowances and associated ancilliary coststhat are directly related to the illness the direct costs inhungary are mainly financed by the national health insurance fund nhif since it is called the national health insurance fund administration nhifa but we must not disregard the cost of sick leave and private costs outside of nhifnhifa financing the latterof which are directly borne by members of societyamong the indirect burdens we include items that constitute a loss to the economy or to society as a result ofthe loss of work caused by a disease there was a significant change in this area during the research periodwhile in and there was a longterm loss ofproduction only in jobs on the skillsshortage list or invery special cases by the number of job vacanciesin hungary reached thousand while by july thisnumber rose to thousand people which is of theworkforce our calculations were based on the following assumptions in and in a labor marketcharacterised by an oversupply of labor a frictional unemployment of months groupbased performance expectations and the market of goods and services beingoversupplied people on average worked days a yearand loss was calculated based on gdp per capita thestudy that inspired our calculations had a similarcalculation but we replaced its assumptions with theabovementioned assumptions and we broadened andtightened formulas and corrected data that had becomefact since then however when calculating the results we had to change the assumptions about the labormarket from the previouslyoutlined conditions as by hungarian labor market had become characterizedwith overdemand therefore we had to increase frictiontime as well monthsanother economic burden is presenteesim which isthe term used for the phenomenon when a sick individual goes to work which results in poorer performanceand thus loss of productionour main goalin this research was to quantify theeconomic burden of diseases for the years and and more specifically the costs to thenational economy directly attributable to physicalinactivity in the market years and during our research we treated as relevant secondarydata eurobarometer and and nhifnhifa data from and []in the course of our resreach we examined the typesof medical conditions related to physical inactivity andtheir possible complications the factual data for whichwas obtained from nhif and nhifawith the help of par method population attributable risk the most commonly used method in international research we were able to obtain quantitativemeasurements that were used uniformly in the analysisof data for all three yearspar ¼ pexp 02 rr¾ ¾ pexp 02 rr°°¾ 02 wherepexp prevalence refers to the section of the populationwhere a given risk is present 0c¡cs bmc public health 20suppl page of rr relative risk describes the risk associated with asedentary lifestylewhen using the index it is necessary to break downthe population into physically active and inactive sections and then by determining the relative risk rate wecan estimate the number and cost of illnesses stemmingfrom a physically inactive lifestyle the physical activity indicators ofthe hungarianpopulation showed fluctuations during the period underreview the situation was the worst in when wesaw of the population as physically inactive in ouropinion the health protection effect does not manifestitself in the case of those who never excercise or only doso times a month by this figure dropped to and at the same time the ratio of those engaging inexercise at least times per week increased threefold inthe following years a more negative trend was observed as the rate of inactive people rose to although this is still significantly better than the basefigure for fig with the help of metaanalysis we calculated the relative risk ratio rr an indicator which is prevalent ininternational literature in order to estimate the futureexpenditure stemming from physical inactivity for all affected disease groups such as cardiovascular diseasestroke hypertension colon cancertype diabetesosteoporosis depression gastrointestinal complicationsobesity high triglyceride diseases and deliberate selfharm []the rr is the proportion of the applicaple diseasesamong people with inactive lifestyles divided by the proportion of the applicable diseases among people with active lifestyles on the basis of the rr values it is possibleto quantify the par indicator by disease group for eachyear table in order to allow the data to be compared over timethe data on the burden of illnesses stemming from physicalinactivity for and was recalculated to prices while the total amount of burden imposedby illnesses was recalculated to prices using thefig the ratio of physical activity and inactivity in hungary in sourcespecial eurobarometer special eurobarometer specialeurobarometer 0c¡cs bmc public health 20suppl page of table the cumulative relative risk rate and par values for theexamined disease types in disease typesheart and coronary diseasespar par rrpar strokehypertensioncolon cancertype diabetesosteoporosisdepressiongastrointestinal complicationsobesityhigh triglyceridesdeliberate selfharmsource katzmarzyk aldoori ewing andersen schuch domestic producer and consumer price index of thehungarian central statistical office hcso the economic burden ofresultsat pricesillnessesamounted to more than billion forints huf in of which the direct burden was billion forintsdirect costs accounted for of the burden of illnessesand the billion huf sickness benefit represented justover of total direct costs indirect burden representeda significantly lower percentage amounting to over billion huf the economic burden imposed by sicknessin was of hungarys gdpby the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden of illnesses that year less than of which amounting to billion huf was forsickness allowance expenditues indirect burdens decreased to billion huf the burden of sicknessamounted to of the gdp in by the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden that year and of it weresick allowances amounting to billion forints indirectburdens fell to billion huf the burden of sicknessdecreased to of the gdp in by the economic burden of illnesses increasedcompared to but it was still below the initial figure huf billion and it decreased in comparison with the gdp the share of direct costs dropped significantly to within which the sickness benefitrepresented to the value of billion forints atthe same time indirect burdens increased significantlyto billion huf all in all the burden of sickness decreased to of the gdp in between and the economic burden of diseases fell by billion huf which is a total decrease of corresponding to an average annual decrease of and in the meantime the countrys gdp increasedsignificantly altogether at current prices obviously the decrease is due to a number of reasons butthe effect of the increase in physical activity is an important factor among them table in the years examined in the case of disease groupslinked to physical inactivity the burden of illnesses onthe state budget excluding sickness allowance amounted to billion huf and billion hufrespectively of which the lowest value was in however only a part of these can be directly linked tophysical inactivity as many other risk factors play a rolein the development of these diseases as regards therelative weight of each disease group cardiovascular disease is the biggest burden followed by hypertension atthe same time type diabetes was only ranked the fifthfor costs in the first year but by it became thethird largest item only slightly behind high blood pressure expenditure on stroke obesity and deliberate selfharm were almost negligible compared to other diseasegroups table based on the results it can be stated that in theexpenditures in the state budgetfor the diseasegroups examined drastically decreased by approximately billion huf compared to the initial starting positionof billion huf but by the expenditures hadsurpassed the base total from by more than billion huf compared to only type two diabetesand osteoporosis showed an increase and respectively compared to although the latter is dueto the relatively low total expenditure for all other disease groups the level of expenditure declined in absoluteterms resulting in a significant decrease of billionhuf in total expenditurehowever in the case of the picture is more varied if we examine the relative position of certain diseasegroups compared to type diabetes showed themost significant increase to the tune of more than billion huf the other diseases lag behind in terms ofexpenditure cardiovascular diseases and colon cancerare next with an increase of billion forints inaddition there is an increase in the costs associated withosteoporosis stagnation or decrease was observed forthe other disease groups but this could not compensatefor the increase in the costs of the aforementioned diseases the most significant drop in expenditure is observed in hypertension billion huf and hightriglyceride diseases billion huf table focusing on the direct burden of physical inactivitywe can conclude that of the total expenditure ofthe disease groups is directly attributable to physical 0c¡cs bmc public health 20suppl page of table economic burdens of diseases in hungary in huf million in real terms in direct costs statefinancedeconomic burdens of diseasesin hungary medicationmedical aidsgeneral practitioner servicesdental servicesoutpatient carect mrimedical centers exluding vd clinicsdialysishome careinpatient carehighcost medical procedurespatient transportspa treatmentsgovernmental health careexpendituresick leavedisability rehabilitation treatmentcharged tonhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifnhifanhifnhifain totalprivate costsprivate health care expenditureindirect costsexpenditure associated withsick leavehealth insurance managementand other costsfriction due to sickness leading toloss of productionof which reduced pay due to sickpay and sick leaveof which tax loss for the statefriction due to disability leadingto loss of productionindividualemployernhifaemployer individualstateindividualstatesocietypresenteeism costsin totalemployerinactivity the major part is the cost of cardiovasculardiseases and hypertension and these were closelyfollowed by type diabetes by due to the factthat the total expenditure for stroke obesity and deliberate selfharm was also insignificant compared to otherdisease groups their expenditure related to physical inactivity is insignificant in the case of deliberate selfharm the costs cannot be measured even in the order ofone hundred thousand forints table compared to the decrease for the year ofthe direct costs stemming from physical inactivity is larger in proportion than the decrease of total expenditurethis is true of each disease group and for those twogroups type diabetes and osteoporosis where therewas an actual increase in costs the increase was less forrespectivelyphysical inactivityrelated expenses than for overall expenses the largest drop in monetary terms can be observed in the case of hypertension and cardiovasculardiseases over billion huf but there was a decreaseof and billion hufin hightriglyceriderelated diseases and colon cancer howeverpercentagewise nhif achieved the highest cost reduction for high triglycerides and colon cancer closely followed by a decrease in stroke expenditure and deliberate selfharm although inthe last two categories the low sum total spent alsomakes this decrease appear larger percentagewise thanwould be the case with larger totalscompared to the expenditure related to physicalinactivity in shows a decrease of billion huf 0ctotal amountinactivitytotal amountinactivitytotal amountinactivitycardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotal¡cs bmc public health 20suppl page of table total cost incured by nhifa nhif of those disease groups that are associated with physical inactivity and costs directlyattributtable to physical inactivity itself in terms of prices million hufdisease typesat the level of the individual disease groups the amountsvary the most significant decline in absolute terms is inthe high blood pressure and high triglyceriderelated illness groups however the burden of type diabetes increased significantly and there was an increase in coloncancer and osteoporosis disease groups the direction andextent of the changes are mostly comparable to the totalexpenditure amounts at the overall level of the diseasegroups although the changes in the physical inactivity ratenaturally lead to differences in the specific values this isso much so that the total expenditure amounts increasedat the level of all disease groups by but overall theexpenditure related to physical inactivity shows a decreaseof table discussionwe can clearly conclude similarly to other international researches [ ] that physical activityand forms of recreational exercise have a protectiveeffect eg a preventive effect against certain types ofchronic diseases cardiovascularlocomotor disordersdiabetes and certain types of tumors the decreasein physical inactivity has a positive effect on productivity as fewer people avail themselves of sick leave atable changes in total expenditure as reported by nhifa nhif and changes in expenditure directly stemming from physicalinactivity compared to the base level expenditure in in real terms adjusted to prices million hufdisease typescardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotaltotal amount inactivity total amount inactivity 0c¡cs bmc public health 20suppl page of study of economic development over the past centuryhas concluded that the advancement of the populations health status is responsible for about ofeconomic growth []in our comparative study we used four samplingpoints between and to demonstrate the burden of diseases at the level of the national economy forthe various loadcarriers in the period under review theeconomic burden decreased significantly overall from of the gdp to the weight of indirect burden increased however as in the currently demanddominated labormarket it is more difficult to replacelost workforce in the period of analysis the number ofemployees in hungary increased with which increased the amount of sick leave and number of sicknessdays but their gdp contribution was significantly higheralthough associated costs and burdens increased innominal terms they decreased in relation to the gdpa large part of diseases burdens are borne by the stateand society followed by households andemployers the proportions are similar to ding in european countries included hungary although we estimate that the burdens on employers arehigher and the burdens on households are lower inhungarysince the physical activity rate of the hungarianpopulation has been fluctuating but overall there is animproving tendency which is also apparent in the savings potential of the examined expenditures categoriescompared to the gdp the amount of spending dependsheavily apart from the physical inactivity rate on thenumber of employees as well as those people who arenot employed can not have sick leavefor exampleoverall government spending depends on the budget ofthe country which is connected to the overall economicsituationcardiovascular diseases accounts for most of the costof physical inactivity in hungary which coincides withmattli in switzerland however of directinactivityto depression inswitzerland while nhifas depression costs account foronly in hungaryattributablecostsarein hungary a number of measures have recently beentaken in order to integrate physical activity and sportinto peoples daily lives such measures include theintroduction of everyday physical education in schoolsor the extensive development of sports infrastructure however the effects of these measures will have amore pronounced effect in the long run several studieshave shown that high physical activity in childhood isnot yet measurable in terms of economic returns lessfrequent use of health care and a lower cost associatedwith using them as some effects such as the high costof sports injuries high rates of childhood illness haveresearch data confirm the facta negative bearing on the rate of return on investmenthowever a longterm change of attitude and opennessto physical activity at later stages in life are where thesemeasures bear a profit so any effort to support childinterhood sports is rewarding [ ] in additionnationalthatthoseparents who are themselves engaged in sport or currently do so are more likely to prefer sporting activitiesamong their children it is important to draw attentionto the fact even minimal physical activity has a healthimproving effect at any stage of life that is whysport and health policies at all times should promoterecreational activities for all ages not just young peoplewe would like to expand the scope of our current research if we could also examine how the patient numbers varied each year by disease group unfortunatelythe data was not available this would be of particularinterest for the year as the expenditure on illnessesshowed a significant increase in real terms compared to which may be due to the fact that more patientsreceived care and treatment but may also be due an increase of the normative provision per person by the government possibly to provide better quality careif we posit based on the eurobarometer data that thephysical activity rate improved compared to wecould also assume that fewer people were treated for theanalysed medical conditions in which would basically have a downward effect on total expenditure at thesame time however the picture is somewhat shaded bythe fact that even if the attitude of the population towards regular physical activity has changed in the lastfew years it is not certain that the number of illnesseswould decrease significantly in such a short period asthe negative effects of a sedentary lifestyle led for decades would not be easily offset by a few years ofexcerciseladen lifestyle this is especially true forolder age groups that is to say a reduction in the number of patients is not realised yet in patient care at thesame time the use of rapidlydeveloping medical technologies is also increasing the financial burden on thebudget as the higher costs of new technologies makemedical care per patient more expensive on the otherhandit should not be fotten that healing can bemade more effective and can lead to higher returns onhuman capitalthe study examined the development and compositionof direct and indirect burdens of disease in hungary andthe costs of physical inactivity to the state budget theseburdens fell in the examined periode which was associated with an increase of gdphowever there was an increase in the economic burinactivity which can beden associated with physical 0c¡cs bmc public health 20suppl page of attributed to the combined effect of two factors changesin total expenditure on specific disease groups whichshowed an increase in the period under review andchanges in the physical activity levels of the hungarianpopulation which showed an improvement over theperiod under review initiatives in hungary aimed at encouraging an active lifestyle from childhood onwardsshould be continued since beyond the initial impactthat has already been felt to some extent in recent years these initiatives will come to their full fruition in thecoming decadesabbreviationsct computed tomography gdp gross domestic product hcso hungariancentral statistical office huf hungarian forint mri magnetic resonanceimaging nhif national health insurance fund nhifa national healthinsurance fund administration oecd anisation for economic cooperation and development par population attributable risk rr relativerisk vd veneral disease who world health anizationacknowledgementsthe authors acknowledge to the nhifas colleagues for their help incollecting the dataset especially to mr zsolt kiss director general to drmihaly palosi head of department to petra fadgyasfreyler head ofdepartment and to valentina beitl analistthe authors would like to express their special thanks to prof attila fabianformer vice state secretary for his cooperative help during the datacollectionabout this supplementthis has been published as part of bmc public health volume supplement level and determinants of physical activity in the v4countries part the full contents of the supplement are available online athttpsbmcpublichealthbiomedcentralcomssupplementsvolume20supplement1authors contributionspa was the leader of the complete research coordinated the different coauthors work systematized the dataset summarised the literature related tothe relative risk ratios of illnesses calculated the par indices and contributedto the s dp has made calculations of par indices the direct costs ofphysical inactivity in the nhifa budget and contributed to the sfrom its results ms has made calculations of the total burdens direct andindirect of illnesses in hungary and contributed to the s from itsresults mh and psz summarised the related literature to the section ak and tsz have revised the results and contributed to the sall authors read and approved the final manuscriptfundingthe research was carried out and the publication costs funded by thesupport of hrdop36216201700003 cooperative research network ineconomy of sport recreation and health the authors declare that thefunding body does not have any role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe data of the state financed direct costs that support the findings of thisstudy are available from national health insurance fund administration butrestrictions apply to the availability of these data which were used underlicense for the current study and so are not publicly available data arehowever available from the authors upon reasonable request and withpermission of national health insurance fundthe datasets of the private ind indirect costs used and analysed during thecurrent study are available from the corresponding author on reasonablerequestethics approval and consent to participatethe ethical approval was granted for the study by ethics committee ofuniversity of p©cs nr participants were informed about theresearch aim and methods before signing the informed consent form theinvestigation conforms to the principles outlined in the declaration ofhelsinkiconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1university of pecs faculty of health sciences pecs hungary 2university ofphysical education budapest hungary 3corvinus university of budapestcorvinus business school budapest hungaryreceived march accepted march published august referencessebestyen a boncz i molnar a korosi l kovi r kriszbacher i olah apentek m sandor j relationship between surgical intervention type and days mortality of elderly femoral neck fracture in the prsence of differentcomorbidities value health 2009123a66kruk j health and economic costs of physical inactivity asian pac j cancerprev reiner m niermann c jekauc d woll a longterm health benefits ofphysical activitya systematic review of longitudinal studies bmc publichealth pratt m norris j lobelo f roux l wang g the cost of physical inactivitymoving into the 21st century br j sports med who global recommendations on physical activity for health switzerlandgeneva who blair sn cheng y holder js is physical activity or physical fitness moreimportant in defining health benefits med sci sports exerc s379tremblay ms colley rc saunders tj healy gn owen n physiological andhealth implications of a sedentary lifestyle appl physiol nutr metab rishiraj n inactivity a bad habit costing our productive lifestyle int j physmed rehabil oecd health status in edited by development ofecoa ¡cs p h©cz r pa¡r d stocker m a fitts©g m©rt©ke a fizikai inaktivit¡snemzetgazdas¡gi terhei magyarorsz¡gon k¶zgazdas¡gi szemle gabnai z m¼ller a b¡cs z b¡ba ©b the economic burden of physicalinactivity at national level [a fizikai inaktivit¡s nemzetgazdas¡gi terhei]eg©szs©gfejleszt©s health dev acs p stocker m fuge k paar d olah a kovacs a economic and publichealth benefits the result of increased regular physical activity eur j integrmed hcso in hcs o editor edn stadat time series of annual data labour market distribution of job vacancies koll¡nyi z imecs o az eg©szs©gbefektet©s budapest demosmagyarorsz¡g special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan powell ke population attributable risk of physical inactivity phys actcardiovasc health katzmarzyk pt gledhill n shephard rj the economic burden of physicalinactivity in canada cmaj 0c¡cs bmc public health 20suppl page of aldoori wh giovannucci el rimm eb wing al willett wc use ofacetaminophen and nonsteroidal antiinflammatory drugs a prospectivestudy and the risk of symptomatic diverticular disease in men arch fammed andersen lb schnohr p schroll m hein ho allcause mortality associatedwith physical activity during leisure time work sports and c | Colon_Cancer |
" 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 |
" the cell proliferative markers are very important in breast cancer since spag5 and numa proteinsplay a significant role in the mitosis regulatory network and cell division we aimed to study their mrna levels aswell as spag5 gene amplification correlated to clinicopathological status in ductal carcinoma of the breastmethods spag5 and numa gene expressions were investigated in breast cancer tissues and normal adjacenttissues via realtime pcr pum1 was selected as the reference gene qmf pcr method was applied to study spag5gene amplification and agbl2 bod1l and por were designated as internal control genes gene amplification wasdetermined by calculating a dosage quotient for each dna fragmentresults increased spag5 mrna expression was detected in breast cancer tissues p and related to tumorsize no significant difference was observed between numa gene expression level in tumor tissue and the normaladjacent tissue p however we observed that numa expression was significantly increased in erpositivetumor tissues there was no clear correlation pattern between spag5 and numa mrna levels r seventeenpercent of tissues showed complete amplification in spag5 gene fragments our results were consistent with the previous publications regarding spag5 gene expression andamplification in breast cancer with an emphasis on the prominent role of this protein in tumor pathogenesis ourresults failed to yield any correlation between spag5 and numa mrna levels which implies independence of thesegenes in breast cancer pathogenesiskeywords spag5 numa gene expression gene amplification breast ductal carcinomaintroductionbreast cancer is the most common cancer type and the second leading cause of cancer death in women worldwidenumerous studies have been conducted to find the cellularand molecular biomarkers to improve early detection andmanagement of the disease and reduce its mortality ratethe study published by abdelfatah has drawn attention to spermassociated antigen spag5 as a new correspondence shshahbazimodaresacir1department of medical genetics faculty of medical sciences tarbiatmodares university tehran iranfull list of author information is available at the end of the prognostic biomarker in breast cancer using the interactome map they showed a prominent role for spag5 in patients response to chemotherapy and survival spag5 gene is located on chromosome 1717q112comprising exons with nine reported mrna splicevariants it is categorized among conserved genes as ithas been present since the common ancestor of chordates the aminoacids protein of spag5 is a homodimer which binds to microtubules in the mitoticspindle and regulates its dynamic function spag5 isone of the essential components required for chromotiming of sister chromatidsome alignment normal 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 0cmohamadalizadehhanjani world of surgical oncology page of segregation and spindle pole formation spag5 proteinis expressed in a cell cycledependent manner with thehighest level at telophase in nonmitotic cells various stimuli upregulate spag5expression spag5 protein plays multiple roles in different pathways of cell growth and differentiation includingpi3kakt upon cell oxidative stress spag5 preventsapoptosis via mtorc1 suppression significant evidenceconfirms thatthe pi3kaktmtor pathway is themost common defected signaling cascade in breast cancer increased spag5 mrna expression in tumortissues was reported in different cancers such as cervixlung and bladder [] indicating theprostatehigher spag5 levels the poorer prognosis and patientsurvivalincreasedspag5 expression in cancer the cellular and molecularevents affected by this increase are still unclear it is notwell elucidated whether spindlassociated proteins areinvolved as much as cell signaling cascades[ ] despite several reports ofliverone of the important spag5 interactors in cytokinesisis the nuclear mitotic apparatus numa protein whichdirectly binds and recruits spag5 to the mitotic spindlethis interaction is reciprocal in a way that the lack ofspag5 interferes with the appropriate numa spindledistribution and function numa gene is located on11q13 with exons that encodes a large aminoacids protein it acts as a microtubulebinding proteininvolved in the arrangement of the spindle poles andproper separation of the chromosomes in a complexcomposed of dyneindynactinnuma ddn dynein is an energy supplier for eukaryotic cellular movements and dynactin serves as its cofactor studies havedemonstrated numadynein role in dna doublestranded break repair by homologous recombination related to brca proteins numa is also involved in different cellular pathways including plk1 activated celldivision and caspasemediated apoptotic response our study aimed to investigate the association betweenspag5 and numa mrna levels in breast cancer tumors since spag5 gene amplification was also involvedin the pathogenesis ofthe breast cancer we alsointended to identify any association between the amplified genome and spag5 numa mrna levelsmethodspatientspatients were selected between january and october by random sampling from farmaniyeh hospitaland sina hospital affiliated to tehran university ofmedical sciences inclusion criteria were as follows anew case of biopsyconfirmed breast invasive ductal carcinoma idc with or without ductal carcinoma in situdcis exclusion criteria were receiving any chemotherapy radiotherapy and hormone therapy the patientssigned a consent form approved by the ethics committeeof tarbiat modares university with the registrationnumber of irtmurec1396681 eighty tissue samplesincluding forty tumors and forty normal tissues adjacentto the tumor nat were collected under the supervision of the breast surgeon patients clinicopathologicalcharacteristics comprising age family history of cancerlymph node involvement tumor size tumor grade estrogen receptor er progesterone receptor pr andhuman epidermal growth factor receptor her2 statuses were recorded clinical stage was determined bytnm system including tumor size lymph node involvement and metastasis lymph node involvement wasconfirmed histologically according to ajcc guidelinethe molecular stage was defined using tnm tumrade and er pr and her2 status we further classified patients to breast cancer subtypes luminal a luminal b her2 positive and basallike ki67 index as amarker for tumor proliferation was applied to subtypeluminal tumors by the cutoff value of since nats were taken from the nontumorous regionof the same patients breast for a more accurate comparison we also included three samples of healthy breasttissues from individuals with no history of breast cancerthe reason for surgery in these healthy individuals wascosmetic restorations and they did not have any historyof cancer in 1st and 2nddegree family relativesmcf7 and mdamb231 cell line culturebreast cancer cell lines were used as the control of realtimepcr and quantitative multiplex fluorescent pcr qmfpcr mcf7 ibrc c10082 and mdamb231ibrcc10684 were obtained from the national cell bank of iranboth cell lines were invasive breast ductal carcinoma bearing an epitheliallike morphology cell lines were culturedin the medium containing dulbeccos modified eaglemedium dmem and fetal bovine serum fbsdna and rna extractionafter homogenization of mg breast tissue dna andrna were simultaneously extracted by allspintm minikit geneall pishgam biotech iran dna and rnaquantities were determined by spectrophotometry at and nm the observation of 18s rrna and28s rrna bands in gel electrophoresis was in favor ofappropriate rna quality cdna synthesis was performed by 2x rt premix kit biofact noavaran tebiran mmlv reverse transcriptase oligodt and random hexamer were simultaneously applied to increasecdna synthesis efficacyrealtime pcrthe expression of spag5 and numa genes were quantified by realtime rt pcr to normalize the results 0cmohamadalizadehhanjani world of surgical oncology page of pum1 was selected as the reference gene pum1 is ahousekeeping gene with high and constant expression innormal and tumor breasttissue primer design forspag5 numa and pum1 genes was conducted by primer3 and oligo analyzer software and checked onblast basic local alignment search tool websites toprevent genomic dna amplification all realtime pcrprimers spanned exonexon junctions the detailedcharacteristics of realtime primers are provided in table realtime pcr was performed by applied biosystemstep one system using sybr green dye pcr efficiencywas calculated by linreg pcr software through linearregression analysis1bod1l atpgtpbinding protein likeqmf pcrspag5 gene amplification was examined through qmfpcr and biorientation of chromosomes in cell division likeagbl2 and cytochrome p450 oxidoreductase porwere selected as internal control genes the primerswere designed for three parts of spag5 gene ² endmiddle and ² end of the gene all internal controls andthree spag5 primers comprised similar tm table to fluorescently label pcr fragments universal primerwas opted for as an efficient and costeffective methoduniversal primer sequence published by roche companywhich does not have any specific complementary site inthe human genome was selected each fragmentwas amplified by primers tailed locusspecific forwardprimerlocusspecific reverse primer and 6famlabeled universal primer in a concentration ratio of due to the lower starting concentration of the tailedlocusspecific primer as pcr progress it was graduallydepleted and the 6fam labeled universal primer wassubsequently included amplitaq goldtm dna polymerase ampliqone pishgam biotech iran with hotstart activity was applied for a multiplex pcr reactioncapillary electrophoresis was performed via μl of thepcr products μl offormamide and μl ofgenescan500 liz size standard applied biosystemscourtaboeuf france the fluorescent pcr productswere heatdenatured chilled on ice and separated on an8capillary sequencer genetic analyzer appliedbiosystems france the results were processed withgenemarker software the peak area values wereimported into an excel microsoft file and the copynumber of each fragment was calculated by the dosagequotient dq methodstatistical analysisthe sample size was calculated by epi info¢ softwarespss statistic version and graphpad prism version were used for realtime data analysis to determine thetype of appropriate statistical method data were analyzed by kolmogrovosmirnov for distribution normaltheity since the distribution of data was normalparametric test was used pairedsample ttest wasemployed to compare spag5 and numa gene expression level between tumors and nats the links betweenclinicopathological status and mrna levels were calculated via independenttest and onewayanova for qmf pcr result analysis spss statisticversion was used to determine the frequency of samples with gene amplificationsample tresultspatients characteristicsthe mean age of the patients was ± years theminimum and maximum ages of the patients were and years thirteen patients had a family history of cancer mainly breast or colon cancer detailedclinicopathological features of the patients are given intable clinical stage was categorized by tnm system and theresults revealed that stage i stage ii and stages iiiivassigned to and of the samples respectively as indicated in table of the sampleswere categorized in the molecular stage i in stageii and in stages iiiiv further classification ofbreast cancer subtypes showed that luminal her2 positive and basallike comprised and of tumorsrespectively using ki67 index analysis weobserved that luminal a allocated to and luminal bto of the tumorstable specific primers designed for quantitative realtime pcrgenepum1sequence ²_3²fcctaccaactcatggtggatgtlengthspag5numaragccagcttctgttcaagactfaaggagaagactgaacaagagaccrtcatctgccactgctgtcaagfagagagcaaactaagcaggtggrcctggacagccttcagcttcttmgcproduct bppcr polymerase chain reaction tm melting temperature gc guanine cytosine bp base pair pum1 pumilio rnabinding family member f forward r reversespag5 spermassociated antigen numa nuclear mitotic apparatus protein 0cmohamadalizadehhanjani world of surgical oncology page of table specific primers designed for qmf pcrgenebod1lsequencefaatgcctccgctttcaggcchagbl2porspag55²spag5middlespag53²ratcacttggcaactcacacatggfgcgagctgcattccatgcgrtcccagctttggaaacgcacfagccactttgtgccagatcartccagcacgtgttcacatcafccctaagaagcccaaaatgcgrtcctggaaagttgggtcgagfaggctgctcatctgattcatgcrcaagctaccatctgcccacgfgttaggaaagggtcgaaagggcracaccctatcaaaagtctgttcclengthtmgcproduct bpqmf pcr quantitative multiplex fluorescent polymerase chain reaction ch chromosome tm melting temperature gc guanine cytosine bp base pair bod1lbiorientation of chromosomes in cell division like agbl2 atpgtpbinding protein like por cytochrome p450 oxidoreductase spag5spermassociated antigenspag5 gene expression levelsmelting curve analysis of the pcr products showed a single peak indicating specific amplification of the desiredgene fragment to normalize the results the ct differenceδct of spag5 and pum1 was computed to comparespag5 gene expression in tumor with nats δδct wasdetermined the fold change was calculated by δδctmethod the spag5 and pum1 gene pcr efficiency was obtained by linregpcr softwarethe statistical analysis revealed a significant differencebetween spag5 gene expression levels in tumor relativeto nats p spag5 gene expression was upregulated in tumor tissues with a mean fold change of and the mean difference of fig using independent sample t test the spag5 mrna levelswere analyzed in correlation to age tumor grade er pr andher2 status as indicated in table the results showed nostatistically significant association between spag5 gene expression and these clinicopathological featureshowever correlation studies revealed a significant linkbetween spag5 gene expression and clinical stage p since the clinical stage was defined by tumor sizelymph node involvement and metastasis we analyzedthese parameters separately the patients tumors werecategorized according to the size cm cm cm and spag5 gene expression was evaluated in the patients groups by oneway anova analysis the resultsdemonstrated a significant link between spag5 mrnalevels and tumor size p spag5 gene expressionlevel was higher in larger tumors lymph node involvement and metastasis revealed no significant correlation tospag5 mrna levels the statistical analysis regardingmolecular stage and molecular subtypes failed to showany significant association table numa gene expression levelsthe mean of ct values for numa and pum1 were analyzed for duplicate samples and modified based on reaction efficiency the results of linreg analysis indicatedpcr efficiency of for numa gene the specificity ofthe pcr amplification was confirmed by melting curvegraphs of each reaction the mean fold change of numagene expression in tumors relative to nats was which was not statistically significant p056 furtherdata analysis disclosed a significant association betweennuma gene expression and er status p0006 ernegative tumors showed eight times less numa gene expression than erpositive tumors er is one of the mainparameters of molecular subtype classification so as expected this significant difference was also observed regardinglink between numa expression andmolecular subtypes p however numa expression did not show any significant difference in the molecular stage or the clinical stage table thethe correlation study to other clinicopathological status revealed no significant link with age tumor gradelymph node involvement and tumor size no significantconnection was observed regarding pr or her2 statustable the correlation between spag5 and numa geneexpression in tumor tissuethe study of the correlation was conducted betweenspag5 and numa genes expression using the pearsontest in fig the x and yaxes show δct of spag5 andnuma genes respectively the test results showed thatthere was no statistically significant correlation betweenthe two mrna levels r 0cmohamadalizadehhanjani world of surgical oncology page of table statistical analysis of clinicopathological variables related to spag5 and numa mrna expressionsspag5agegradelnfherprher2numaagegradelnfherprher2n t years yearsi and iiiiipositivenegativepositivenegativepositivenegativepositivenegativepositivenegative years yearsi and iiiiipositivenegativepositivenegativepositivenegativepositivenegativepositivenegative ssigtailedstderror cilower upper independent sample t test was applied to compare the spag5 and numa gene expression levels according to clinicopathological features normal distribution ofvariables and equality of variances were checked by kolmogorovsmirnov and levenes test respectively the significance level was selected at n number t t test sig significance p value std standard ci confidence intervals spag5 spermassociated antigen numa nuclear mitotic apparatus protein lnlymph node involvement fh family history er estrogen receptor pr progesterone receptor her2 human epidermal growth factor receptor qmf pcr resultsthe fragment analysis was performed following verifiedmultiplex pcr pcr products should have at least bplength differences to be distinguishable in capillary electrophoresis fig we applied universal primer toavoid nonspecific pcr products and amplify multiplelociin one pcr reaction the copy number of eachspag5 fragment was determined by calculating the dqobtained by dividing the fragment peak area by the meanpeak area of control genes as demonstrated in fig the x and yaxes represents dna fragments length inbase pair bp and signal intensity as a relative fluorescent unit rfu the 3sd and 3sd values were considered as cutoff points values greater than wasconsidered amplified and values less than as deletionaccording to previous publications mcf7 and mdamb231 cell lines showed completespag5 gene amplification while three normal breasttissues and two healthy donor blood samples which usedas normal controls did not show any gene amplificationwe calculated mean dq of and for mcf7and mdamb231showed complete gene amplification in all three spag5gene fragments with mean dq of comparing samples which had no amplification in any fragment of spag5 gene mean dq the rest of thesamples showed amplification in one or two parts of thegene with a mean dq of respectively six samples 0cmohamadalizadehhanjani world of surgical oncology page of table analysis of staging related to spag5 and numa mrna expression clinical stage was determined by the tnm systemtumor size lymph node involvement metastasis according to ajcc guideline the molecular stage was defined using tnm tumrade and er pr and her2 statusspag5clinical stagemolecular stagemolecular subtypesnumaclinical stagemolecular stagemolecular subtypesstage istage iistages iii and ivstage istage iistages iii and ivluminal aluminal bher2 positivebasallikestage istage iistages iii and ivstage istage iistages iii and ivluminal aluminal bher2 positivebasalliken sum of squaresdfmean squarefp valueoneway anova was employed to analyze the difference between spag5 and numa gene expression levels related to clinical stage molecular stage andmolecular subtypes normal variable distribution and equality of variances were checked by kolmogorovsmirnov and levenes test respectively our chosensignificance level was n number df degree of freedom spag5 spermassociated antigen numa nuclear mitotic apparatus proteinfig fold changes of spag5 and numa genes calculated by the relative expression software tool rest spag5 mrna expression wasincreased times in breast tumors than the normal adjacent tissues p the mean fold change of numa gene expression in tumorrelative to normal adjacent tissue was 0cmohamadalizadehhanjani world of surgical oncology page of fig pearson correlation of spag5 and numa expression levels in tumor samples the x and yaxes show δct of spag5 and numagenes respectivelydiscussionthe main purpose ofthis study was to investigatespag5 gene expression and amplification related tonuma mrna levels in breast cancer tissues as thenormal control sample we used the same patients nontumor breastthisit can be assumed thattissuefig gel electrophoresis of multiplex pcr first well is loaded byladder bp and wells are loaded with dna of tumor tissuesbands arrangement from biggest to smallest are ² end of spag5 bp middle of spag5 bp por bp agbl2 bpbod1l bp and ² end of spag5 bpseemingly healthy tissue which was adjacent to a tumormay have altered gene expression or amplification toprevent this drawback we attempted to sample at thefarthest possible distance from the tumor as recommended by the cancer genome atlas tcga tcga mentioned that nat sampling must be performed cm from the tumor margin it has been reported that genetic changes could be detected in thenormal margin of the tumor but are rarely evident atdistances greater than cm in our study we further evaluated the nats histopathologically and we observed no morphological or molecular alterations toevaluate possible changes in spag5 and numa geneexpression in the nats three healthy breast tissuesfrom women referred for cosmetic surgery were included in the study the δct of spag5 and numagenes in these three samples were the same as the meanof δct of spag5 and numa genes in nats indicatingthatthe nats did not bear noticeable expressionchanges in the studied genesour results showed that spag5 mrna expressionwas increased times in breast tumors than thenats correlation studies revealed a significant link between spag5 gene expression and tumor size withhigher levels in larger tumors our results also showed asignificant correlation between spag5 gene expressionand clinical stagespag5 knockdown has shown cell growth suppressionand apoptosis enhancement while the increased spag5expression promotes cell proliferation and inhibits apoptosis as previously mentioned spag5 preventsapoptosis through pi3kaktmtor pathway and issimilarly involved in wntβcatenin signaling pathwayactivation via stimulation of wnt and βcatenin expression clinical studies have confirmed the role ofspag5 in breast cancer prognosis and survival it has 0cmohamadalizadehhanjani world of surgical oncology page of fig dna fragments electropherograms in genemarker® software the x and yaxes represent dna fragments length in bp and signals intensity as arelative fluorescent unit rfu respectively the orange peaks were generated by standard size fragments and the blue peaks by spag5 dna fragmentsand control genes the order of the peaks from the largest to the smallest corresponds to ²spag5 bod1l agbl2 por middle spag5 and ²spag5been shown that patients with higher spag5 gene expression were at high risk of cancer spag5 was also proposed as a proliferative marker in earlystage of cancer li reported spag5 as an oncogene in basalliketriplenegative breast cancer by direct interaction withmycbinding protein mycbp which results in an increased cmyc transcriptional activity cmyc regulatesthe expression of several genes involved in the process ofcell growth and dna repair exhibiting high expression inbasallike tumors based on the obtained results they considered spag5mycbpcmyc axis as a potential therapeutic target in the triplenegative patients howeverour study did not reach similar results and we observedno significant difference in spag5 gene expression inbasallike tumorsthe previous study had suggested a link between spag5and numa during cell division we also analyzed thecorrelation between spag5 and numa gene expression inpairwise comparisons our results yielded no significantcorrelation between spag5 and numa indicating independent expression of these two genes in the process ofbreast tumorigenesis although no link was found betweenthe expression levels of these two genes further studiescould investigate the association of their proteinswe investigated numa transcript levels expression inbreast cancer tissues relative to nats and we observeddecreased numa expression however it was not statistically significant an interesting finding of our studywas that numa expression in erpositive was higherthan ernegative tumor specimens it can be justifiedthat numa expression increases in erpositive cellssince the er requires nuclear matrix components suchas numa for its transcriptional activity previous studieshave suggested the association between er intranuclearmobility and the dynamics of the nuclear matrix the main function of the er is localized in the nucleuswhere it acts as a liganddependent transcription factorit interacts with multiple intranuclear components including nuclear matrix proteins to regulate transcriptionof the estrogenresponsive genes in the presence of aligand er distribution shifts from the diffused to thediscrete state indicating the binding of the receptor tothe nuclear matrix elements since numa is a part ofthe nuclear matrix components it could be concludedthat it plays a pivotal role in the mobility of er our results showed a significant association betweennuma mrna levels and molecular subtypes which areclassified based on er pr and her2 expression 0cmohamadalizadehhanjani world of surgical oncology page of recently using machine learning algorithms kothari attempted to find potential genes discriminatingbasallike from other subtypes they reported tbc1d9as a candidate gene that its expression was linked to improved patients survival further analysis revealed differentroles for tbc1d9 in anelle biogenesis andlocalization as well as recruitment of numa to the mitotic centrosome it has been shown that brca deficiency causes cortical asymmetry of numadyneincomplex in dividing cells which provoke genomic instability and aneuploidy a new role for numa wasrevealed related to diffusion regulation of p53bindingprotein 53bp1 which plays a prominent role in dnarepair higher numa mrna levels were significantlylinked to increased breast cancer patients survival evenwhen specifically analyzed in basallike tumors association study using snps in nearly genesshowed that a 300kb region on chromosome 11q13encompassing the numa gene influences breast cancerrisk they suggested a794g variant as a susceptibility allele for breast cancer however additional investigations did not support the role of numa variants asbreast cancer susceptibility alleles the other phenomenon of interest in our study wasthe spag5 gene amplification applying qmf pcr toevaluate gene copy number variation cnv we observed complete amplification in of tumor samplesthese results were in line with the results of the abdelfatah that reported spag5 gene amplification in breast tumors defects in dna replicationor telomere dysfunction promote gene amplificationwhich can lead to tumor progression in any stage ofbreast cancer gene amplification confers a selectiveadvantage during earlystage of cancer by increasing oncogenes expression in amplified regions it has beenshown that gene amplification is associated with moreaggressive tumors as well as resistance to chemotherapywhich can predict disease prognosis despite the significant results our study also has particular limitations that should be addressed in the futurethe association of spag5 and numa at the proteinlevels and the molecular mechanism of their interactionneed to be clarified in vitro and in vivo furthermoredue to the limited sample size we included idc samplesin the study without considering the presence or absenceof dcis it would be interesting to study the gene expression differences between pure idc and idcdcisour study highlights the role of increased spag5 geneexpression and gene amplification in breast cancer theresults showed that in largersized tumors the spag5mrna wasthatspag5 may be further studied as a therapeutic target inincreased this evidence suggeststhe treatment of breast cancer therefore complementary studies can be suggested to investigate the role ofthe proteins that interact with spag5 we did not observe any significant correlation between spag5 andnuma mrna levels however our results indicated anassociation between numa and tumor er expressionstatus this finding reported for the first time in ourstudy could be the starting point for further studies oner and cell division apparatus in breast canceracknowledgementsthe authors would like to thank the patients who participated in this surveyauthors contributionsauthors contributions are as follows zmh project development datacollection and management data analysis and manuscript writingeditingss project development data collection and management data analysismanuscript writingediting lg data collection and management dataanalysis manuscript writingediting all authors read and approved the finalmanuscriptfundingnot applicableavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding authors upon reasonable requestethics approval and consent to participatethis study was approved by the ethics committee of tarbiat modaresuniversity all patients provided written informed consent and the studywas conducted in accordance with the declaration of helsinkiconsent for publicationnot applicablecompeting intereststhe authors declare no competing interestsauthor details1department of medical genetics faculty of medical sciences tarbiatmodares university tehran iran 2department of surgery sina hospitaltehran university of medical sciences tehran iranreceived may accepted august referencesabdelfatah tma agarwal d liu dx russell r rueda om liu k spag5 as a prognostic biomarker and chemotherapy sensitivity predictor inbreast cancer a retrospective integrated genomic transcriptomic andprotein analysis lancet oncol mack gj compton da analysis of mitotic microtubuleassociated proteinsusing mass spectrometry identifies astrin a spindleassociated protein procnatl acad sci u s a thein kh kleyleinsohn j nigg ea gruneberg u astrin is required for themaintenance of sister chromatid cohesion and centrosome integrity j cellbiol thedieck k holzwarth b prentzell mt boehlke c klasener k ruf s inhibition of mtorc1 by astrin and stress granules prevents apoptosis incancer cells cell yang yf zhang mf tian qh fu j yang x zhang cz spag5 interactswith cep55 and exerts oncogenic activities via pi3kakt pathway inhepatocellular carcinoma mol cancer valk k vooder t kolde r reintam ma petzold c vilo j geneexpression profiles of nonsmall cell lung cancer survival prediction andnew biomarkers oncology liu jy zeng qh cao pg xie d yang f he ly spag5 promotesproliferation and suppresses apoptosis in bladder urothelial carcinoma by 0cmohamadalizadehhanjani world of surgical oncology page of upregulating wnt3 via activating the aktmtor pathway and predictspoorer survival oncogene liu h hu j wei r zhou l pan h zhu h spag5 promoteshepatocellular carcinoma progression by downregulating scara5 throughmodifying betacatenin degradation j exp clin cancer res chu x chen x wan q zheng z du q nuclear mitotic apparatus numainteracts with and regulates astrin at the mitotic spindle j biol chem seldin l poulson nd foote hp lechler t numa localization stability andfunction in spindle orientation involve and cdk1 interactions mol biolcell vidi pa liu j salles d jayaraman s d | Colon_Cancer |
s studies were published in a language otherthan english and studies that reprocessed data from public databasesquality assessment and data extractiontwo investigators yyw and yjz independently assessed all potentially eligible studies based on thenewcastleottawa scale nos s with nos ¥ were regarded as highquality studies any discrepancywas resolved by discussion we extracted the following required information first author and publication year patient characteristics country ethnicity sample size type of specimen histological grade stage of disease hormonereceptor including progesterone receptor pr and estrogen receptor er status human epidermal growth factor receptor her2 status lymphoid node status and followup detection methods of malat1 and cutoff valueand hrs and the corresponding ci of upregulated malat1 for overall survival os metastasisfree survival mfs diseasespecific survival dss diseasefree survival dfs or recurrencefree survival rfs we alsoutilized engauge digitizer version to estimate the hrs based on the graphical survival plots when hrs were notdirectly available in the study we also corresponded with relevant original authors for additional data requiredfor metaanalysisbreast cancer patient cohort analysis based on public datasetsthe gene expression profile of gse19615 containing chemotherapytreated breast cancer samples was obtained fromthe gene expression omnibus geo database wwwncbinlmnihgovgeo the genome expression dataof breast cancer samples from the cancer genome atlas tcga database portalgdccancergov were alsodownloaded malat1 expression levels were compared between the two groups according to whether the includedpatients received chemotherapy kaplanmeier survival analysis was then employed to compare the survival outcomesbetween groups given the vital roles of immune infiltration in the tumorigenesis and metastasis we applied thecibersort algorithm to determine the association between malat1 and infiltrating immune cells from geneexpression profiles the correlation was evaluated using the pearsons correlation coefficient an estimated pvalue of was considered statistically significant 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 bsr20200215101042bsr20200215table main characteristics of the studies included in the present metaanalysisauthors name yearcountrynumbertypecutofffollowupmonthssampledetectionmethodjin xu huang jadaliha chinachinachinausamediannr expressionnrnrnrtissuetissuetissuetissuertpcrrtpcrrtpcrrtpcrexpressionlevelhrscnrreportedreportedsurvivaloutcomesmultivariateanalysisnosscoreosnrrfsdssnonononotnbcmixeder and ertnbc andher2mixedmixedmixedmixedtnbcmixedmixedmixedmeseure miao wang zhang zuo li wang zhang abbreviations er er positive er er negative her2 her2 positive nm not mentioned nr not reported qrtpcr quantitative reverse transcriptionpolymerase chain reaction sc survival curvenrscscreportedscscreportedsc302foldmediannrmediannrmedianmedianmfsdfsosososrfsosrfsosnononoyesnonoyesnofrancechinachinachinachinachinachinachinanrnrnrtissuetissuetissueserumtissuetissuetissuetissuertpcrrtpcrrtpcrrtpcrrtpcrrtpcrrtpcrrtpcrstatistical analysishrs and their associated cis for os mfs dfs dss and rfs were used to evaluate the prognostic effect ofmalat1 in breast cancer an hr and ci not including suggested a significant association between poorprognosis and elevated malat1 expression cochrans q test and pvalues were applied to find the heterogeneityamong studies if the calculation suggested significant heterogeneity i2 ¥ andor p the randomeffectsmodel was utilized otherwise the fixedeffects model was more appropriate ors and their corresponding ciswere calculated to determine the association of malat1 with the clinicopathological variables of breast cancer weused a sensitivity analysis to evaluate the stability of the results the possibility of publication bias was also testedthrough eggers test and beggs funnel plot all statistical analyses were performed using the stata softwareresultssummary of the included studiesbased on the web of science pubmed and embase databases we initially retrieved papers of which wereexcluded due to duplication after careful screening of the titles and s potentially eligible s werereviewed in detail due to the lack of key information regarding survival outcomes of the s were subsequently excluded hence we finally included a total of s in the metaanalysis [] figure summarizes the details of the literature selection procedurea total of patients from china france and the united states were investigated with a minimum and maximum sample size of and patients respectively malat1 expression levels were assessed using reverse transcriptase polymerase chain reaction rtpcr in all studies with studies employing cancerous tissue and usingserum specimens the cutoff estimates for malat1 expression including the median fold change and expression level were inconsistent among these studies elevated malat1 expression levels in breast cancer werereported in most studies except in one study that reported the downregulation of malat1 among the different cancer subtypes jadaliha found that malat1 expression was consistently higher in erpositive patientsthan in those with the triplenegative breast cancer tnbc subtype they also suggested that tnbc subtypecells express relatively lower levels of malat1 than other subtypes of breast cancer cells there were two studieswhich focused only on tnbc of these huang divided all patients into groups based on receptor status with erpositive or negative groups and jadaliha did not report a negative result in all patients butmentioned the survival outcomes in the lymph nodenegative tnbc and her2positive breast cancer subsets eight of all included studies described the clinicopathological features of breast cancer ofthe s studies only described the survival outcomes of breast cancer without providing necessary data toobtain hr hr was reported directly in four studies and estimated indirectly for the remainingsix studies [] table shows the detailed information of the included studies 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 bsr20200215101042bsr20200215figure flow chart depicting the literature selection procedure in this metaanalysisassociation between malat1 expression and ossix studies evaluated the connection between malat1 expression levels and os no statisticallysignificant heterogeneity was observed among the six studies i2 p0458 thus the fixedeffects modelwas applied collectively the results indicated that increased malat1 expression in breast cancer was related to aworse os hr ci p00001 figure next we performed subgroup analysis according tocancer type sample size and the method of variance analysis as shown in table the subgroup analysis for cancertype suggested that malat1 upregulation was a strong prognostic biomarker in both patients with tnbc hr ci p00001 and in those with mixed type breast cancer hr ci p00001 next we performed subgroup analysis according to the sample size with a threshold for patientsthe results showed that the association of malat1 dysregulation and os was not significantly influenced by samplesize n¥ hr ci p00001 n100 hr ci p0006 similarlypooled results of subgroup analysis according to the analysis method revealed that elevated malat1 expression the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20200215101042bsr20200215figure forest plots of studies evaluating the association between malat1 expression and os in patients with breastcancertable results of subgroup analyses of pooled hrs for os with elevated malat1 expressioncategoriesnumber of studiesnumber of patientspooled hr ci heterogeneityi2 pvaluecancer typemixedtnbcsample size¥analysis methodunivariatemultivariate was significantly associated with worse os in both the multivariate hr ci p00001 andunivariate groups hr ci p00001association between malat1 expression and dfsrfsdsssix studies investigated the connection between malat1 expression levels and dfsmfsdssrfs []li s study suggested that low malat1 expression predicted favorable rfs in their cohort hr ci p0001 their data were insufficient for quantitative analysis but were still included in thequalitative analysis meseure s study suggested that the impact of malat1 expression level on mfs was notsignificant unfortunately this study did not provide useful data for hr calculation eventually a total of fourstudies compromising six groups of data reported the prognostic significance of malat1 for dfs rfs and dss 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 bsr20200215101042bsr20200215figure forest plots of studies evaluating the association between malat1 expression and dfsrfsdss in patientswith breast cancerthe pooled hr was ci p00001 using the fixedeffects model figure on account of thelimited number of studies a subgroup analysis for dfsrfsdss was not performedassociation between malat1 expression and clinicopathologicalparameterswe also evaluated the relationship between the expression level of malat1 and the clinicopathological features bypooling the clinicopathological data as shown in table we found that high expression of malat1 was significantly associated with pr status or ci p00006 fixedeffects model unlike the prstatus the er status did not display a significant correlation with malat1 expression as there was great heterogeneity or ci p019 randomeffects model in addition a significant correlation wasnot found at tnm stage or ci p057 randomeffects model tumor size or ci p075 fixedeffects model distant metastasis or ci p083randomeffects model lymph node metastasis or ci p07 randomeffects modelhistological grade or ci p074 randomeffects model and her2 status or ci p056 fixedeffects modelsensitivity analysis and publication biaswe performed sensitivity analysis to examine the stability of the metaanalysis when any one study was sequentiallyomitted no individual study dominantly affected the overall hr for os figure 4a or dfs rfs or dss figure 4beggers test and beggs funnel plot were used to test publication bias as shown in figure 5ab the funnel plotdemonstrated basic symmetrical shape in os studies eggers test p0071 and in dfsrfsdss studies eggerstest p0142 hence there was no noticeable publication bias in the current metaanalysis 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 bsr20200215101042bsr20200215table metaanalyses of malat1 expression with respect to clinicopathologic parametersvariablesnumber ofstudiesnumber ofpatientstnm stage iiiiv vs iiitumor size vs cm distant metastasispositive vs negativelymph node metastasispositive vs negativehistological grade iii vsiiier status positive vsnegativepr status positive vsnegativeher2 status positive vsnegativemodelrandomfixedrandomrandomrandomrandomfixedfixedor cipvalueheterogeneity i2pvalue figure sensitivity analysis of the pooled hrs of malat1 expressiona os b dfsrfsdssfigure funnel plot of publication bias analysisa os b dfsrfsdssbreast cancer patient cohort analysis based on public datasetswe also checked malat1 expression levels in chemotherapytreated patients based on publicly available microarraydatasets our analysis showed that the expression level of malat1 was markedly lower in the chemotherapytreatedgroups than that in the nonchemotherapy group supplementary figure s1 indeed malat1 could not predict 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 bsr20200215101042bsr20200215figure correlation between malat1 and inï¬ltrating immune cellsa association between malat1 and b cells b association between malat1 and cd8 t cells c association between malat1and cd4 t cells d association between malat1 and dendritic cells e association between malat1 and nk cells f association between malat1 and macrophages m0prognosis in these groups data not shown these findings suggested that the prognostic value of malat1 forpatients receiving chemotherapy requires further investigationto further explore the possible reason why malat1 is responsible for poor survival in patients with breast cancerwe analyzed the association between malat1 and tumor infiltrating immune cells based on tcga dataset asshown in figure malat1 expression levels showed a significant positive correlation with immune infiltrationby b cells r p00065 and a negative association with cd8 t cells r p000071 cd4 tcells r p17e dendritic cells r p26e nk cells r p27e andmacrophages m0 r p000013 these findings strongly implied that malat1 may be implicated inimmune infiltration in patients with breast cancerdiscussionmalat1 also referred to as noncoding nuclearenriched abundant transcript was initially identified to promotemetastasis in nonsmallcell lung cancer to date aberrant expression of malat1 has been found in multiplecancers such as ovarian breast colorectal and bladder cancers [] some studies have shown that elevatedmalat1 expression levels contribute to breast cancer carcinogenesis whereas a few studies have demonstrated thatmalat1 may serve as a tumorsuppressing gene hence the role of malat1 and its influence on survivaloutcomes in patients with breast cancer remain controversial in addition although previously published reviewsand metaanalyses have reported that malat1 could function as a potential prognostic biomarker in cancers nostudies have focused on its prognostic significance in breast cancer here we pooled published data to highlight theprognostic and clinical value of malat1 in breast cancer it is wellknown that breast cancer is a highly heterogeneous disease based on the heterogeneity of er and her2 expression breast cancer can be classified into three mainsubtypes luminal luminal a and luminal b her2positive and tnbc these subtypes display distinct histologicalfeatures molecular etiologies and clinical behaviors our comprehensive metaanalysis of cases from cohorts showed that high malat1 expression levels in patients with breast cancer were observed in most studieswith no obvious subtype or cell specificity patients with breast cancer having elevated malat1 expression levels 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 bsr20200215101042bsr20200215displayed worse survival outcomes both os and dfsrfsdss the association between malat1 and poor prognosis in patients with breast cancer was consistent in most of the original studies as well as in the reprocessed dataobtained from tcga database the biological behavior of malat1 in cancer may explain this correlationfirst malat1 is involved in premrna alternative splicing through interactions with serine and argininerichproteins second malat1 participates in transcriptional regulation malat1 actively interacts with the3cid6 end of the gene body and overlaps with h3k36me2 peaks a marker of active transcriptional elongation indicatingits role in gene expression third malat1 can function as a posttranscriptional regulator of gene expressionthrough a cerna mechanism malat1 utilizes mirnaresponsive elements in mirna sequences as a languageto communicate with mrnas and pseudogenes thereby resulting in phenotypic alterations such as cell invasionand metastasis multiple studies have suggested that malat1 plays oncogenic roles in breast cancer forinstance malat1 modulates cdc42 expression by sponging mir1 in cell lines thereby triggering migration andinvasion malat1 can promote angiogenesis by interacting with mir145 malat1 also contributesto the maintenance of stem celllike phenotypes in breast cancer cells by regulating selfrenewalassociated factors moreover malat1 exerts a crucial role in tumor progression and metastasis in both tnbc and luminal cells immune cell infiltration and the tumor microenvironment have been verified to play essential roles in theinitiation and progression of cancers low levels of immuneinfiltrating cells in the tumor microenvironmentmay confer worse prognosis in breast cancer in our study high malat1 expression levels were associated with low immune cell infiltration eg cd4 and cd8 t cells which may explain the correlation betweenmalat1 and poor prognosis in patients with breast cancer taken together the extensive range of functions ofmalat1 enables it to predict survival outcomes in patients with breast cancernotably our results suggested that high malat1 expression levels were significantly associated with pr statusor ci er status also had a tendency for correlation with malat1 expression albeit notsignificant p005 due to the evident heterogeneity previous studies have shown that malat1 regulates the effectof 17βestradiol treatment on breast cell lines and is associated with er and its target genes moreover malat1might confer tamoxifen resistance by affecting the er signaling pathway these studies demonstrate thatmalat1 is closely related to hormone receptor status in breast cancer which is consistent with our results nosignificant association was observed between malat1 expression and advanced clinical parameters such as distantmetastasis lymph node metastasis and tnm stage this negative result seems reasonable given the limited numberof enrolled patients as well as the diverse enrollment criteria among studies in addition kim and kwok recently demonstrated a metastasissuppressing rather than a metastasispromoting role of malat1 inbreast cancer moreover multiple molecular networks and not malat1 alone contribute to tumor developmenttherefore further studies should be conducted to identify the function of malat1 in breast cancer and verify theassociation between clinical parameters and malat1 expressionalthough the prognostic value of malat1 was identified in this metaanalysis there are a few limitations of thestudy first marked heterogeneity existed owing to the difference in methodology such as in the determination ofcutoff values sample selection rtpcr primer sets and statistical analyses we applied the randomeffects modelto minimize the influence of these differences when heterogeneity was large second hr may have been overestimated because only two studies provided multivariate hr estimate for survival outcomes in addition the hr valueextracted from the survival curve might cause slight error for prognosis third we should carefully consider thepreference for publications with significant results as studies showing significant results are easier to publish thannegative ones notably messeur did not report the negative result regarding malat1 and breast cancerprognosis whereas jadaliha and zuo only reported significant results for specific groups fourththe sample size of some included studies was small and smaller studies could obtain higher effect size as wellas heterogeneity compared with larger studies finally language bias could also have been implicated as the selectedstudies were only published in englishin conclusion our study indicated that elevated malat1 expression levels are predictive of unfavorable prognosis not only for os but also dfs rfs and dss in breast cancer therefore it could potentially function as a prognosticbiomarker for patients with breast cancer furthermore our results demonstrated that the level of malat1 tendedto correlate with hormone receptor status nevertheless due to the limitations mentioned above additional comprehensive welldesigned multicenter studies should be performed to confirm and strengthen our findings regardingthe prognostic role of malat1 in breast cancercompeting intereststhe authors declare that there are no competing interests associated with the manuscript the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc by 0cbioscience reports bsr20200215101042bsr20200215fundingthis work was supported by the nature science foundation of zhejiang province [grant numbers lq19h060002 lq19h160041]and the medical and health science and technology project of zhejiang province [grant numbers 2018ky089 2020ky143]author contributionyyw yjz and szz conceived and designed the research yyw yjz kmh and jlq searched the literature and analyzed the data yyw and yjz wrote the manuscript yh and mqz proofread the manuscript szz finally revised themanuscript yyw and yjz contributed equally to this work as first authorsabbreviationscerna competitive endogenous rna ci confidence interval dfs diseasefree survival dss diseasespecific survivaler estrogen receptor her2 human epidermal growth factor receptor hr hazard ratio h3k36me2 histone h3 dimethylation at lysine lncrna long noncoding rna malat1 metastasisassociated lung adenocarcinoma transcript mfsmetastasisfree survival nk natural killer nos newcastleottawa scale or odds ratio os overall survival pr progesterone receptor rfs recurrencefree survival rtpcr reverse transcriptase polymerase chain reaction tcga the cancergenome atlas tnbc triplenegative breast cancer tnm tumor node metastasis zeb zincfinger eboxbinding homeoboxreferences allemani c matsuda t di carlo v global surveillance of trends in cancer survival concord3 analysis of individual recordsfor patients diagnosed with one of cancers from populationbased registries in countries lancet 101016s0140673617333263 siegel rl miller kd and jemal a cancer statistics ca cancer j clin 103322caac21551 slamon dj clark gm wong sg human breast cancer correlation of relapse and survival with ampliï¬cation of the her2neuoncogene science 101126science3798106 prat a parker js fan c and perou cm pam50 assay and the threegene model for identifying the major and clinically relevant molecularsubtypes of breast cancer breast cancer res treat 101007s1054901221430 slamon dj leylandjones b shak s use of chemotherapy plus a monoclonal antibody against her2 for metastatic breast cancer thatoverexpresses her2 n engl j med 101056nejm200103153441101 perou cm sorlie t eisen mb molecular portraits of human breast tumours nature 10103835021093 prat a lluch a albanell j predicting response and survival in chemotherapytreated triplenegative breast cancer br j cancer 101038bjc2014444 quinn jj and chang hy unique features of long noncoding rna biogenesis and function nat rev genet 101038nrg201510 jandura a and krause hm the new rna world growing evidence for long noncoding rna functionality trends genet 101016jtig201708002 huarte m the emerging role of lncrnas in cancer nat med 101038nm3981 chen x yang y cao y lncrna pvt1 identiï¬ed as an independent biomarker for prognosis surveillance of solid tumors based ontranscriptome data and metaanalysis cancer manag res 102147cmars166260 li j wen w zhao s prognostic role of hotair in four estrogendependent malignant tumors a metaanalysis onco targets ther 102147otts84687 zhang x hamblin mh and yin kj the long noncoding rna malat1 its physiological and pathophysiological functions rna biol 1010801547628620171358347 ji p diederichs s wang w malat1 a novel noncoding rna and thymosin beta4 predict metastasis and survival in earlystagenonsmall cell lung cancer oncogene 101038sjonc1206928 zhuo m yuan c han t a novel feedback loop between high malat1 and low mir200c3p promotes cell migration and invasion inpancreatic ductal adenocarcinoma and is predictive of poor prognosis bmc cancer 101186s1288501849549 wang y zhou y yang z mir204zeb2 axis functions as key mediator for malat1induced epithelialmesenchymal transition in breastcancer tumour biol 1011771010428317690998 li j cui z li h clinicopathological and prognostic signiï¬cance of long noncoding rna malat1 in human cancers a review andmetaanalysis cancer cell int 101186s129350180606z xu s sui s zhang j downregulation of long noncoding rna malat1 induces epithelialtomesenchymal transition via the pi3kaktpathway in breast cancer int j clin exp pathol wang z katsaros d biglia n high expression of long noncoding rna malat1 in breast cancer is associated with poor relapsefreesurvival breast cancer res treat 101007s1054901848392 lang s and kleijnen j quality assessment tools for observational studies lack of consensus int j evid based healthc 101111j17441609201000195x the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc by 0cbioscience reports bsr20200215101042bsr20200215 tierney jf stewart la ghersi d burdett s and sydes mr practical methods for incorporating summary timetoevent data intometaanalysis trials 10118617456215816 jin c yan b lu q lin y and ma l reciprocal regulation of hsamir1 and long noncoding rna malat1 promotes triplenegative breastcancer development tumour biol 101007s1327701546056 huang ns chi yy xue jy long noncoding rna metastasis associated in lung adenocarcinoma transcript malat1 interacts withestrogen receptor and predicted poor survival in breast cancer oncotarget 1018632oncotarget9364 jadaliha m zong x malakar p functional and prognostic signiï¬cance of long noncoding rna malat1 as a metastasis driver in ernegative lymph node negative breast cancer oncotarget 1018632oncotarget9622 meseure d vacher s lallemand f prognostic value of a newly identiï¬ed malat1 alternatively spliced transcript in breast cancer br jcancer 101038bjc2016123 miao y fan r chen l and qian h clinical signiï¬cance of long noncoding rna malat1 expression in tissue and serum of breast cancerann clin lab sci zhang p luo q hu t and yan d serum malat1 expression predicts clinical outcome in breast cancer patients receiving cyclophosphamidebased treatment int j clin exp med zuo y li y zhou z ma m and fu k long noncoding rna malat1 promotes proliferation and invasion via targeting mir1295p intriplenegative breast cancer biomed pharmacother 101016jbiopha201709005 li z xu l liu y lncrna malat1 promotes relapse of breast cancer patients with postoperative fever am j transl res zhang p zhou h lu k exosomemediated delivery of malat1 induces cell proliferation in breast cancer onco targets ther 102147otts155134 zhou y xu x lv h the long noncoding rna malat1 is highly expressed in ovarian cancer and induces cell growth and migrationplos one e0155250 101371journalpone0155250 li pl zhang x wang hy malat1 is associated with poor response to oxaliplatinbased chemotherapy in colorectal cancer patientsand promotes chemoresistance through ezh2 mol cancer ther 10115815357163mct160591 li c cui y liu lf high expression of long noncoding rna malat1 indicates a poor prognosis and promotes clinical progression andmetastasis in bladder cancer clin genitourin cancer 101016jclgc201705001 kim j piao hl kim bj long noncoding rna malat1 suppresses breast cancer metastasis nat genet 101038s4158801802523 kwok z roche v chew x fadieieva a and tay y a noncanonical tumor suppressive role for the long noncoding rna malat1 in colonand breast cancers int j cancer 101002ijc31386 zheng l zhang y fu y long noncoding rna malat1 regulates blcap mrna expression through binding to mir3395p andpromotes poor prognosis in breast cancer biosci rep 101042bsr20181284 tripathi v ellis jd shen z the nuclearretained noncoding rna malat1 regulates alternative splicing by modulating sr splicingfactor phosphorylation mol cell 101016jmolcel201008011 tripathi v shen z chakraborty a long noncoding rna malat1 controls cell cycle progression by regulating the expression ofoncogenic transcription factor bmyb plos genet e1003368 101371journalpgen1003368 thomson dw and dinger me endogenous microrna sponges evidence and controversy nat rev genet 101038nrg201620 chou j wang b zheng t malat1 induced migration and invasion of human breast cancer cells by competitively binding mir1 withcdc42 biochem biophys res commun 101016jbbrc201602102 huang x xia y he g zheng l cai y yin y malat1 promotes angiogenesis of breast cancer oncol rep zeng l cen y and chen j long noncoding rna malat1 contributes to maintenance of stem celllike phenotypes in breast cancer cellsoncol lett sharma p hulieskovan s wargo j and ribas a primary adaptive and acquired resistance to cancer immunotherapy cell 101016jcell201701017 kato t noma k ohara t kashima h katsura y sato h cancerassociated ï¬broblasts affect intratumoral cd8 and foxp3 tcells via il6 in the tumor microenvironment clin cancer res 10115810780432ccr180205 bense rd sotiriou c piccartgebhart mj haanen j van vugt m de vries ege relevance of tumorinï¬ltrating immune cellcomposition and functionality for disease outcome in breast cancer j natl cancer inst 101093jncidjw192 tawï¬k o kimler bf karnik t and shehata p clinicopathological correlation of pdl1 expression in primary and metastatic breast cancerand inï¬ltrating immune cells hum pathol 101016jhumpath201806008 zhao z chen c liu y and wu c 17βestradiol treatment inhibits breast cell proliferation migration and invasion by decreasing malat1rna level biochem biophys res commun 101016jbbrc201402006 sherene l benjamin hk christine d deï¬nition of clinically distinct molecular subtypes in estrogen receptorpositive breastcarcinomas through genomic grade j clin oncol 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 0c' | Colon_Cancer |
" autism spectrum disorder asd is a developmental disorder and the effective pharmacologicaltreatments for the core autistic symptoms are currently limited increasing evidence particularly that from clinicalstudies on asd patients suggests a functional link between the gut microbiota and the development of asdhowever the mechanisms linking the gut microbiota with brain dysfunctions gutbrain axis in asd have not yet beenfull elucidated due to its genetic mutations and downregulated expression in patients with asd ephb6 which alsoplays important roles in gut homeostasis is generally considered a candidate gene for asd nonetheless the role andmechanism of ephb6 in regulating the gut microbiota and the development of asd are unclearresults here we found that the deletion of ephb6 induced autismlike behavior and disturbed the gut microbiota inmice more importantly transplantation of the fecal microbiota from ephb6deficient mice resulted in autismlikebehavior in antibiotictreated c57bl6j mice and transplantation of the fecal microbiota from wildtype miceameliorated the autismlike behavior in ephb6deficient mice at the metabolic level the disturbed gut microbiota inephb6deficient mice led to vitamin b6 and dopamine defects at the cellular level the excitationinhibition eibalance in the medial prefrontal cortex was regulated by gut microbiotamediated vitamin b6 in ephb6deficient mices our study uncovers a key role for the gut microbiota in the regulation of autismlike social behavior byvitamin b6 dopamine and the ei balance in ephb6deficient mice and these findings suggest new strategies forunderstanding and treating asdkeywords gut microbiota asd ephb6 vitamin b6 dopamine ei balance correspondence tgaosmueducn lijming3sysueducn ying li and zhengyi luo contributed equally to this work2state key laboratory of an failure research key laboratory of mentalhealth of the ministry of education guangdonghong kongmacao greaterbay area center for brain science and braininspired intelligenceguangdong province key laboratory of psychiatric disorders collaborativeinnovation center for brain science department of neurobiology school ofbasic medical sciences southern medical university guangzhou peoples republic of china1department of pathology sun yatsen memorial hospital sun yatsenuniversity guangzhou peoples republic of chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cli microbiome page of autism spectrum disorder asd which affects approximately of the population around the worldis mainly characterized by impaired social interactionand communication and restricted and repetitive behavior although early behavioral and educationalinterferences have shown effective ameliorative roleson autistic symptoms of asd patients the effectivepharmacological therapies for the treatment of coreautistic symptoms remain limited [ ]thataccumulating evidence showsthe gutbrainmicrobiota axis plays a key role in regulating homeostasis of the human body gut microanisms reportedlyparticipate in many neuropsychiatric disorders suchas anxiety disorders depression and epilepsy in most asd patients changes in gut microanismsand serious gastrointestinal problems have been observed [] interestingly several studies have foundthat the gut microbiota play important role in modulating the asdlike phenotypes of mice [] aclinical study showed that microbiota transfer therapycan improve gastrointestinal problems and autisticsymptoms in asd patients aged to years and thisbenefit can last for years [ ] these studiessuggest that the gutbrainmicrobiota axis might havea significantimpact on the development of asdhowever the contribution of the gut microbiota tothe dysregulation of brain function has not been fullyelucidatedephb6 which belongs to the eph family of receptortyrosine kinases is located on chromosome 7q in twostage genome research on susceptibility lociinautism found transcripts mapped to the chromosome 7qregion that are associated with a predisposition to autincluding ephb6 more recent studies haveismsuggested that ephb6 is a candidate asdassociatedgene [] and recent genomic studies have foundthat ephb6 is mutated in some asd patients [ ]most importantly transcriptome analyses have shownthat ephb6 is downregulated in asd patients [ ]although ephb6 plays an important role in regulatingeph receptor signaling networks t cellfunctionsintestinal epithelium and epithelialdevelopment ofhomeostasis []the role and mechanisms ofephb6 involved in regulation of the gut microbiota andasd remain unclearin our study we found that ephb6 is functionallyassociated with asd and regulates autismlike socialbehavior by gut microbiotamediated vitamin b6 anddopamine moreestablished thefunctionallink between dysregulated gut microbiotaand excitationinhibition ei imbalance in the medial prefrontal cortex mpfc a key gutbrain functional axis in ephb6deficient miceimportantly weresultsthe deletion of ephb6 led to autismlike behavior andgut microbial disturbance in micealthough ephb6 has been identified as a candidate geneassociated with asd whether and how ephb6 functions inasd remain unclear to address these unanswered questions we established ephb6knockoutko mice andfound that ephb6 was deleted in different tissues includingthe colon brain lung and spleen in these mice comparedwith ephb6 wildtype wt mice additional file figure s1cd however the brain and body weights thebody length and the daily dietary consumption were similarbetween the two groups of mice despite the deletion ofephb6 additional file figure s1ehthan the wt mice fig 1bpatients with asd often display repetitive stereotypedbehavior and social deficits interestingly we found thatthe ko mice spent more time selfgrooming than thewt mice fig 1a in the marble burying test the komice buried similar marbles as the wt mice additionalfile figure s1j and in the social partition test the komice spent less time sniffing at the partition regardlessof whether a familiar or novel mouse was placed in thecagein the threechambered social approach task both the wt and komice spent similar lengths of time in bilateral chambersduring the first 10min trial which indicated that the experimental environment was normal fig 1c howeverthe ko mice spent a similar length of time in chamberswith an unfamiliar mouse or inanimate object fig 1dand also showed less preference for the social mousestranger over the object than the wt mice fig 1fgif a novel social partner stranger was placed in theempty wire cage the ko mice still spent a similar lengthof time in the two chambers fig 1e and showed lesspreference for the novel mouse over the familiar mousethan the wt mice fig 1h these results sufficientlyconfirmed that the ko mice exhibited abnormal socialinteraction olfactory cues have generally been consideredto be of the utmost importance in communication amongmice [ ] in the olfactory habituationdishabituationtest repeated presentation of cotton swabs saturated withthe same odor resulted in increasingly decreased lengthsof time spent sniffing at cotton swabs and the presentation of cotton swabs saturated with a new odor increasedthe time spent sniffing these findings were obtained withboth the wt and ko mice however the ko miceshowed less interest in cotton swabs saturated with socialodor than the wt mice fig 1i these results indicatedthat the ko mice exhibited communication deficits eventhough their ability to discriminate and habituate differentodors was normalasd is often accompanied by other mental diseasessuch as hyperactivity anxiety and intellectual disabilityin the open field test the ko mice showed the same 0cli microbiome page of fig see legend on next page 0cli microbiome page of see figure on previous pagefig the deletion of ephb6 led to autismlike behavior and gut microbial disturbance in mice a the 8weekold male ko mice spent more time selfgrooming than wt mice n mice for each group b in social partition test ko mice spent less time sniffing the partition than wt mice n mice respectively ch in threechambered social approach task time spent in chambers during different 10min trials ce trajectory diagram duringthe second 10min trial f were showed ko mice showed less preference for the social mouse over the object g and less preference for the novelsocial mouse over the familiar social mouse h than wt mice n mice respectively i in olfactory habituationdishabituation test ko mice spentless time sniffing social odors than wt mice n mice for each group j in elevated plus maze test ko mice spent less time in open arm and moretime in closed arm than wt mice n mice respectively k the intestinal permeability of 8weekold wt and ko mice was detected using fitcdextran n mice for each group l the mrna expressions of tight junction molecules were detected in colon of 8weekold wt and ko mice n mice respectively m the mrna expressions of cytokines were detected in colon of 8weekold wt and ko mice n mice respectively nr 16srrna gene sequencing of gut microbiota of 8weekold wt and ko mice the species richness n and diversity o of gut microbiota were similarwhile the microbial composition p was different between the two groups relative abundance of different bacteria in phylum level was showed qat genus level the relative abundance of mucispirillumn was decreased in ko mice r n mice for each group data shown are mean ± sem ormedian ± iqr twotailed unpaired students t test a ce gh jm mannwhitney test no q r mixed design anova with genotype asindependent factor and stimulitrials as repeatedmeasure factor b i anosim analysis p p p p wt ephb6 mice koephb6 mice fitc fluorescein isothiocyanate statistical values are presented in additional file table s2locomotor activities and spent almost the same time inthe center area as the wt mice additional file figures1kl in the elevatedplusmaze testthe ko micespent less time in the open arm and more time in theclosed arm than the wt mice fig 1j which impliedthat the ko mice displayed anxietylike behavior in themorris water mazethe ko mice exhibited normalspatial learning and memory similarly to the wt miceadditional file figure s1mo collectively the resultsshowed that the deletion of ephb6 in mice resulted inautismlike behavior including stereotyped behavior andsocial deficits accompanied by anxietylike behavior butdid not result in any evidence of intellectual disabilityephephrin signaling reportedly modulates gut epithelial development and homeostasis and it is also generallyaccepted that many asd patients present gastrointestinal gi symptoms [ ] and a changed gut microbiota composition we then questioned whether komice would suffer from gi problems measurement ofthe intestinal permeability by fluorescein isothiocyanatefitcdextran revealed that the intestinal permeabilityof ko mice was significantly increased compared withthat of the wt mice fig 1k accordingly the mrnaexpression of cldn4 a tight junction molecule in thecolon of ko mice was lower than that in the colon ofwt mice fig 1l in addition we found that the colonof the ko mice presented substantially increased mrnaexpression of il1β a proinflammatory factor and decreasedexpression of il6 which exerts an antiinflammatory effectcompared with that of the wt mice fig 1m the gi problems in the ko mice were not accompanied by morphological changes in the distal ileum proximal colon liver orlung additional file figure s1pthe integrity of the intestinal mucosa is important formaintaining the balance of the ecological environmentin the animals gut we then detected the fecal microbialpopulations of mice by 16s rrna gene sequencing nodifferences in the microbial species richness and diversitywere found between the two groups fig 1n o notablya principal coordinates analysis of the braycurtis distanceshowed that the fecal microbiota of the ko mice clustereddifferently from that of the wt mice fig 1p which indicated that the gut microbial composition differed betweenthe two groups at the phylum level the differencesbetween the two groups were caused by a decreased abundance of deferribacteres in the fecal microbiota of the komice fig 1q at the genus level mucispirillum which isa genus belonging to the phylum deferribacteres wasdecreased in the fecal microbiota of the ko mice fig 1rin general our results indicated that the deletion of ephb6in mice resulted in increased intestinal permeability andchanges in the gut microbial compositionmany studies have indicated that gi problems and thebehavioral abnormalities associated with asd always appearin parallel in patients we thus questioned which of thesesymptoms appears first in the ko mice and found that themicrobial species richness and diversity did not differ between the 34weekold wt and ko mice additional file figure s2ab the principal coordinates analysis revealedthat the gut microbiota of 4weekold ko mice clustered differently from that of 4weekold wt mice additional file figure s2d whereas the gut microbiota of 3weekold komice clustered similarly to that of 3weekold wt miceadditional file figure s2c in addition 4weekold butnot 3weekold ko mice showed increased selfgroomingand decreased interest in social odors compared with sameaged wt mice additional file figure s2eh these resultsfurther implied a possible relationship between the abnormalbehavior and gut microbial dysbiosis in mice with deletion ofephb6transplantation of the fecal microbiota from ephb6deficient mice caused autismlike behavior in spf c57bl6j miceasd is generally considered a neurodevelopmental disorder postnatal developmental disorder can also causeautism in patients and the postnatal mutation ofnrxn1 in neurons leads to autismlike behavior in mice 0cli microbiome page of additionally the gut microbiota of asd patientscould induce autismlike behavior in mice thereforeto study the relationship between gut microbial dysbiosisand autismlike behavior in mice with deletion of ephb6we gavaged the fecal microbiota from 8weekold malewt or ko mice to 3weekold spf male c57bl6j micefor week fig 2a three weeks after the gavage offecal microbiota the gut microbial composition of spfc57bl6j mice treated with the fecal microbiota fromthe ko mice differed from that of spf c57bl6j micetreated with the fecal microbiota from the wt micefig 2bd more interestingly c57bl6j mice that weregastrically perfused with the fecal microbiota from theko mice displayed increased selfgrooming fig 2e andpartially decreased social behavior fig 2fi comparedwith the control mice the two groups of mice showedsimilar behaviors in the open field test and elevated plusmaze test additional file figure s3ad furthermorewe orally gavaged the suspending solution offecalmicrobiota from the wt or ko mice to antibioticpretreated spf male c57bl6j mice after pretreatmentwith antibiotics for days 3weekold spf male c57bl6j mice was gavaged orally with the fecal microbiota of8weekold male wt or ko mice for days fig 2japproximately weeks after fecal microbial colonizationwe similarly found that the gut microbiota of spf c57bl6j mice treated with the fecal microbiota from the ko miceclustered differently from that of the control mice fig 2km we subsequently found that c57bl6j mice that weregastrically perfused with the fecal bacteria from the komice showed increased selfgrooming fig 2n and partiallydecreased social behavior fig 2or additionally the twogroups of mice showed similar behaviors in the openfield test and elevatedplusmaze test additional file figure s3eh moreover the fecal microbiota from weekold but not 3weekold ko mice induced increasedselfgrooming and partial social deficits in 3weekold spfc57bl6j micecompared with c57bl6j micegavaged with fecal microbiota from sameaged wtmice additional file figure s4ah collectively thefecal microbiota from ephb6deficient mice caused increased selfgrooming and partially impaired socialbehavior in c57bl6j micewe subsequently questioned whether the gut microbiota continue to play a role in autismlike behavior inadult mice first we orally gavaged a mixture of antibiotics to 6weekold male spf c57bl6j mice for weekand found that this antibiotic treatment greatly disruptedthe gut microbiota and induced decreased selfgroomingand partial social deficits in young adult c57bl6j miceadditional file figure s5ai these results indicatedthat the gut microbiota was related to autismlike behavioreven in adult mice and that different gut microbiota compositions likely contributed to different behaviors such asselfgrooming and social behavior we then gavaged thefecal microbiota from 8weekold male wt or ko micedirectly to 6weekold spf male c57bl6j mice for week and found that the fecal microbiota from ko micealso induced a disturbed gut microbiota increased selfgrooming and partial social deficits in adult c57bl6jmice fig 3ai unexpectedly we also found that metabolites of the gut microbiota from the ko mice inducedpartial social deficits in c57bl6j mice fig 3jm thegut microbiota without metabolites from the ko mice stillcaused partial social deficits in c57bl6j mice additionalfile figure s5jmoverall our results indicated that the gut microbiotaplays an important role in autismlike behavior even inadult micetransplantation of the fecal microbiota from wildtypemice ameliorated autismlike behavior in adult ephb6deficient miceno previous study has focused on the effectiveness ofmicrobiota transplantation in adult asd patients wesubsequently orally gavaged the fecal microbiota from weekold male wt mice to 8weekold ko mice for week a week later we found that the gut microbiota ofthe ko mice gavaged with the fecal microbiota of thewt mice clustered differently from that of the ko micegavaged with sterile pbs fig 4b the phylumlevelanalysis revealed that the relative abundance of deferribacteres was increased in the ko mice gavaged with thefecal microbiota of the wt mice fig 4c at the genuslevel mucispirillum which is a genus belonging to thephylum deferribacteres was also increased in the komice treated with the fecal microbiota of the wt micefig 4da functional analysis revealed that the ko mice exhibited increased social behavior fig 4fi after gavage withthe fecal microbiota from the wt mice and a decreasedtendency of selfgrooming fig 4e these results indicated that gut microbial dysbiosis was responsible forautismlike behavior in mice with deletion of ephb6gut microbiotamediated vitamin b6 homeostasisregulated social behavior in ephb6deficient micebecause the abnormal behaviors were likely due tobrainrelated problems we attempted to determine howthe gut microbiota affected the brain and subsequentlycaused autismlike behavior in ephb6deficient micefirst we attempted to identify the key region of thebrain affected by the dysregulated gut microbiota inmice with deletion of ephb6 and that was responsiblefor the resulting autismlike behavior studies on asdpatients or mouse models have shown that the hippocampus cerebellum and mpfc are implicated in asd[ ] after processed with a threechambered social 0cli microbiome page of fig see legend on next page 0cli microbiome page of see figure on previous pagefig transplantation of the fecal microbiota from ephb6deficient mice caused autismlike behavior in 3weekold spf c57bl6j mice aischematic of the fecal microbiota transplantation a the 3weekold spf male c57bl6j mice were orally gavaged with the fecal microbiotafrom 8weekold male wt or ko mice each contained eight healthy mice from at least three cages for week after weeks the fecalmicrobiota of the treated c57bl6j mice were sequenced bd eight treated c57bl6j mice of each group were selected randomly from at leastthree cages and selfgrooming test e olfactory habituationdishabituation test f threechambered social approach task gi open field testand elevated plus maze test were conducted with an interval of at least days ei n mice respectively jr schematic of the fecalmicrobiota transplantation j the 3weekold spf male c57bl6j mice were orally gavaged with antibiotics ampicillin vancomycin neomycinmetronidazole for days and then orally gavaged with the fecal microbiota from 8weekold male wt or ko mice each contained eight healthymice from at least three cages for another days after days the fecal microbiota of the treated c57bl6j mice were sequenced km sixtreated c57bl6j mice of each group were selected randomly from at least two cages and selfgrooming test n olfactory habituationdishabituation test o threechambered social approach task pr open field test and elevated plus maze test were conducted with an intervalof at least days nr n mice respectively data shown are mean ± sem or median ± iqr twotailed unpaired students t test e h i nq r mannwhitney test b c k l mixed design anova with genotype as independent factor and stimulitrials as repeatedmeasure factor f oanosim analysis d m p wt col or ko col colonized with the fecal microbiota from ephb6 or ephb6 mice abx pretreated withantibiotics ampicillin vancomycin neomycin metronidazole statistical values are presented in additional file table s2approach task the protein expression of cfos in thempfc of the ko mice was significantly higher than thatin the mpfc of the wt mice additional file figures6ac asd is generally considered to be caused by anincreased ratio of synaptic excitation and inhibition andasd children exhibit elevations in the restingstateneuronal activity therefore whether the mpfc ismodulated by the gut microbiota of the ko mice needsto be further investigated because the mpfc tissue wastoo small for some experiments we used pfc tissuefrom mice in our subsequent studythe first question we asked was whether the bacteriacould directly modulate the mpfc undoubtedly we didnot detect any bacterial dna or colonies in the pfctissues of the wt or ko mice additional file figures6de because metabolites of the gut microbiota fromthe ko mice also induced social deficits in c57bl6jmice we hypothesized that some substances that hadbeen affected by gut microbial dysbiosis caused socialdeficits in the ko miceto identify the significantly changed metabolites wedetected the metabolites in the target tissue that is thepfc of the ko mice using nontargeted metabolomicsstrategies surprisinglythe metabolites in the pfcshowed significant differences between the two groupsof mice as demonstrated by orthogonal partialleastsquares discriminant analysis fig 5a a kegg pathwayanalysis identified four pathways that were significantlyenriched in the differentially changed metabolites andthese included the vitamin b6 metabolism pathway dueto the decreased relative abundances of pyridoxaminepm and pyridoxal ²phosphate plp in the pfc ofthe ko mice fig 5bdvitamin b6 in the body is mainly derived from dietand gut bacteria synthesis and is then absorbed in theintestine we then detected the levels of vitamin b6 inthe feces blood and pfc of mice and found that theephb6deficient mice presented increased fecal levels ofpyridoxine pn decreased plasma levels of pm andplp and decreased levels of plp in the pfc fig 5ekone week after gavage the ko mice gavaged with thefecal microbiota from the wt mice exhibited decreasedlevels of pn in feces and tended to show increased levelsof pm in plasma and increased levels of plp in plasmaand the pfc compared with the ko mice gavaged withsterile pbs fig 5ek these results indicated that thegut microbiota regulated the level of vitamin b6 in thefeces blood and pfc of mice probably by regulatingthe absorption of vitamin b6 in intestinewe subsequently supplied vitamin b6 to the ko mice toclarify its effect on autismlike behavior however intragastric supplementation with vitamin b6 did not amelioratethe social deficits in the ko mice additional file figures7ac one hour after the intraperitoneal injection of mgplp the ko mice presented higher levels of plp in plasmafig 6b and increased social behavior fig 6df comparedwith the control mice no changes in selfgroomingfig 6c and social novelty fig 6g were detected inthe ko mice after the injection of plp additionallythe intraperitoneal injection of or mg of plp exerted noeffect on the social behavior of c57bl6j mice fig 6hjmoreover after being fed without vitamin b6 for weeksc57bl6j mice presented lower plasma plp levels anddecreased social behavior fig 6kn conclusively ourresults proved the existence of a relationship between gutmicrobiotamediated defects of vitamin b6 and socialdeficits in ephb6deficient micegut microbiotamediated vitamin b6 homeostasisregulated dopamine in the pfc of ephb6deficient micevitamin b6 as a cofactor has been implicated in morethan biochemical reactions in cellsincluding thebiosynthesis and catabolism of amino acids and neurotransmitters as the most important active substances in the brain we first detected neurotransmittersin the pfc of mice by highperformance liquid chromatography hplc and found similar levels of glutamategammaaminobutyric acid gaba glycine aspartic 0cli microbiome page of fig fecal microbiota transplantation from ephb6deficient mice partially induced social deficits in 6weekold spf c57bl6j mice aischematic of the fecal microbiota transplantation a the 6weekold spf male c57bl6j mice were orally gavaged with fecal microbiota from weekold male wt or ko mice for week after week the fecal microbiota of the treated c57bl6j mice were sequenced bd n mice foreach group and selfgrooming test e olfactory habituationdishabituation test f threechambered social approach test gi were conductedwith an interval of at least days ei n mice respectively jm fecal metabolites from 8weekold male wt and ko mice were orallygavaged to 6weekold spf male c57bl6j mice for week j after week olfactory habituationdishabituation test k and threechamberedsocial approach task l m were conducted with an interval of at least days n mice respectively data shown are mean ± sem ormedian ± iqr twotailed unpaired students t test e h i m mannwhitney test b c mixed design anova with genotype as independentfactor and stimulitrials as repeatedmeasure factor f k anosim analysis d p p wt col or ko col colonized with fecalmicrobiota or fecal metabolites from ephb6 mice or ephb6 mice statistical values are presented in additional file table s2acid serine and glutamine in the wt and ko micegavaged with sterile pbs or the fecal microbiota fromthe wt mice fig 7a b interestingly the pfc of theko mice exhibited decreased dopamine levels and increased 5hydroxytryptamine 5ht levels than that ofthe wt mice fig 7c treatment with the fecal microbiota from the wt mice increased the level of dopaminebut did not affect the level of 5ht in the pfc of theko mice compared with the levels found in the komice gavaged with sterile pbs the levels of noradrenaline epinephrine and dihydroxyphenyl acetic aciddopac did not differ among the three groups of micefig 7c more excitingly the level of dopamine in thepfc of spf c57bl6j mice gavaged with the fecalmicrobiota from the ko mice was lower than that in thepfc of c57bl6j mice gavaged with the fecal microbiotafrom the wt mice additionalfile figure s8aeadditionally the intraperitonealinjection of plp increased the level of dopamine in the pfc of the komice fig 7d and vitamin b6 deficiency decreased thelevel of dopamine in the pfc of spf c57bl6j micefig 7e brieflyindicated that gutthese results 0cli microbiome page of fig transplantation of the fecal microbiota from wildtype mice ameliorated autismlike behavior in adult ephb6deficient mice a schematic ofthe the fecal microbiota transplantation the 8weekold male wt and ko mice were orally gavaged with the fecal microbiota from 8weekoldmale wt mice eight healthy mice from at least three cages or sterile pbs for week after week the fecal microbiota of the treated wt andko mice were sequenced bd and behavioral tests were conducted with an interval of at least days ei bd 16s rrna gene sequencing ofthe fecal microbiota from mice principal coordinates analysis of braycurtis distance b the relative abundance of deferribacteres c at phylumlevel and the relative abundance of mucispirillum d at genus level were showed at phylum level the range of on x axis was used for therelative abundance of p_bacteroidetes p_firmicutes and p_proteobacteria and the range of on x axis was used for other bacteria n mice respectively ei selfgrooming test e olfactory habituationdishabituation test f and threechambered social approach task giwere performed n mice respectively data shown are mean ± sem or median ± iqr oneway anova e h i kruskalwallis testc d mixed design anova with genotype as independent factor and stimulitrials as repeatedmeasure factor f anosim analysis b p p p wt ephb6 mice ko ephb6 mice fmt fecal microbiota transplantation pbs phosphatebuffered saline statisticalvalues are presented in additional file table s2microbiotamediated vitamin b6 homeostasisaffect the level of dopamine in the pfc of micecouldto determine whether the decrease in dopamine contributed to the autismlike behavior of ephb6deficientmice and considering the fast metabolism of dopaminein the brain we injected agonists of dopamine receptorsinto the mpfc of mice the deletion of ephb6 had noeffect on the mrna expression of dopamine receptorsor tyrosine hydroxylase th in the mpfc or ventraltegmental area vta fig 7f we then injected anagonist of dopamine d1 receptor d1r skf38393or dopamine d2 receptor d2r quinpirole into thempfc of mice the results showed that the ko miceexhibited increased social behavior fig 7gj afterinjection with skf38393 compared with the ko miceinjected with artificial cerebrospinalfluid acsfhowever no differences were found between c57bl6j mice injected with acsf and c57bl6j mice 0cli microbiome page of fig gut microbiota regulated vitamin b6 in ephb6deficient mice ad in nontargeted metabolomics analysis the metabolites in pfc of weekold male wt and ko mice were differently clustered by orthogonal partial least squares discriminant analysis a the enriched keggpathways associated with differential metabolites b the relative abundance of pyridoxamine pm c and pyridoxal ²phosphate plp d wereshowed n mice respectively ek the fecal microbiota from 8weekold wt mice or pbs were gavaged to 8weekold wt or ko mice for week e one week later the level of pm f plp g and pyridoxine pn h in feces of mice were detected n mice respectively thelevel of pm and plp in plasma i j n mice respectively and level of plp in pfc k n mice respectively of mice were alsodetected data shown are mean ± sem r bd oneway anova fk p p p wt ephb6 mice ko ephb6mice fmt fecal microbiota transplantation pbs phosphatebuffered saline pfc prefrontal cortex pm pyridoxamine plp pyridoxal ²phosphate pnpyridoxine statistical values are presented in additional file table s2injected with skf38393 fig 7kmin contrastquinpirole did not increase social behavior in the komice additionalfile figure s8fh and the d1rantagonistinc57bl6j mice fig 7nq in short these results indicated that dysregulated gut microbiota and vitaminb6 defect led to autismlike behavior via the d1rmediated pathway in ephb6deficient miceinduced decreased social behaviut microbiota regulated the ei balance in the mpfc ofephb6deficient miceit is generally thought that d1rs modulate ga | Colon_Cancer |
" tumor microenvironment tme plays an important role in malignant tumors our study aimed toinvestigate the effect of the tme and related genes in osteosarcoma patientsmethods gene expression profiles and clinical data of osteosarcoma patients were downloaded from the targetdataset estimate algorithm was used to quantify the immune score then the association between immune scoreand prognosis was studied afterward a differential analysis was performed based on the high and lowimmunescores to determine tmerelated genes additionally cox analyses were performed to construct two prognosticsignatures for overall survival os and diseasefree survival dfs respectively two datasets obtained from the geodatabase were used to validate signaturesresults eightyfive patients were included in our research the survival analysis indicated that patients with higherimmune score have a favorable os and dfs moreover genes were determined as tmerelated genes theunsupervised clustering analysis revealed two clusters were significantly related to immune score and t cells cd4memory fraction in addition two signatures were generated based on three and two tmerelated genesrespectively both two signatures can significantly divide patients into low and highrisk groups and were validatedin two geo datasets afterward the risk score and metastatic status were identified as independent prognosticfactors for both os and dfs and two nomograms were generated the cindexes of os nomogram and dfsnomogram were and respectively tme was associated with the prognosis of osteosarcoma patients prognostic models based on tmerelated genes can effectively predict os and dfs of osteosarcoma patientskeywords tumor microenvironment osteosarcoma prognosis immune features nomogram osteosarcoma is the most common bone tumor especiallyin children and adolescents it was reported that approximately of patients are between and yearsold and osteosarcoma is considered as the second leadingcause of death in this age group currently surgery and correspondence 407404159qqcom4wenzhou medical university wenzhou chinafull list of author information is available at the end of the chemotherapy are still major treatments for osteosarcomapatients and these therapies are constantly improving inrecent years however due to the susceptibility of localaggressiveness and lung metastasis in osteosarcoma patients the prognosis of osteosarcoma remains unfavorable previous studies indicated that the 5years survivalrates were and in metastatic and nonmetastaticpatients respectively thereforeit is necessary to the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0chu bmc cancer page of investigate the mechanism of pathogenesis and progressionof osteosarcoma and accurately classify the risk of patientsrecently an increasing number of diagnostic and prognostic biomarkers of osteosarcoma patients have beenidentified for example chen reported that tumorsuppressor p27 is a novel biomarker for the metastasis andsurvival status in osteosarcoma patients moreover huang discovered that dysregulated circrnas serve asprognostic and diagnostic biomarkers in osteosarcomapatients and the relative potential mechanism mainly attributes to the regulation of downstream signaling pathwaysby sponging microrna in addition lncrna microrna and many clinical data were also identified asprognostic biomarkers for osteosarcoma patients however osteosarcoma is one of the malignant cancers entitiescharacterized by the high level of heterogeneity in humanstherefore it is necessary to find accurate biomarkers forosteosarcomain recent years researchers have paid more and moreattention to the role of the tumor microenvironmenttme in malignant tumors the function of tme inthe tumorigenesis progression and therapy of tumorshave been initially understood [ ] more importantly estimation of stromal and immune cells in malignant tumor tissues using expression data estimate an algorithm to quantify the score of immune cellsand stromal cells by analyzing the gene expression datawas developed in based on the algorithm theprognostic value of immune and stromal cells in bladdercancer acute myeloid leukemia gastric cancer cervicalsquamous cell carcinoma adrenocortical carcinomaclear cell renal cell carcinoma hepatocellular carcinomathyroid cancer and cutaneous melanoma have beenreported [] generally the above research indicatedthat tme can serve as the prognostic biomarker in tumorsand many tmerelated genes were determined as the prognostic genes however the role of tme and tmerelatedgenes in osteosarcoma patients remains unclearin the present study gene expression data and corresponding clinicopathologic data were obtained from thetherapeutically applicable research to generate effectivetreatments target dataset then the estimatealgorithm was performed to quantify the immune score ofosteosarcoma and the tmerelated genes were identifiedby the differential expression analysis subsequently theprognostic value of tme and tmerelated genes weredetermined by a series of bioinformatics methodsmethodsgene expression datasetslevel data of gene expression profiles and correspondingclinical data of osteosarcoma patients were downloadedfrom target dataset ocgcancergovprogramstarget accessed on oct the correspondingclinicopathologic data included in the present study wereage gender race ethnicity tumor site and metastaticstatus after data were extracted from the public domainthe estimate an algorithm inferring tumor puritystromal score and immune cell admixture from expression data was performed to evaluate the immune score byusing the estimate package in r software version meanwhile the messenger rnamrna expressionprofiles and clinical data ofincludinggse21257 and gse39055 were obtained fromthe gene expression omnibus as external validationcohortstwo cohortssurvival analysis and correlation analysisafter scores were obtained patients were divided intohighscore group and lowscore group according to themedian of the immune score the kaplanmeier survivalanalysis with logrank test was performed to estimatethe differences of overall survival os and diseasefreesurvival dfs between high and lowscore cohorts inaddition the association between clinicopathologic dataand tme score was also studied mannwhitney signedrank test was performed to compare the differences ofimmune score between each clinical group all statisticalanalyses in the present study were performed using rsoftware except for the special instructions p value twoside was identified as statistically significantin the present studydegexpressed genedifferentially expressed gene analysisdifferentiallyanalysis wasperformed by comparing the proteincoding genesexpression between the lowimmune score group andthe highimmune score group the limma package in rsoftware was used to perform the differential analysisand genes with log fc and adjusted pvalue qvalue were identified as degs to further understand the function of degs identifiedin the present study gene ontology goincludingbiological processes bp molecular functions mf andcellular componentscc and kyoto encyclopedia ofgenes and genomes kegg analysis were performedby clusterprofiler package in r software evaluation of association with immune cellsto further investigate the association between degs andimmune cells the cibersort package was used toestimate the relative proportions of types of immunecells meanwhile the consensusclusterplus package was used to cluster in an unbiased and unsupervisedmanner based on the overlapping degs cumulative distribution function cdf and relative change inarea under the cdf curve were used to determine theoptimal number of clusters k then mannwhitney 0chu bmc cancer page of signedrank test was performed to study the differenceof immune cells proportion between the clusters and theviolin plot was established to show the differences ofimmune cells among clusters survival analysis of degsbased on the degs the univariate cox analysis was performed to determine the prognostic value of immunerelated genes then the osrelated genes were validatedin the gse21257 dataset while the dfsrelated geneswere validated in the gse39055 dataset only genes successfully validated were selected for further analysis afterward based on the validated genes the multivariate coxanalysis was performed to establish the prognostic signature for predicting the prognosis of osteosarcoma patientsthe risk score for each patient was calculated based onthe coefficient from the multivariate cox analysis and thecorresponding gene expression meanwhile all patientswere divided into the high and lowrisk groups accordingto the median of the risk score the survival receiver operating characteristic roc curve was used to show the discrimination of signatures and the kaplanmeier survivalcurve with the logrank test was generated to show thedifferences of os and dfs between high and lowriskgroups in addition the risk score of patients in the validation cohort was also calculated according to the aforementioned risk signature the kaplanmeier survivalcurve and survival roc curve were generated to show thepredictive ability of the signature in the validation cohortdevelopment of a nomogram for osteosarcoma patientsnomogram is a tool to visualize the predictive model andconvenient for clinical practice therefore we attemptedto develop a nomogram based on the tmerelated genessignature and clinicopathologic data to predict the prognosis of osteosarcoma patients firstlythe univariatecox analysis was performed to filter prognostic variableswhich will be further included in the multivariate coxanalysis secondly based on independent prognostic variables two nomograms were established for predicting theos and dfs respectively the cindex was used to assessthe discriminatory performance of the nomogram whichrange from to a cindex of means agreement by chance and a cindex of represents perfectdiscriminatory performance the higher value of the cindex the better performance of the nomogram is furthermore the calibration curves of and 3year weredeveloped to evaluate the effectiveness of nomogramsresultsimmune significantly associated with the prognosis ofosteosarcoma patients osteosarcoma patients were included in the presentstudy including males and females the immunescore of the cohort range from to tostudy the relationship between the immune score and theprognosis of osteosarcoma patients patients wereincorporated into the lowimmune score group while theremaining patients were incorporated into the highimmune score group the survival analysis indicated thatpatients with higher immune score had a favorable osand dfs fig 1a and b after adjusted age tumor siteand metastatic status the immune score still was a prognostic variable for both os and dfsfig 1a and b inaddition the relationship between immune score and clinical features was also investigated however there was nosignificant relationship between immune score and clinicalvariables supplementary figure 1a1cdifferential expression analysisaccording to the median of the immune score patients were divided into highscore n and lowfig association between immune score and prognosis in osteosarcoma patients a kaplanmeier survival analysis of overall survival for patientswith high vs low immune score b kaplanmeier survival analysis of diseasefree survival for patients with high vs low immune score 0chu bmc cancer page of score group n there were differentiallyexpressed genes between two groups which include upregulated genes and downregulated genesfig 2a b and supplementary table to furtherunderstand the function of degs go analysisand kegg analysis were performed the top significant results of go analysis among three types wereillustrated in fig 2c interestingly we can find that theresults of go analysis are mostly associated with immunity which further verify that the immunerelated degsare associated with immune features in addition the results of kegg also confirmed it such as phagosomeautoimmune thyroid disease antigen processing andpresentation b cell receptor signaling pathway intestinal immune network for iga production inflammatorybowel disease primary immunodeficiency th1 andth2 cell differentiation th17 cell differentiation natural killer cell mediated cytotoxicity and nfkappa bsignaling pathway fig 2dconsensusunsupervisedevaluation of degs and immune cellsto further understand the molecular heterogeneity ofosteosarcomaanalysis wasperformed to divide patients into subgroups to explorewhether immunerelated genes presented discernable patterns based on the consensus matrix heat map patientswere clearly divided into two clustersfig 3a in additionby comprehensively analyzing the relative change in areaunder the cumulative distribution function two clusterswere determined fig 3bc the immune score betweentwo clusters was significantly different fig 3d in additionthe proportion of types of immune cells in osteosarcomapatients was illustrated in a barplot fig 3e interestinglywe can see that the t cells cd4 memory activated ofcluster is significantly higher than cluster fig 5fprognostic value of tmerelated genesprevious studies indicated that tmerelated genes canserve as the prognostic biomarker for tumor patientsfig differentially expressed genes with the immune score in osteosarcoma patients a heatmap of significantly differentially expressed genesbased on immune score b the volcano figure to show the upregulated and downregulated genes c go analysis of differentially expressedgenes d kegg of differentially expressed genes go gene ontology kegg kyoto encyclopedia of genes and genomes 0chu bmc cancer page of fig the immune landscape of the tumor microenvironment ac unsupervised clustering of all samples based on the overlapping degs dcomparison of immune score between two clusters e the distribution of types of immune cells in osteosarcoma patients f the comparisonof types of immune cells between clusters deg differentially expressed genehence we performed the univariate cox analysis toidentify prognostic degs the results showed that and genes were identified as os and dfsrelateddegs respectively supplementary table and afterward five osrelated genes were successfully validated inthe gse21257 data set and five dfsrelated genes were successfully validated in the gse39055 cohort furthermoremultivariate cox analysis was performed and two prognostic signatures were generated for predicting the os anddfs respectively the risk score for predicting the os wasasrisk score fcgr2b0766 gfap0702 mpp70387 in addition the risk score for predicting thedfs was as follows risk score cyp2s10574 icam3 the auc values of osrelated signature were follows 0chu bmc cancer page of and in and 3year respectively fig 4aand the auc values of dfsrelated signature were and in and 3year respectively fig 5amoreover survival curves showed that patients in the highrisk group had worse os and dfs compared with the lowrisk patients figs 4b and 5b heat maps risk score plotsand survival status were generated to show the distinctionbetween highrisk patients and lowrisk patients figs 4ceand 5ce then both signatures were validated in independent cohorts for os signature the auc values ofvalidation cohort were and at and3year fig 4f for dfs signature the auc values ofvalidation cohort were and at and3year fig 5f additionallyin both validation cohortssurvival curves showed that lowrisk patients were favorableprognosis than highrisk patients figs 4g and 5gheat maps risk score plots and survival status of validation cohorts were also generated to show the distinction between highrisk patients and lowrisk patientsfigs 4hj and f 5hjdevelopment of a nomogram for osteosarcoma patientsto generate a nomogram for clinical use the cox analysiswas performed to select the clinical prognostic variables infig establishment and validation of the prognostic model for overall survival based on significant degs a receiver operating characteristiccurves of prognostic signature in the training cohort b the survival curve showed the different overall survival status between high and lowriskpatients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each sample reordered by riskscore e the scatter plot showed the overall survival status of osteosarcoma patients in the training cohort f receiver operating characteristiccurves of prognostic signature in validation cohort g the survival curve showed the different overall survival status between high and lowriskpatients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reordered by riskscore j the scatter plot showed the overall survival status of osteosarcoma patients in the validation cohort 0chu bmc cancer page of fig establishment and validation of the prognostic model for diseasefree survival based on significant degs a receiver operatingcharacteristic curves of prognostic signature in the training cohort b the survival curve showed the different diseasefree status between highand lowrisk patients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each samplereordered by risk score e the scatter plot showed the diseasefree status of osteosarcoma patients in the training cohort f receiver operatingcharacteristic curves of prognostic signature in validation cohort g the survival curve showed the different diseasefree status between high andlowrisk patients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reorderedby risk score j the scatter plot showed the diseasefree status of osteosarcoma patients in the validation cohortthe univariate cox analysis risk score and metastatic status were identified as both os and dfsrelated variablesfig 6a and e afterward risk score and metastatic statuswere determined as both independent os and dfsrelated variables in the multivariate cox analysis fig 6band f based on independent variables two nomogramswere established for predicting the os and dfs in osteosarcoma patients respectively fig 6c and g the cindexvalues were and in os nomogram and dfsnomogram respectively the results of cindex mean thatboth two nomograms have good discrimination meanwhile to evaluate the calibration of nomograms six calibration curves were generated and the results showed thatthe predictive curves were close to the ideal curve fig 6dand h which indicated a good calibrationdiscussionthe relationship between tme and tumor have beenwidely studied in recent years in the present study estimate algorithm was utilized to quantify the immunescore based on gene expression profiles in osteosarcomapatients from target database we confirmed that thetme is significantly associated with the prognosis ofosteosarcoma patientsinadditionfunctional enrichment analyses of tmerelated genes indicated that immunerelated processesincluding os and dfs 0chu bmc cancer page of fig nomograms based on the tumor microenvironment related genes for osteosarcoma patients a univariate cox analysis of overall survivalrelated variables b multivariate cox analysis of overall survivalrelated variables c nomogram for predicting the overall survival in osteosarcomapatients d1 and 3year calibration curves of overall survival nomogram e univariate cox analysis of diseasefree survivalrelated variables fmultivariate cox analysis of diseasefree survivalrelated variables g nomogram for predicting the diseasefree survival in osteosarcoma patientsh1 and 3year calibration curves of diseasefree survival nomogramknown to contribute to tumor progression more importantly degs based on the tme were identified asimportant prognostic biomarkers for osteosarcoma patients and two nomograms were developed for predicting the os and dfs of osteosarcoma patientsrespectivelyin recent years an increasing number of studiesfocused on the carcinogenesis and progression of tumorsbased on the tme and the estimate algorithm is oneof the most important quantitative tools for this researchfield based on the estimate algorithm the association between the prognosis and tme has been initially 0chu bmc cancer page of elucidated in some tumors such as cervical squamouscell carcinoma gastric cancer cutaneous melanomaacute myeloid leukemia bladder cancer and clear cellrenal carcinoma [ ] however previousstudies indicated that tme scores serve as a differentrole in different tumors for example for hepatocellularcarcinoma gastric cancer acute myeloid leukemiabladder cancer and clear cell renal carcinoma patientswith high immune score have a worse prognosis [ ] however for cervical squamous cell carcinoma adrenocortical carcinoma and cutaneous melanoma patients with high immune score have a favorableprognosis [ ] therefore we can find great heterogeneity among different tumors from the perspectiveof tme for osteosarcoma patients the present studyindicated that patients with higher immune score had abetter os and dfs hence the present study indicatedthat immune cells infiltrating tumor tissue may play animportant role in suppressing tumor progressionin our research tmerelated genes were identified by comparing the highscore and lowscore osteosarcoma patients the functional enrichment includinggo and kegg analyses showed that tmerelated geneswere mainly involved in the immune features such asregulation of leukocyte activation mhc protein complex mhc protein and complex binding more importantly the unsupervised cluster analysis based on degswas performed and all patients were divided into twoclusters immune score and t cell cd4 memory activated fraction were significant difference between twoclusters which further elucidated the relationship between degs and immune featuresdue to the poor prognosis of osteosarcoma patientsidentifying robust prognostic biomarker is very importantthe tumor immune microenvironment is closely relatedto the prognosis of bone tumor patients emilie etal performed the first genomewide study to describe therole of immune cells in osteosarcoma and found thattumorassociated macrophages are associated with reduced metastasis and improved survivalin highgradeosteosarcoma recently the prognostic signature based ontmerelated genes have been established for many tumors [ ] but only one study focused on osteosarcoma patients compared with the study performedby zhang we think that our research have someadvantages firstly our signatures were established basedon several validated genes and both two signatures weresuccessfully validated in independent cohorts secondlythe outcome of dfs was not reported in the previousstudy as reported in published studies tumor recurrenceis a terrible medical problem for osteosarcoma patientsand the 5year survival rate for osteosarcoma patients withmetastasis or relapse remains disappointing [ ]hence the dfs nomogram can improve the managementof osteosarcoma patients finally two nomograms incorporated tmerelated signature and clinical variables wereestablished in our research which further facilitated theclinical application of our findingsin our research five genes were incorporated into thefinal prognostic signatures fcgr2b gfap and mpp7were identified and validated as osrelated biomarkerswhile cyp2s1 and icam3 were dfsrelated biomarkersthe role of these genes in tumor prognosis had beenwidely reported in previous studies [] fcgr2bhas been confirmed as an immunerelated gene previously although the relationship between fcgr2band prognosis in sarcoma patients had not been reported the prognostic value of fcgr2b had been widelyconfirmed in other cancerssuch as hepatocellularcarcinoma and glioblastoma [ ] in addition newm etal demonstrated that mpp7 is novel regulatorsof autophagy which was thought to be responsible forthe prognosis of pancreatic ductal adenocarcinomacyp2s1 described as cytochrome p450 family subfamily s member was reported significantly associatedwith colorectal cancer in primary colorectal cancercyp2s1 was present at a significantly higher level ofintensity compared with normal colon more importantly the presence of strong cyp2s1 immunoreactivity was associated with poor prognosis the roleof icam3 in cancer was also widely reported in published studies and the akt pathway plays an importantrole in the impact of icam3 on tumors yg kim etal reported that icam3 can induce the proliferationof cancer cells through the pi3kakt pathway additionally jk park etal showed that the icam3 can enhancethe migratory and invasive potential of human nonsmall celllung cancer cells by inducing mmp2 andmmp9 via akt pathway showed that the icam3can enhance the migratory and invasive potential ofhuman nonsmall celllung cancer cells by inducingmmp2 and mmp9 via akt pathwayalthough the role of tme and tmerelated genes inosteosarcoma patients have been initially studied by bioinformatic and statistical analyses in our research somelimitations should be elucidated firstly the treatmentinformation cannot be obtained from the target database which may influence the prognosis of osteosarcomapatients secondly two nomograms were generated andshowed good performance in our study however externalvalidation by a large cohort is needed thirdly many independent prognostic genes for osteosarcoma patients wereidentified in the present study but the potential mechanism to influence osteosarcoma remains unclear finally inthe training cohort and degs were identified asos and dfsrelated degs respectively however onlyfive os and five dfsrelated genes were identified in thevalidation cohort the different age structures smaller 0chu bmc cancer page of sample sizes and the platform covering only part of thegenes may contribute to this resultreceived february accepted july in tme plays an important role in osteosarcoma patients and related with the progression of thetumor moreover tmerelated genes can serve as prognostic biomarkers in osteosarcoma patients howeverfurther researches are needed to study the potentialmechanism and validate the nomogram that developedin our present studysupplementary informationsupplementary information accompanies this paper at doi101186s12885020072162additional file additional file additional file additional file abbreviationstme tumor microenvironment deg differentially expressed genesos overall survival dfs diseasesfree survival roc receiver characteristiccurve estimate estimation of stromal and immune cells in malignanttumor tissues using expression data target therapeutically applicableresearch to generate effective treatments go gene ontology bp biologicalprocesses mf molecular functions cc cellular components kegg kyotoencyclopedia of genes and genomes cdf cumulative distribution functionacknowledgementsnoneauthors contributionsc h l y sq t c l and yh w conceived of and designed the study c h r sand c l performed literature search r s l y and b c generated the figuresand tables l y hl r x y and jy l analyzed the data c h wrote themanuscript and sq t and l y critically reviewed the manuscript l ysupervised the research all authors have read and approved the manuscriptfundingwe received no external funding for this studyavailability of data and materialsthe data of this study are from target and geo databaseethics approval and consent to participatethe research didnt involve animal experiments and human specimens noethics related issuesconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of joint surgery the affiliated hospital of qingdao universityqingdao china 2department of medical oncology the first hospital ofchina medical university shenyang china 3department of nursing sir runrun shaw hospital affiliated to zhejiang university hangzhou china4wenzhou medical university wenzhou chinareferencesjaffe n bruland os bielack s pediatric and adolescent osteosarcoma vol new york springer science business media vander rg osteosarcoma and its variants orthopedic clin north am biermann js adkins d benjamin r brigman b chow w conrad eu 3rdfrassica d frassica fj gee s healey jh bone cancer j natl comprcancer netw simpson s dunning md de brot s grauroma l mongan np rutland cscomparative review of human and canine osteosarcoma morphologyepidemiology prognosis treatment and genetics acta vet scand chen x cates jm du yc jain a jung sy li xn hicks jm man tkmislocalized cytoplasmic p27 activates pak1mediated metastasis and is aprognostic factor in osteosarcoma mol oncol huang x yang w zhang z shao z dysregulated circrnas serve as prognosticand diagnostic markers in osteosarcoma by sponging microrna to regulatethe downstream signaling pathway j cell biochem liu m yang p mao g deng j peng g ning x yang h sun h long noncoding rna malat1 as a valuable biomarker for prognosis in osteosarcoma asystematic review and metaanalysis int j surg xu k xiong w zhao s wang b microrna106b serves as a prognosticbiomarker and is associated with cell proliferation migration and invasionin osteosarcoma oncol lett zheng w huang y chen h wang n xiao w liang y jiang x su w wens nomogram application to predict overall and cancerspecific survival inosteosarcoma cancer manag res kahlert c kalluri r exosomes in tumor microenvironment influence cancerprogression and metastasis j mol med binnewies m roberts ew kersten k chan v fearon df merad m coussenslm gabrilovich di ostrandrosenberg s hedrick cc understanding thetumor immune microenvironment time for effective therapy nat med yoshihara k shahmoradgoli m martÃnez e vegesna r kim h torresgarcia wtreviño v shen h laird pw levine da inferring tumour purity and stromaland immune cell admixture from expression data nat commun yang s liu t nan h wang y chen h zhang x zhang y shen b qian pxu s comprehensive analysis of prognostic immunerelated genes inthe tumor microenvironment of cutaneous melanoma j cell physiol deng z wang j xu b jin z wu g zeng j peng m guo y wen z miningtcga database for tumor microenvironmentrelated genes of prognosticvalue in hepatocellular carcinoma biomed res int zhao k yang h kang h wu a identification of key genes in thyroid cancermicroenvironment med sci monit xu wh xu y wang j wan fn wang hk cao dl shi gh qu yyzhang hl ye dw prognostic value and immune infiltration of novelsignatures in clear cell renal cell carcinoma microenvironment agingalbany ny chen b chen w jin j wang x cao y he y data mining of prognosticmicroenvironmentrelated genes in clear cell renal cell carcinoma a studywith tcga database dis markers li x gao y xu z zhang z zheng y qi f identification of prognostic genesin adrenocortical carcinoma microenvironment based on bioinformaticmethods cancer med pan xb lu y huang jl long y yao ds prognostic genes in the tumormicroenvironment in cervical squamous cell carcinoma aging albany ny wang h wu x chen y stromalimmune scorebased gene signature aprognosis stratification tool in gastric cancer front oncol huang s zhang b fan w zhao q yang l xin w fu d identification ofprognostic genes in the acute myeloid leukemia microenvironment agingalbany ny yan h qu j cao w liu y zheng g zhang e cai z identification ofprognostic genes in the acute myeloid leukemia immunemicroenvironment based on tcga data a | Colon_Cancer |
" african americans aa are at high risk for colorectal cancer crc studies report a increasein crc risk with physical inactivity obesity and metabolic syndrome activation of the wntcatenin ctnnb1signaling pathway plays a critical role in colorectal carcinogenesis accumulating evidence also indicates a role ofwntctnnb1 signaling in obesity and metabolic diseasesaim to examine the association between obesity catenin expression and colonic lesions in african americansmethods we reviewed the pathology records of colorectal specimens from to crcs advanced adenomas and normal colon tissues tissue microarrays tma were constructed from these samplesimmunohistochemistry ihc for ctnnb1 catenin clone catenin1 was performed on the constructed tmasthe ihc results were evaluated by pathologists and the nuclear intensity staining was scored from to bmisex age location of the lesion and other demographic data were obtainedresults positive nuclear staining in normal advanced adenoma and crc was and respectively p crc was asso ciated with positive status for nuclear ctnnb1 intensity adjusted or 95ci p for positive nuclear staining compared to noncrc samples normal or advanced adenoma nuclearstaining percentage has a fair diagnostic ability for crc with an auc of 95ci overweightobese patients bmi did not show a significant difference in p nuclear ctnnb1 staining positive in normal weight vs positive in overweightobese the association between nuclear intensityand crc was not different between normal and overweight patients p for interaction the positive nuclearctnnb1status in crc stage iii and iv of all crc was not different from stage i and ii vs respectively p continued on next page correspondence b_shokranihowardedu hashktorabhowardedu1department of medicine department of pathology and cancer centerhoward university college of medicine geia avenue nwwashington dc usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cshokrani bmc gastroenterology page of continued from previous page in our study advanced adenoma and crc were associated with activation of catenin in physically fitoverweight and obese patients thus obesity and wntcatenin pathway seem to be independent in africanamerican patients wntcatenin signaling pathway has a potential to be used as an effector in coloncarcinogenic transformation whether or not bmi is a modifier of this pathway needs to be investigated furtherkeywords catenin colorectal cancer advanced adenoma african americans colorectal cancer crc is one of the most commoncancers in the industrialized world lifestyle and epidemiological factors associated with an increased risk ofcrc include physical inactivity obesity and metabolicsyndrome in the united states approximately twothirds of the adult population are overweight or obesewhich represents a putative risk factor for multiple target an malignancies including crc there is evidence to suggest that excess adiposity is associated with up to greater risk of crc comparedwith normal weight individuals and that physical activity may decrease colorectal cancer risk althoughexcessive accumulation of white adipose tissue wat isthe key feature of adiposity obesity is clinically definedby a bmi over kgm2 which does not take fat contentinto account it is also known that most crcs arise froma genetic and morphological adenoma to carcinomatransition also it is widely accepted that both crcsand colorectal adenomas cras share similar etiologicalcauses which explains why cras which are amongstthe mostfindings in all crcscreening participants are present in more than ofgeneral asymptomatic populations consequentlyrisk algorithms have been applied to use bmi as a predictor variable to stratify individuals according to theirrisk of colorectal neoplasia however the underlyingmechanisms that might explain the association and themagnitude ofthe connection between excess bodyweight and crc remain unclearfrequent pathologicalin the obesitycancer relationship multiple biologicalprocesses including insulininsulinlike growth factorigf1 insulin resistance sexual hormones estrogensand proinflammatory cytokines tnfα il6 and crpactively participate all these elements create a favorable environment for carcinogenesis and a decrease inapoptosisas a separate molecular pathway activation of thewnt signaling pathway plays a critical role in colorectalcarcinogenesis wnt ligands are a family of proteinsfor normal cell development that are importantcatenin ctnnb1 is a major mediator of the wntpathway that is traditionally classified into canonical catenindependent and noncanonical cateninindependent wnt canonical pathway utilizes a group ofcell surface receptors called frizzled frz to activateseveral pathways the most important one involving catenin and apc in the absence of wnt signalingapc causes degradation of catenin preventing its accumulation in the cytoplasm by forming a complex withcatenin which leads to the phosphorylation and eventually destruction of catenin by the proteasome signaling by wnt blocks this process allowing cateninto migrate from the cytoplasm to the nucleus once inthe nucleus catenin upregulates cmyc cyclin d1and other genes which increase cellular proliferation therefore continuous wnt signaling can be seenin cells with loss of apc metabolic syndromeassociated conditionssuch asobesity and type ii diabetes are influenced by geneticand functional variations in the wnt signaling pathway wnt signaling when activated represses the terminal differentiation during adipogenesis whereby preadipocytes take on the characteristics of mature adipocytes a cascade of transcriptional events like the induction of catenin ensues which in turn inducesenhancer binding proteinα cebpa and peroxisomeproliferatoractivated receptorÎ pparg the excessive accumulation of wat features adiposity butobesity does not take fat content into account recently genetic factors linked to fat mass and adipositywere reported to be associated with increased obesityrisk in young obese individuals wholeexome sequencing revealed rare gainoffunction mutations inctnnb1catenin the cateninregulated transcription of an adipocytederived chemokine calledserum amyloid a3 saa3 leads to the formation of a catenintcf complex in mature adipocytes that promotes the proliferation of preadipocytes in wat andthereby increases obesity and the risk for metabolic syndrome other data also suggest that obesity and lack ofphysical activity are associated with a higher risk forcolorectal cancer [ ] these findings have importantimplications especially in the obese and physically inactive african american population that may haveunderlying predisposing mutations to colorectal cancer the aim of this study is to assess the catenin expression profile in colorectal premalignant and malignant lesions in correlation with obesity as determined by 0cshokrani bmc gastroenterology page of body mass index bmi or waist circumference wc inafrican american populationmethodspatients and clinical datacolorectaltissue samples submitted to the surgicalpathology laboratory at howard university hospitalfrom january to december were retrieved from the pathology archive system powerpath¢a total of samples were included in the study consisting of tissue samples from crc n advancedadenoma n and normal colon n patientsdata included age sex height weight and waist circumference body mass index bmi was calculated in thestudy table the protocol of this study was approvedby the howard university institutional review boardirbtissue microarray tma constructionhematoxylineosin stained slides he slides weremade from paraffinembedded blocks the he slideswere reviewed by two pathologists to confirm the pathological diagnosis and to mark areas of interest multipleareas from more than one block were marked to ensurea good representability of the sample on the tma fivetma paraffin blocks each containing cores of mmin diameter each and mm distance from each otherwere constructed tissuespecific and an system controls were included in each tmaimmunohistochemical ihc analysis of ctnnb1 cateninthe constructed tmas were stained for catenin theimmunostaining was carried out as follows dako monoclonal mouse antihuman betacatenin cateninc1intended for laboratory application to identify qualitatively by light microscopy catenin positive cells in normal and neoplastic tissues was used at a dilution of using the envision dab code k4006 detectionsystem the deparaffinized tmas were treated prior tothe ihc staining procedure target retrievalinvolvedimmersion of tissue sections in a preheated buffer solution and maintaining heat in a water bath °cfor greater adherence of tissue sections to glass slidessilanized slides dako code s3003 were used targetretrieval solution code s1700 or 10x concentratecode s1699 using a 20min heating protocol was usedthe cellular staining pattern of anticatenin is mainlymembranous especially at the cellcell boundaries positive nuclear staining and diffuse cytoplasmic staining arealso reported in cancer cells fig evaluation and assessment of the catenin expressiontwo pathologists interpreted the ihc slides cateninexpression status was assessed based on the pattern ofstaining nuclear cytoplasmic and membranous intensity to and percentage of staining to the staining would be considered negative if there wasweak or no nuclear expression or positive if there wasmoderate or strong nuclear expressionstatistical analysisdistribution of continuous and categorical variables weretested by kruskalwallis and chisquare test betweendifferent groups respectively we used logistic regression analysis to test association between the staining andrisk of crc after adjusting for age and gender areaunder the curve auc was calculated for variables withsignificant association with crc using receiver operative characteristics curve all statistical analyses wereperformed by stata statacorp college station txresultsepidemiological characteristics and bmi in normaladvanced adenoma and crcthe bmi was calculated for individual patients and normal subjects as represented in table crc patientswere older p while our healthy normal population was mostly overweight higher bmi was moreclosely associated with advanced adenoma and crchowever the differences were not significant table advanced adenomas and crcs were associated withpositive nuclear ctnnb1we assessed whether alterations in wntctnnb1 catenin signaling plays any role colon carcinogenesispositive catenin nuclear stains were seen in normaladvanced adenomas and crcs were and respectively p table based on the designationtable epidemiological characteristics and bmi in normal advanced adenoma and crc patientsage median iqrmale n bmi kgm2 median iqroverweight n normaln advanced adenoman crcn p value 0cshokrani bmc gastroenterology page of fig immunostain for catenin in three individuals normal a à advanced adenoma b à and cancer c d à and à respectively showing membranous staining in the normal cytoplasmic and membranous staining in adenoma with no evidence of nuclearexpression arrow showing lack of nuclear staining and nuclear and cytoplasmic staining in cancer arrow showing nuclear stainingof n intensity which is associated with higher risk ofcancer crcs were associated with positive status fornuclear ctnnb1 intensity age gender adjusted or 95ci p for positive nuclearstaining compared to noncrc samples normal or advanced adenoma fig nuclear staining percentagehas also a fair diagnostic ability for crc with an auc of 95ci table fig overweight and obese patients show a trend withpositive nuclear ctnnb1 expressionpositive nuclear ctnnb1 staining was in normalweight and in overweightobese bmi patientsthis difference pointed to trend that was not statisticallysignificantassociation between nuclear intensity and crc in normaland overweight patientsthe association between nuclear intensity and crcwas not statistically significant different between normal weight and overweight patientsinteraction tables and the positive nuclearctnnb1 status in crc stage iii and iv of allcrc was not different from stage i and ii vs respectively p p fortable catenin nuclear and cytoplasmic expression tabulated as intensity and percentage in normal advanced adenoma andcrcnormaln cnc intensity n intensity advanced adenoma n crcn c cytoplasmic n nuclearc intensity and n intensity mean intensity and aboveoverall p valuep value for advanced adenoma vs normalp value for crc vs other 0cshokrani bmc gastroenterology page of fig catenin nuclear and cytoplasmic expression in normal advanced adenoma and crcdiscussionone of the important risk factors in colorectal cancer isobesity [ ] catenin is an ecadherin binding proteinthat mediates cellcell adhesion and plays a role inthe canonical wnt signaling pathway that controls thecoordinated expansion and differentiation of the intestinal crypt stem cells degradation of catenin byphosphorylation followed by alteration of destructioncomplex apc gsk3 and axin results in inactivation if wnt pathway in our study we found thatwas associated with an increased adjusted or of 95ci p for positive nuclear staining compared to noncrc samples normal or advanced adenomathe gatekeeper gene apc is a negative regulator of catenin and is mutated in approximately of sporadic and hereditary colon cancers there are severalmutations that can cause an accumulation of cateninin tumor cells such as mutations of the apc gene pointmutations in gsk3 or mutations in catenin gene itself []our positive nuclear staining in crc and its association with the positive status for nuclear ctnnb1intensity compared to noncrc samples are in contrastto a study by brabletz which showed that catenin is localized in the cytoplasm and membrane ofthe tumor cells whereas in our study it was mainly concentrated in the cytoplasm and the nucleus they alsomentioned that there was positive nuclear staining at theinvasive front as catenin is involved in tumor progression such is not the case in our studyindeed evenwhen considering nuclear staining in our specimensthere was no statistically significant differences betweenstage iiiiv cases staining versus stages iii crc caseslevels of staining the fact that catenin is expressedearly in the african american specimens analyzed heremight partially explain the aggressive nature of crc inthis population in addition we showed that there is uniform membranous staining in normal and increasingcytoplasmic and nuclear staining in advanced adenomasand crcs this confirms that the decrease in membranous staining begins with dysplastic changes leading to atable association of bmi with catenin nuclear intensity in advanced adenoma casesadvanced adenoma with catenin expression n in intensity and no nuclear intensity n bmi median interquartileoverweight n nuclear intensity negativen nuclear intensity n p value 0cshokrani bmc gastroenterology page of table association of bmi with catenin nuclear intensity in crc casescrc with catenin expression with high nuclear intensity and without negativebmi median interquartileoverweight n nuclear intensity negativen nuclear intensity n p valueprogressive disappearance atcrcsthe membrane levelinas we mentioned above a major risk factor for crc isobesity which continues to expand as a pandemicworldwide the american cancer society cancerprevention study ii states that there is an increased incidence of crc esophageal adenocarcinoma and othercancers with obesity in our study we showed that of advanced adenoma patients and of crc patients were overweight with bmi in comparison toadvanced adenoma the percentage was lower in cancerperhaps due to the late stage of cancer and weight lossin the interim table there are several mechanismsby which obesity is believed to promote crc this includes increase in leptin levels that cause an increase ingrowth and proliferation of colon cancer cells altered adipokine levels altered gut microbiome apartfrom increased steroid hormones and growth factors insulin is however the established biochemical linkand the main pathway involved is pik3aktmtorpathway elevated igf1 and insulin act through the insulin receptors and phosphotidylinositide3 kinase in addition to the above findings we also found thatoverweight and obese patients bmi did not showa significantly increased expression p of nuclearctnnb1 positive in normal weight vs positive in overweightobese morikawa found that inobesepositivity wasnuclear ctnnb1patientsassociated with significantly better cancerspecific survival suggesting that wnt signaling acts as a switch andwhen it is on adipogenesis is repressed kennell demonstrated that activated frz1 frizzled promotes catenin stabilityinhibits apoptosis and adipogenesisross also showed that wnt signaling acts as a molecular switch that controls adipogenesis upregulationof wnt signaling maintains preadipocytes in an undifferentiated state and when wnt signaling is prevented theydifferentiate into adipocytes [ ]although in our study there was no association between nuclear intensity and crc between normal andoverweight patients p for interaction there is accumulating evidence to show that the state of chronicinflammation incited by obesity might play a role in promoting colorectal carcinogenesis [ ] of the manymarkers tnfα is important [ ] as it activateswnt signaling through the induction of gsk3 phosphorylation resulting in increased nuclear localization ofcatenin in addition to tnfα other humoralagents associated with obesity might also be contributingto the activation of wnt signaling like il1 and adiponectin which is decreased in the obese state and is notan inflammatory cytokine that can modulate gsk3catenin signaling pathway although multiplemechanisms may be operating in parallel and contributing to the protumorigenic milieu wnt is a pivotaltumorigenic pathway aberrations of which isfig the putative relationship between obesity and colorectal cancer evolution pathways by cellular ctnnb1 status based on the data by thecurrent study our study suggests that there is no association between obesity and ctnnb1 expression 0cshokrani bmc gastroenterology page of important in the evolution of most sporadic crc insummary there is positive nuclear staining in crcs which was associated with the positive status fornuclear ctnnb1 intensity adjusted or 95ci p for positive nuclear staining compared to noncrc samples normal or advanced adenoma this shows that advanced adenomas and crcswere associated with activation of catenin in physically fit overweight and obese patients fig advanced adenoma and crc were associated with activation of catenin in physically fit overweight andobese patients thus participation of obesity and wntpathway seem to be independent crc factors in africanamerican patientsinflammationdriven activation ofwnt signaling as a potential pathway linking obesity tothe development of crc needs to be investigated furtherin the african american population this might provideinsights into the identification of new therapeutic targetsto reduce the burden of obesityassociated crcabbreviationscrc colorectal cancer wat white adipose tissue aa african americansacknowledgementsnot applicableauthors contributionsconceived and designed experiments bs ha performed experiments bsel ha hb analyzed data ha hb mn th aa and zs contributed reagentsmaterialsanalysis tools zs ha hb bs and el wrote and edited manuscriptbs and ha provided statistical analysis mn all authors have read andapproved the manuscriptfundingthis project was supported in part by grant from the national institute onminority health and health disparities of the national institutes of healthunder award numbersg12md007597 the funder had no role in designing or execution of thisstudyavailability of data and materialsall data generated or analyzed during this study are included in thispublished ethics approval and consent to participatethis retrospective and chart review study was conducted according to theworld medical association declaration of helsinki and was approved by theinternal review board of howard university since the chart review was donethrough unidentifiable approach no consent form needed for this studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests related to thismanuscriptauthor details1department of medicine department of pathology and cancer centerhoward university college of medicine geia avenue nwwashington dc usa 2division of pulmonary allergy and criticalcare medicine university of pittsburg pittsburg pa usareceived april accepted august referencesresearch iafco estimated cancer incidence mortality and prevalenceworlwide in lyon globocan haggar fa boushey rp colorectal cancer epidemiology incidencemortality survival and risk factors clin colon rectal surg oxentenko as body size and incident colorectal cancer a prospectivestudy of older women cancer prev res phila ma y obesity and risk of colorectal cancer a systematic review ofprospective studies plos one 201381e53916slattery ml physical activity and colorectal cancer sports med wong mc lam ty tsoi kk hirai hw chan vc ching jy chan fk sung jja validated tool to predict colorectal neoplasia and inform screening choicefor asymptomatic subjects gut sung jjy wong mcs lam tyt tsoi kkf chan vcw cheung w ching jyla modified colorectal screening score for prediction of advanced neoplasiaa prospective study of subjects jgastroenterolhepatol liu z dietinduced obesity elevates colonic tnfalpha in mice and isaccompanied by an activation of wnt signaling a mechanism for obesityassociated colorectal cancer j nutr biochem najdi r holcombe rf waterman ml wnt signaling and coloncarcinogenesis beyond apc j carcinog macdonald bt tamai k he x wntbetacatenin signaling componentsmechanisms and diseases dev cell denysenko t wntbetacatenin signaling pathway and downstreammodulators in low and highgrade glioma cancer genomics proteomics willert k nusse r betacatenin a key mediator of wnt signaling curr opingenet dev vella a camilleri m pharmacogenetics potential role in the treatment ofdiabetes and obesity expert opin pharmacother christodoulides c adipogenesis and wnt signalling trendsendocrinol metab world health anization [httpwwwwhointmediacentrefactsheetsfs311en] chen m lu p ma q cao y chen n li w zhao s chen b shi j sun yshen h sun l shen j liao q zhang y hong j gu w liu r ning g wangw wang j ctnnb1betacatenin dysfunction contributes to adiposity byregulating the crosstalk of mature adipocytes and preadipocytes sci adv20206eaax9605 morikawa t kuchiba a yamauchi m meyerhardt ja shima k nosho kchan at giovannucci e fuchs cs ogino s association of ctnnb1 betacatenin alterations body mass index and physical activity with survival inpatients with colorectal cancer jama morikawa t prospective analysis of body mass index physical activityand colorectal cancer risk associated with betacatenin ctnnb1 statuscancer res ashktorab h kupfer ss brim h carethers jm racial disparity ingastrointestinal cancer risk gastroenterology kaler p augenlicht l klampfer l activating mutations in betacatenin incolon cancer cells alter their interaction with macrophages the role of snailplos one 201279e45462enzo mv the wntbetacatenin pathway in human fibroticlikediseases and its eligibility as a therapeutic target mol cell ther stamos jl weis wi the betacatenin destruction complex cold springharb perspect biol 201351a007898 chung gg tissue microarray analysis of betacatenin in colorectalcancer shows nuclear phosphobetacatenin is associated with a betterprognosis clin cancer res miyoshi y activation of the betacatenin gene in primaryhepatocellular carcinomas by somatic alterations involving exon cancerres fukuchi t betacatenin mutation in carcinoma of the uterineendometrium cancer res brabletz t variable betacatenin expression in colorectal cancersindicates tumor progression driven by the tumor environment proc natlacad sci u s a 0cshokrani bmc gastroenterology page of berger na obesityassociated gastrointestinal tract cancer from beginningto end cancer calle ee the american cancer society cancer prevention study iinutrition cohort rationale study design and baseline characteristicscancer frezza ee wachtel ms chirivainternati m influence of obesity on the riskof developing colon cancer gut berger na obesity and cancer pathogenesis ann n y acad sci guo s insulin signaling resistance and the metabolic syndrome insightsfrom mouse models into disease mechanisms j endocrinol 20142202t1t23 ross se inhibition of adipogenesis by wnt signaling science kennell ja macdougald oa wnt signaling inhibits adipogenesis throughbetacatenindependent and independent mechanisms j biol chem liu z yingka y inflammation driven activation of wnt pathway a potentialmechanism responsible for obesity associated colorectal cancer obes resopen j tilg h moschen ar adipocytokines mediators linking adipose tissueinflammation and immunity nat rev immunol fischerposovszky p wabitsch m hochberg z endocrinology of adiposetissue an update horm metab res oguma k activated macrophages promote wnt signalling throughtumour necrosis factoralpha in gastric tumour cells embo j wang y adiponectin modulates the glycogen synthase kinase3betabetacatenin signaling pathway and attenuates mammary tumorigenesis ofmdamb231 cells in nude mice cancer res renehan ag roberts dl dive c obesity and cancer pathophysiologicaland biological mechanisms arch physiol biochem publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Colon_Cancer |
to the human gut microbiota biochem taxonomic hierarchy of the phylum firmicutes and novel firmicutes species originated from various environments in korea j microbiol front microbiol filippidou s a combination of extreme environmental conditions favor the prevalence of endosporeforming firmicutes bintsis t lactic acid bacteria as starter cultures an update in their metabolism and genetics aims microbiol verma d k bioprocessing technology in food and health potential applications and emerging scope apple academic caulier s overview of the antimicrobial compounds produced by members of the bacillus subtilis group front microbiol lopetuso l r scaldaferri f petito v gasbarrini a commensal clostridia leading players in the maintenance of gut homeopress burlington stasis gut pathogens rood j i general physiological and virulence properties of the pathogenic clostridia in clostridial diseases of animals eds uzal fa prescott jf songer jg and popoff mr wiley wells c l wilkins t d medical microbiology 4th edn university of texas medical branch galveston kho z y lal s k the human gut microbiomea potential controller of wellness and disease front microbiol koliada a association between body mass index and firmicutesbacteroidetes ratio in an adult ukrainian population bmc microbiol yuan j antibacterial compoundsmacrolactin alters the soil bacterial community and abundance of the gene encoding pks front microbiol 103389fmicb scientific reports 101038s41598020706868vol1234567890wwwnaturecomscientificreports 0c romerotabarez m 7omalonyl macrolactin a a new macrolactin antibiotic from bacillus subtilis active against methicillinresistant staphylococcus aureus vancomycinresistant enterococci and a smallcolony variant of burkholderia cepacia antimicrob agents chemother moldenhauer j chen x h borriss r piel j biosynthesis of the antibiotic bacillaene the product of a giant polyketide synthase complex of the transat family angew chem int ed engl 101002anie20070 butcher r a the identification of bacillaene the product of the pksx megacomplex in bacillus subtilis proc natl acad wu l difficidin and bacilysin from bacillus amyloliquefaciens fzb42 have antibacterial activity against xanthomonas oryzae chen xh structural and functional characterization of three polyketide synthase gene clusters in bacillus amyloliquefaciens sci rice pathogens sci rep fzb j bacteriol yang j genomicsinspired discovery of three antibacterial active metabolites aurantinins b c and d from compostassociated bacillus subtilis fmb60 j agric food chem kang h k seo c h park y marine peptides and their antiinfective activities mar drugs 103390md130 sur s romo t d grossfield a selectivity and mechanism of fengycin an antimicrobial lipopeptide from molecular dynamics j phys chem b 101021acsjpcb7b118 knight c a the first report of antifungal lipopeptide production by a bacillus subtilis subsp inaquosorum strain microbiol peypoux f bonmatin j m wallach j recent trends in the biochemistry of surfactin appl microbiol biotechnol grangemard i wallach j magetdana r peypoux f lichenysin a more efficient cation chelator than surfactin appl biores s0025 chem biotechnol zhou m bacillibactin and bacillomycin analogues with cytotoxicities against human cancer cell lines from marine bacillus sp pkuma00093 and pkuma00092 mar drugs hertlein g production of the catechol type siderophore bacillibactin by the honey bee pathogen paenibacillus larvae one e108272 gu q bacillomycin d produced by bacillus amyloliquefaciens is involved in the antagonistic interaction with the plantpathogenic fungus fusarium graminearum appl environ microbiol e0107517 barsby t kelly m t andersen r j tupuseleiamides and basiliskamides mew acyldipeptides and antifungal polyketides produced in culture by a bacillus l aterosporus isolate obtained from a tropical marine habitat j nat prod mthethwa b c comparative analyses of cytochrome p450s and those associated with secondary metabolism in bacillus species int j mol sci 103390ijms1 podust l m sherman d h diversity of p450 enzymes in the biosynthesis of natural products nat prod rep 101039c2np2 0020a greule a stok j e de voss j j cryle m j unrivalled diversity the many roles and reactions of bacterial cytochromes p450 in secondary metabolism nat prod rep 101039c7np0 0063d medema m h minimum information about a biosynthetic gene cluster nat chem biol 101038nchem bio1890 ngwenya m l blooming of unusual cytochrome p450s by tandem duplication in the pathogenic fungus conidiobolus coronatus int j mol sci 103390ijms1 matowane r g in silico analysis of cytochrome p450 monooxygenases in chronic granulomatous infectious fungus sporothrix schenckii special focus on cyp51 biochim biophys acta proteins proteom 101016jbbapa p201710003 qhanya l b genomewide annotation and comparative analysis of cytochrome p450 monooxygenases in basidiomycete biotrophic plant pathogens one e0142100 101371journ alpone01421 kgosiemang i k r syed k mashele s s comparative genomics and evolutionary analysis of cytochrome p450 monooxygenases in fungal subphylum saccharomycotina j pure appl microbiol jawallapersand p cytochrome p450 monooxygenase cyp53 family in fungi comparative structural and evolutionary analysis and its role as a common alternative antifungal drug target one e107209 101371journ alpone01072 syed k shale k pagadala n s tuszynski j systematic identification and evolutionary analysis of catalytically versatile cytochrome p450 monooxygenase families enriched in model basidiomycete fungi one e86683 101371journ alpone00866 suzuki h comparative genomics of the whiterot fungi phanerochaete carnosa and p chrysosporium to elucidate the genetic basis of the distinct wood types they colonize bmc genom 1011861471216413444 akapo o o distribution and diversity of cytochrome p450 monooxygenases in the fungal class tremellomycetes int j mol sci 103390ijms2 sello m m diversity and evolution of cytochrome p450 monooxygenases in oomycetes sci rep 101038srep1 b biol 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 furumichi m tanabe m kegg as a reference resource for gene and protein annotation nucleic acids res d457d462 101093nargkv10 nelson d r the p450 superfamily update on new sequences gene mapping accession numbers early trivial names of enzymes and nomenclature dna cell biol 101089dna1993121 nelson d r cytochrome p450 nomenclature methods mol biol clifton nj 101385089603 nelson d r cytochrome p450 nomenclature methods mol biol clifton nj 101385159259 scientific reports 101038s41598020706868vol0123456789wwwnaturecomscientificreports 0c de lima procpio r e da silva i r martins m k de azevedo j l de araºjo j m antibiotics produced by streptomyces braz j infect dis munro a w p450 bm3 the very model of a modern flavocytochrome trends biochem sci li h poulos t l the structure of the cytochrome p450bm3 haem domain complexed with the fatty acid substrate palmitoleic acid nat struct biol bm3 biochem soc trans noble m miles c reid g chapman s munro a catalytic properties of key activesite mutants of flavocytochrome p450 lee ds crystallization and preliminary xray diffraction analysis of fattyacid hydroxylase cytochrome p450bsβ from bacillus subtilis acta crystallogr sect d biol crystallogr lee ds substrate recognition and molecular mechanism of fatty acid hydroxylation by cytochrome p450 from bacillus subtilis crystallographic spectroscopic and mutational studies j biol chem girvan h m structural and catalytic properties of the peroxygenase p450 enzyme cyp152k6 from bacillus methanolicus bower s cloning sequencing and characterization of the bacillus subtilis biotin biosynthetic operon j bacteriol j in biochem cryle m j schlichting i structural insights from a p450 carrier protein complex reveal how specificity is achieved in the p450bioi acp complex proc natl acad sci cryle m j bell s g schlichting i structural and biochemical characterization of the cytochrome p450 cypx cyp134a1 from bacillus subtilis a cyclolleucyllleucyl dipeptide oxidase biochemistry cimermancic p insights 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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" mucinous colon cancers mcc are characterized by abundant production of mucin muc2 proteinand are less sensitive to standard systemic chemotherapy we postulated that severepersistent endoplasmicreticulum stress ers aggravation in mcc would overwhelm compensatory cytoprotective pathways and induceapoptosisresults basal levels of ers markers were higher in mcc and dntcfls174t cells than nonmucinous tumors andthese levels were significantly increased by combinatorial treatment with ers aggravators celecoxib orlistatcombination treatment inhibited cell viability and synergistically induced apoptosis treatmentinduced cell deathwas ersdependent apoptotic pathways were not activated following knockdown of ers protein chop dual drugtreatment significantly reduced mucinous tumor growth in vivo and induced ers and apoptosis consistent within vitro experimentss novel therapies are needed since mcc are more resistant to standard systemic chemotherapy thisstudy suggests ers aggravation is a viable therapeutic strategy to reduce tumor growth in mcckeywords muc2 mucinous colon cancer xenograft colonoids endoplasmic reticulum stress mucinous colon cancers mcc account for only of colorectal cancers in the usa each year casesyear they arise from rapidly proliferating neoplastic cells with goblet celllike features thatproduce large quantities of mucin muc2 protein [] from a treatment standpoint they are less responsiveto systemic chemotherapy in the neoadjuvant and palliative setting chemoresistance of mcc has been attributed to the abundant extracellular muc2 protein thatmay act as a barrier against drug delivery or immune infiltration and forms an immunosuppressivehypoxic correspondence choudrymhupmcedu1department of surgery university of pittsburgh medical center hillmancancer center centre avenue suite pittsburgh pa usa2department of pharmacology chemical biology university of pittsburghmedical center pittsburgh pa usamicroenvironment that impairs treatment efficacy andallows cancer cells to thrive therefore our prior research has focused on targeted therapies that simultaneouslyinducemechanisms for neoplastic cell death []inhibit muc2productionandthe endoplasmic reticulum er is a major site forbiosynthesis posttranslational modification and properfolding of proteins like muc2 that are destined to be secreted from cells proteins that fail to undergo correct foldingmaturation undergo erassociated proteindegradation erad via ubiquitinproteasomeandautophagymediated pathways a variety of conditionsthat disrupt normal protein processing eg calcium imbalance hypoxia can overwhelm the ability of cells tomaintain proper protein processing in the er therebytriggering er stress ers and its associated molecularthe unfolded proteinsignaling pathways known as 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 0cdilly orphanet of rare diseases page of protective mechanismsresponse upr the upr pathways represent a coordinated effort by cells to decrease overall protein synthesisand improve protein foldingmodification or degradation however severe and persistent ers can overwhelm thesetriggermolecular pathways associated with cell death []we therefore hypothesized that mucinous colon cancerscharacterized by high muc2 protein turnover wouldexhibit elevated basal ers levels and be vulnerable totherapies that aggravate ers thereby inducing ersassociated cell death pathwaysandduring low to moderate levels of ers various prosurvival upr pathway proteins are activated these include heat shock protein grp78 glucose regulated protein also called bip immunoglobulin heavy chainbinding protein and three er transmembrane proteinsperk protein kinase activated by doublestrandedrnalike er kinase ire1 inositolrequiring enzyme and atf6 activating transcription factor duringers grp78 disassociates from these upr proteins tochaperone terminally misfolded proteins for degradationat the same time the freed upr proteins become activated and initiate signaling pathways that serve to correct or neutralize ers conversely during severe andpersistent ers proapoptotic upr signaling pathwaysare activated these include elevated expression of transcription factor chop cebp homologous protein alsocalled gadd153 inhibition of antiapoptotic proteinseg bcl2 stimulation of proapoptotic bh3only proteins eg bim and activation of caspases [ ]byinhibitionofersaggravationin this study we first characterized basal ers levels inmcc and then investigated whether aggravation of ersin these tumors would induce ersassociated cell deathwe studied a combination of two federal drug administration fda approved drugs to aggravate ers celecoxibnoncyclooxygenase [cox2] target sarcoplasmicendoplasmic reticulum calcium atpase [serca] and orlistaters activation by fatty acid synthase [fasn] inhibition[ ] serca inhibition on the er membrane activates ers by disrupting calcium homeostasis withinthe er while fasn inhibition triggers ers by a varietyof mechanisms including the disruption of protein lipidation an important process for normal protein foldingstability membrane association localization traffickingand secretion [] additional rationale for the useofthese drugs include the basal overexpression ofcox2 and fasn in mcc and prior studies demonstrating inhibition of muc2 expressionsecretion by targeting these two cancer pathways [ ] bothcelecoxib and fasn inhibitors have demonstrated effective in vitro and in vivo cellular growth suppressionin a variety of malignancies including colorectal cancerhowever clinicalthese drugs have beentrials ofdisappointing [ ] we postulate that the irrational use of such drugs against unselected cancers inclinical trials is likely responsible for lack of clinical efficacy to date and that mcc are more likely to be susceptible to such targeted therapies given their high basalers levelsresultsmcc and high muc2 producing cells exhibit elevatedbasal ers that correlates with muc2 expression levelswe compared basal ers levels in explant tissue from patients with mcc and nmcc we found significantlyhigher basal muc2 grp78 bip atf4 and chop protein expression levels in explant tissue from mcc compared to nmcc fig 1a similar results were foundwhen comparing high versus low muc2 expressingls174t cells dntcf4ls174t cells exposed to doxycycline for h high muc2 expressing cells expressedhigher levels of muc2 and upr proteins grp78 bipatf4 and chop compared to dntcf4ls174t cellslacking doxycycline exposure low muc2 expressingcells fig 1b or wildtype ls174t cells low muc2 expressing cells data not shownwe confirmed the association of muc2 expressionand upr pathway activity by demonstrating a decreasein grp78 bip levels following stable muc2 knockdown muc2 kd in ls174t cells compared to lentiviral lv control cells fig 1c these data demonstratehigher basal ers levels in mcchigh muc2 producingcells compared to nmcclow mucin producing cellsand suggest a correlation between muc2 expressionand cellular basal ers levels these results support ourhypothesis that mcc with high basal ers may be susceptible to ersaggravation as a therapeutic strategy toinduce ersmediated apoptosisers is aggravated by dual celecoxib plus orlistat drugtherapy in mcc and correlates with muc2 expressionlevelswe demonstrated a dosedependent increase in uprprotein grp78 bip levels in ls174t cells when exposed to increasing doses of celecoxib μm ororlistat μm for h consistent with theirknown role as ers aggravators fig 2a similarlymrna expression levels for grp78 bip chop andatf4 increased in ls174t cells exposed to combinationof celecoxib μm and orlistat μm for h figs2b explant tissues derived from mcc demonstratedsimilaratf4 andchop following combination therapy for h fig 2cwe demonstrated a significant increase in upr proteinsgrp78 bip atf4 peif2 [phosphorylatedeukaryotic initiation factor ] and chop in dntcf4ls174t cells exposed to doxycycline for h highincrease in upr protein levels 0cdilly orphanet of rare diseases page of fig see legend on next page 0cdilly orphanet of rare diseases page of see figure on previous pagefig mucinous colon cancers and high muc2 producing cell lines exhibit elevated basal ers that correlates with muc2 expression levels arepresentative pictures from immunofluorescence if analysis of human explant tissue comparing muc2 and ers proteins grp78 [bip] atf4chop between mucinous colon cancer mcc and nonmcc nmcc the bar graphs demonstrate mean intensity difference brepresentative pictures from if analysis of muc2 and ers proteins grp78 [bip] atf4 chop comparing highmuc2 expressing cells dntcf4ls174t cells exposed to doxycycline for h versus lowmuc2 expressing cells dntcf4ls174t cells without doxycycline exposure the bargraphs demonstrate mean intensity difference c representative pictures from if analysis of muc2 and grp78 bip comparing stable muc2knockdown kd in ls174t cells compared to lentiviral lv control cells western blot shows muc2 protein expression in muc2 kd cellscompared to lentiviral control cells error bars represents standard deviation sd from triplicate experiments p muc2 expressing cells following treatment with com μm and celecoxib μmbination of orlistatcompared to low mucin producing dntcf4ls174tcells lacking doxycycline exposure conversely combination therapyinduced ers aggravation was significantlyreduced following stable muc2 kd in ls174t cellscompared to lv control cells we found much higherlevels of grp78 atf4 peif2 and chop in lv controlcells exposed to dual drug therapy compared to levelsseen in muc2 kd cells figs 2d these results suggestthat the degree of druginduced ers aggravation correlates with muc2 expression levels and that high mucinproducing cells are more susceptible to drugmediateders aggravationtreatmenttreatment of ls174t cells with celecoxib μm ledto rapid decrease in ercytoplasmic calcium ratio at s consistent with inhibition of calcium atpase channelon the er membrane and the likely mechanism for ersaggravation fig 2e we evaluated changes in muc2sinceprotein secretion following orlistatfasn is essential for the posttranslationallipidationand secretion of muc2 protein we found that orlistat μm inhibited muc2 secretion from cos7 cellstransfected with psnmuc2mg vectorexpressingmuc2 nterminal fig 2f mechanistically we foundthat exposure to orlistat reduced fasn enzyme activitybut not enzyme expression levels fig 2g h moreover we performed abe assay in cos7 cells stably expressing muc2 nterminal our data demonstrated asignificant reduction in muc2 nterminal palmitoylation and secretion following orlistat therapy a likelymechanism for ers aggravation fig 2iers aggravation by celecoxib plus orlistat combinationtherapy induces ersmediated apoptosiswe treated ls174t cells with celecoxib μm andorlistat μm alone and in combination at varying doses for h fig 3ac the ic50 doses for celecoxib alone and orlistat alone were μm and μm respectively cell viability was significantly reduced at h following combination therapy with celecoxib μm and orlistat μm demonstrating acombination index of calculated using the computer software compusyn suggested synergy betweenin mitochondrialthe two drugs in reducing cell viability combinationtherapy induced significant apoptosis as demonstratedby tunel assay at h figs 3d ls174t cells exposedto combination therapy with celecoxib μm and orlistat μm for h demonstrated significant increasein upr proteins grp78 bip and chop proapoptoticmolecules bim noxa and puma and activatedcleaved caspase 3parp1 while single drug therapy atthese doses had minimal effect figs 3e we assessedchangestransmembrane potentialδÏm following dual drug therapy using mitochondrialmembranepermeant fluorescence dye jc1 jc1 aggregates reduced redfluorescent jaggregates were significantly reduced following cotreatment consistent withmitochondrial membrane damage and activation of intrinsic apoptosis fig 3f involvement of intrinsic mitochondrial apoptotic pathway was confirmed by cleavageof caspase but not caspase following dual drugtherapy fig 3g we also confirmed the induction ofupr and apoptotic proteins in colonoid cultures and explanttissue exposure to combination of celecoxib μm and orlistat μm for h resulted in apoptosis tunel assay in colonoid cultures fig 3h andmuch higher chop and cleaved caspase levels in explant tissue from mcc compared to nmcc suggestingtheirto ers aggravation and ersmediated apoptosis fig 3isusceptibilitysimilarly we demonstrated a significant increase inapoptotic markers puma cleaved caspase 3parp1in dntcf4ls174t cells exposure to doxycycline for h high muc2 expressing cells following combinationtreatment with orlistat μm and celecoxib μmcompared to low mucin producing dntcf4ls174tcellslacking doxycycline differentiation converselycombination therapyinduced apoptosis was significantlydampened following stable muc2 kd in ls174t cellscompared to lv control cells we found high levels ofpuma cleaved caspase 3parp1 in lv control cellsexposed to combination of orlistat and celecoxib whilethese were suppressed in muc2 kd cells figs 3j ourresults showed that combination treatmentinducedapoptosis correlated with muc2 expressionto determine whether combination therapy inducedapoptosis was ers dependent we tested this drug 0cdilly orphanet of rare diseases page of fig ers is aggravated by dual celecoxib plus orlistat drug therapyin mucinous colon cancer and correlates with muc2 expressionlevels a western blot assay of ers protein grp78 bip followingtreatment of ls174t cells with celecoxib μm or orlistat μm for and h qpcr assay of mrna expression for ersmarkers following treatment with celecoxib orlistat or combinationfor h in ls174t cells b and mcc explant tissue c representativepictures of if assay with bar graph demonstrating mean intensitydifference d western blot assay of ers markers comparing highmuc2 expressing cells dntcf4ls174t cells exposed to doxycyclinefor h versus low muc2 expressing cells dntcf4ls174t cellswithout doxycycline exposure and comparing stable muc2 kd inls174t cells compared to lv control cells following treatment withcelecoxib orlistat or combination for h e ratio of ercytoplasmiccalcium concentration at s in ls174t cells following combinationtreatment celecoxib orlistat f elisa assay of muc2 secretionfrom cos7 cells transfected with psnmuc2mg vector expressingmuc2 nterminal following treatment with celecoxib orlistat orcombination for h g fasn enzymatic activity assay in ls174tcells following combination treatment celecoxib orlistat for hand h western blot analysis of fasn expression levels i nterminalmuc2palmitoylation in cos7 cells stably expressing muc2 nterminal following single or dual drug therapy for h wasdetermined by abe assay and quantified by western blot assayhydroxylamine ham a strong reducing agent that cleavespalmitate from cysteine residues is necessary for biotinylation theomission of ham cleavage ham serves as negative control errorbars represents standard deviation sd from triplicate experiments p p p combination in chop knockdown ls174t cells chopkd we demonstrated a significant reduction in cleavedcaspase 3parp1 in chop kd cells compared to lvcontrol cells fig 3kcombination of celecoxib and orlistat reduced mucinoustumor growth in vivowe evaluated the therapeutic efficacy of this combination in vivo using ip pdx models ofluciferaselabelled ls174t cells seven days following ip tumorinoculation animals were treated ip with vehiclepbs celecoxib c alone mgkg orlistatoalone mgkg or combination of celecoxib andorlistat c o every other day for weeks animals per group drug dose selection for ip celecoxiband orlistat was based on prior publications and ourpilot studies [ ] treatmentrelated drug toxicitywas not encountered in the in vivo experiments dualdrug therapy resulted in significant reduction in mucinous tumor growth compared to either drug aloneas demonstrated by luciferase intensity measured byivis bioluminescent imaging system fig 4a tumortissue harvested from euthanized animals following weeks of therapy demonstrated a significant increasetunel positive staining with dualin apoptosistherapy fig 4b changesin upr protein levelschop and atf were consistent with those seen inin vitro studies fig 4c 0cdilly orphanet of rare diseases page of fig ers aggravation by celecoxib plus orlistat combinationtherapy induces ersmediated apoptosis mts cell proliferation assayin ls174t cells treated with increasing doses of celecoxib μm a or orlistat μm b or combination of celecoxib andorlistat for h c representative pictures of tunel assay in ls174tcells treated with single or dual drug therapy for h d westernblot analysis for ers and apoptotic markers at h e f ls174t cellswere treated with single or dual drug therapy and stained with jc1diffuse green jc1monomers indicate mitochondrial depolarizationdamage and punctate red jc1aggregates indicates intactmitochondrial membrane potential δÏm g western blot assay forcaspases in ls174t cells following single and dual drug therapy for h h representative pictures of tunel assay in colonoid culturesfrom mcc following single and dual drug therapy for h the bargraph demonstrates mean intensity difference i representativepictures from immunofluorescence if analysis of human explanttissue comparing caspase and chop between mcc and nmccfollowing single and dual drug therapy for h the bar graphdemonstrates mean intensity difference j western blot assay ofapoptotic markers comparing highmuc2 expressing cells dntcf4ls174t cells exposed to doxycycline for h versus lowmuc2expressing cells dntcf4ls174t cells without doxycycline exposureand comparing stable muc2 knockdown kd in ls174t cellscompared to lv control cells following treatment with celecoxiborlistat or combination for h k western blot assay of chop andapoptotic markers comparing stable chop knockdown kd inls174t cells compared to lv control cells following treatment withcelecoxib orlistat or combination for h asterisk represents astatistically significant difference compared with the control group p p discussionmcc are a unique histologic subtype in which greaterthan of the tumor mass is composed of extracellularmuc2 protein they demonstrate unique clinicalphenotype and molecular genotype when compared totheir nonmucinous counterparts nmcc clinicallymcc are more likely to occur in younger patients havea predilection for the proximal colon are bulkier at presentation and have a higher propensity for peritonealand distant lymph node metastases molecularly theytend to follow the serrated pathway for carcinogenesisand are more likely to have rasgnas mutationsmicrosatellite instability msi and cpg island methylator phenotype cimp positive [ ]in general many moleculartargeted therapies havedemonstrated disappointing results against advancedsolid cancers in clinical trials despite promising preclinical data this may largely be attributed to irrational useof targeted drugs against unselected cancers major considerations to improve the efficacy of targeted therapiesinclude identification of ideal drugs and drug combinations optimizing dosing schedules and enriching for patient factors eg clinical and molecular phenotypes andgenotypes that are more likely to respond to specifictargeted therapies in this study we focused on specificvulnerabilities of mucinous tumors that may make themsusceptibletherebytargeted therapiesto specific 0cdilly orphanet of rare diseases page of fig see legend on next page 0cdilly orphanet of rare diseases page of see figure on previous pagefig combination of celecoxib and orlistat reduced mucinous tumor growth in vivo murine xenograft models of intraperitoneal ip luciferaselabelled ls174t cells were treated with pbs alone control or celecoxib c alone mgkg bw or orlistat o alone mgkg bw orcombination of c o every other day starting on day following tumor implantation until they were euthanized at day gross intraabdominal tumor burden is depicted pictorially on day serial weekly changes in mean luciferase intensity during treatment for weeks areshown a error bars represent standard error of the mean among the xenografts in each treatment group harvested tumor tissue fromeuthanized mice day was subjected to tunel if assay b and if for ers markers c the bar graphs demonstrate mean intensity differenceasterisk represents a statistically significant difference compared with the control group p p applying a rational approach to treating wellselectedcancers we hypothesized that high basal ers of mccwould make them vulnerable to ers aggravation andersmediated cell death processes in this study we aggravated ers via two wellestablished pathways sercainhibition celecoxib to impair normal calcium homeostasis and fasn inhibition orlistat to exacerbate protein misfolding by inhibiting normal muc2 secretionour rationale for utilizing celecoxib was that serca is aknown noncox2 target of celecoxib and would therefore aggravate ers while at the same time celecoxib hasbeen shown to inhibit muc2 expression and decreasemucinous tumor growth in ex vivo and in vivo modelsof mucinous colonappendix cancers [ ] ourrationale for utilizing orlistat was that it inhibits fasnrequired for muc2 secretion which would induce ersthe same time reducing mucinous tumorwhile atgrowth [ ] moreoverthere is considerablecrosstalk between cox2 and fasn signaling pathwaysfasn plays a role in the conversion of excess carbohydrates to arachidonic acid a polyunsaturated fatty acidcox2 is responsible for converting arachidonic acid toprostanoids and noncox2 target pdk1 regulatesfasn activity [ ] both cox2 and fasn activitiesmodulate cancer signaling and have been shown tomodulate cell proliferation and survival [ ]in this study we first demonstrated that mcc andhigh muc2 producing cells dntcf4ls174t exposedto doxycycline had elevated basal ers levels comparedto nmcc and low muc2 producing cancer cells andthat the mucinous nature of these tumors excessivemuc2 protein production was responsible for the highbasal ers levels this laid the foundation for our targeted therapeutic approach since severe and persistenters ers aggravation can overwhelm normal protectivemechanisms upr to cope with this excessive stress andtrigger molecular pathways associated with cell deathwe then tested the therapeutic efficacy of dual drugtherapy with celecoxib and orlistat since they are bothers aggravators have demonstrated preclinical efficacyagainst a variety of cancers have mechanisms of actionthat would inhibit muc2 protein expressionsecretionand target cox2fasn cancer pathways with significant crosstalk ers aggravation as a potential mechanism to induce cell death in cancers has been tested incancer models by other investigators and we felt this approach would be especially effective in mucinous tumorsgiven the elevated basal ers levels ie rational application in wellselected tumors celecoxib is an fda approved drug used to prevent cancer progression in highrisk patients with familial adenomatous polyposis fapand continues to be investigated in clinical trials to improve the efficacy of standard chemotherapeutic drugs[ ] orlistat a reduced form of the natural productlipstatin is fda approved as an oral antiobesity drugsince it inhibits gastricpancreatic lipases and therebydecreases absorption due to its poor oral bioavailabilityintravenous formulations of orlistat are under development and have shown efficacy in preclinical modelswhile other fasn inhibitors continue to be tested asanticancer drugs in clinical trials in our studycombination treatment celecoxib orlistat aggravateders and synergistically induced markers associated withapoptotic cell death in mcc explants tissues and mccderived colonoid cultures treatmentinduced cell deathwas ersdependent since proapoptotic cell death pathways were not activated following knockdown of ersprotein chop dual drug treatment significantly reduced mucinous tumor growth in vivo and inducedmarkers of ers and apoptosis consistent with in vitroexperimentsour study has a number of limitations mechanistic studies assessing changes in ubiquitinproteosomeand autophagy markers following ers aggravationwould be importantto elucidate cellular changesthat underlie transition from cytoprotective upr tocytodestructiveapoptotic pathways such studieswould also help quantitate the magnitude of ers aggravation required to induce this transition additional studies with other ers aggravating agentswould confirm findings of this study and the proposed therapeuticthein vivo studies celecoxib and orlistat were administered ip which is not ideal for clinical trials whilecelecoxib may be given orally orlistat has poor bioavailability intravenous formulations of orlistat havebeen developed and other parenterally administeredfasn inhibitors are in clinical trials these alternate drugs and routes of administration will needto be tested in our modelsapproach for mcc for 0cdilly orphanet of rare diseases page of sin summary we found that combination of celecoxiband orlistat significantly aggravated ers and this wasmore pronounced in mcc and high muc2 producingcells dntcf4ls174t exposed to doxycycline compared to nmcc and low muc2 producing cancers andthat the mucinous nature of these tumors excessivemuc2 protein production was responsible for the highaggravated ers levels we also demonstrated that ersaggravation by dual therapy with celecoxib and orlistatinduced apoptosis and suppressed mucinoustumrowth providing a preclinical rationale for the use ofthis therapeutic strategy in the clinical settingrockfordmaterials and methodsreagentsdmem dulbeccos modified eagles medium was obtained from invitrogen carlsbad ca fetal bovineserum fbs was obtained from hyclone laboratorieslogan ut cellculture plates were purchased fromdenville scientific charlotte nc celecoxib and orlistat were obtained from cayman chemical ann arbormi cell titer aqueous one solution cell proliferation assay was obtained from promega madison withe enhanced chemiluminescence reagents ecl kitand pierce bca protein assay were obtained from thermoscientificil bd pharmigen fitcannexin v apoptosis detection kit i was obtained frombd biosciences san jose ca control muc2 andchop shrna lentiviral ps were obtained fromsanta cruz biotechnology santa cruz ca for westernblotting betaactin a1978 was obtained from sigmaaldrich st louis mo parp1 cst 46d11 grp78cst c50b12 atf4 cst d4b8 puma cstd30c10 noxa cst d8l7u chop cstl63f7and cleaved caspase 3cst asp175 were obtainedfrom cell signaling technologiesdanvers masytox orange for nucleic acid labeling was obtainedfrom life technologies grand island ny antirabbitand antimouse horseradish peroxidase hrpconjugated secondary antibodies were purchased from jacksonimmunology research west grove pacell culture and treatmentls174t cell line muc2 producing colon cancer cellswith goblet celllike characteristics was obtained fromamerican type culture collection high muc2 producing cells dntcf4 cell line under teton control system was kindly gifted by hans cleversutrechtnetherlands are able to differentiate into gobletlikecells following doxycycline induction h cellswere grown in dmem supplemented with fetal bovine serum 100iu penicillin and μgml streptomycin in co2 at °c in culture platesstable cell line generationls174t cells were incubated with muc2 or chopshorthairpin rna shrna h lentiviral ps and μgml final concentration polybrene sigmaaldrich following h of incubation the medium wasreplaced with complete dmem for h and then puromycin was added at a final concentration of μgmlcell were subcultured for weeks under puromycin selection to eliminate nontransduced cellscos7 cells were transfected with psnmuc2mgvector expressing muc2 nterminal a gift from gunnar hansson gothenburg sweden followingovernight incubation the medium was replaced withcomplete dmem for h and then g418 was added at afinal concentration of microgram per ml to eliminate nontransfected cellsconfocal imagingtumortissues were embedded in oct mediumcontaining cryomolds and immediately frozen in methylbutane then μm frozen tissue sections werecut using a cryostat and layered on super frost plusslides cells were grown on glass coverslips in wellplates for in vitro experiments the covers were incubated in paraformaldehyde for min and thenwashed and blocked for min at room temperaturethe cells were then incubated with muc2 or grp78 orchop antibody they were then washed times with1x pbs and incubated with antirabbit alexa andantimouse alexa and sytox orange for nucleicacid staining at room temperature for min repeat times washing with 1x pbs was performed glass slipswere mounted on slides using proliong gold antifadesolution from invitrogen life technologies grand island ny confocal images were taken from differentfields at random at x63 magnification using a leicaconfocal tcs sl dmre microscopecell proliferation assayscell lines were counted and plated in a 96well plateovernight the next day the cells were treated with varying concentrations of celecoxib and orlistat for or h following this treatment cell viability was determined by celltiter aqueous one solution cell proliferation mts assay according to the manufacturersprotocol promega madison wi cells were incubatedwith the combined solution of a tetrazolium compoundmts [345dimethylthiazol2yl53carboxymethoxyphenyl24sulfophenyl2htrazolium inner salt] andelectron coupling reagent pms phenazine methosulfatefor h at °c the absorbance of the product was measured at mm directly with an enzymelinked immunosorbent assay plate reader all cell treatments wereperformed in triplicate 0cdilly orphanet of rare diseases page of apoptosis analysiscells were counted and plated into a well plate for h cells were then treated with varying concentrations of celecoxib and orlistat alone and in combinationfor to h following treatment cells and mediumwere collected in ml culture tubes and apoptosis wasanalyzed using flow cytometry and bd pharmigen fitcannexin v apoptosis detection kit i bd biosciencessan jose ca per manufacturers protocol briefly cellswere washed twice with cold pbs and then resuspendedin μl of 1x binding buffer five μl of both fitcannexin v and propidium iodide pi were added twoeach tube for each cell line one tube was collected andresuspended in μl 1x binding buffer and incubatedunstained without annexin v and pi or with onlyannexin v or pi cells were incubated for min atroom temperature in the dark following this the samples were analyzed by flow cytometry using an accuric6 flow cytometer combination index was measuredusing the compusyn software combo syn inc paramus njcollected bywestern blottingcells were treated for h with varying concentrations of celecoxib and orlistat alone or in combination following treatment cells were scrapped andcollected in ml conical tubes and kept on ice acell pellet wascentrifugation andwashed twice with cold pbs cell pellets were resuspended in 1x ripa solution cell signaling technology with 1x protease inhibitorcomplete minisigma aldrich in pbs and lysis was performed usingsonication and then centrifuged for min atÃg at °c supernatant was collected proteinconcentration was determined in nonreduced samples using bca reagent thermo scientific proteinwas run on sds gels miniprotean tgxgels bio rad an | 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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0cmatsuoka bmc nephrology page of 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 |
this study hypothesizes that bromelain bl acts as radiosensitizer of tumor cells and that it protects normal cells from radiation effects in vitro and in vivo studies have been carried out to prove that assumption in vitro mtt cell proliferation assay has shown that the irradiated ehrlich ascites carcinoma eac cell line could be sensitized by bl pretreatment in vivo animals were randomly divided into groups group control pbs ip for days group ehrlich solid tumor est bearing mice group est Îradiation fractionated dose gy group est bl mgkg ip daily for days group est bl for days followed by Îirradiation gy the size and weight of tumors in gammairradiated est bearing mice treated with bl decreased significantly with a significant amelioration in the histopathological examination besides bl mitigated the effect of Îirradiation on the liver relative gene expression of poly adp ribose polymerase1 parp1 nuclear factor kappa activated b cells nfκb and peroxisome proliferatoractivated receptor α pparα and it restored liver function via amelioration of paraoxonase1 pon1 activity reactive oxygen species ros content lipid peroxidation lpo and serum aspartate transaminase ast alanine transaminase alt and albumin alb it is concluded that bl can be considered as a radiosensitizer and radioprotector suggesting a possible role in reducing radiation exposure dose during radiotherapykeywordsbromelain tumor Îradiation radiosensitizer radioprotectorsubmitted april revised july accepted july introductionradiotherapy has been used for a long time in treating cancer1 however from the clinical perspective radiotherapy provides inadequate success due to the radioresistance of many tumors as well as the high risk of recurrence and effects on normal cells may occur23 radioresistance occurs as the microenvironment of solid tumors is hypoxic compared with normal tissue4 in addition some tumors have either an intrinsic resistance to ionizing radiation or can attain this property through accumulation of genetic mutations causing an increased survival and proliferation5 thus strategies to improve radiation therapy could include increasing resistance of normal tissues to radiation andor increasing sensitivity of the tumor cells6radiosensitizing agents increase the sensitivity of tumor cells via enhancing the generation of reactive oxygen species ros increasing lipid peroxidation depletion of glutathione which leads to dna damage inhibition of dna repair inhibition of dna synthesis induction of cell cycle arrest induction of apoptosis and inhibition of proliferation7 numerous nutritive cancer chemopreventive compounds having antioxidant properties have been recognized to potentiate radiation therapyinduced cytotoxic 1drug radiation research department national centre for radiation research and technology egyptian atomic energy authority nasr city cairo egypt2biochemistry department alazhar university cairo egyptcorresponding authorhanan a fahmy drug radiation research department national centre for radiation research and technology atomic energy authority p o box nasr city cairo egypt email fahmyhananyahoocomcreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c integrative cancer therapies effects on cancer cells inversely decreasing its toxicity on normal adjacent tissues89 in this regard much research has aimed to develop numerous antioxidant drugs of both natural and synthetic origin tested in both in vitro and in vivo models and also human clinical trials to overcome injuries caused by ir exposure and to induce killing of cancer cells at the same time previous studies have reported that phytochemical soy isoflavones genistein daidzein and glycitein which exhibit anticarcinogenic properties through their antioxidant activities could be used as potent radiosensitizers to enhance the efficacy of radiotherapymediated suppression of the growth and metastatic ability of cancers1011 along parallel lines resveratrol and piperine which possess antitumor activity have been shown to augment ionizing radiation irinduced apoptosis and loss of mitochondrial membrane potential in murine colon carcinoma and melanoma cells via enhancing irinduced ros generation12 moreover pentoxifylline ptx a methylxanthine that possesses antioxidant properties is known for improving tumor tissue oxygenation in murine hypoxic tumors and inhibiting post radiation induced normal tissue injury in mice1314 consequently searching for a natural product possessing anticancer activity that increases radiosensitivity of tumor cells and radioresistance of normal cells may lead to a potential future drug in cancer therapyamong the natural products bromelain bl extract attracts interest due to its anticancer antioxidant as well as antiinflammatory effects1517 bl an extract from pineapple stem ananas comosus belongs to a group of protein digesting enzymes it is a mixture of diï¬erent thiol endopeptidases and other components like phosphatase glucosidase peroxidase cellulase escharase calcium and several protease inhibitors1819 the anticancer activity of bl has been examined in various types of gastrointestinal and breast cancers cell lines in in vivo models bl has shown antimetastatic effect and reduction in local tumor growth2023 it is also used for reducing the severity of such radiation therapy side effects as mucositis skin reactions and dysphagia in patients24 hence this study was aimed to evaluate the radiosensitizing and radioprotective effect of bl using in vivo and in vitro approachesmaterials and methodin vitro studiesmtt cell proliferation assay the growth and viability of ehrlich ascites carcinoma eac cell line were tested in vitro by 345dimethylthiazol2yl25diphenyltetrazolium bromide mtt assay according to freimoser and buch 2526 to verify the antitumor and radiosensitizing effect of bl two plates were designed for this study the first one contained eac cells maintained by serial subculturing at the national cancer institute egypt incubated for hour before irradiation irr gy alone and with different concentrations of bromelain bl in phosphate buffer saline pbs the second one contained eac cells serving as a control and eac with different concentrations of bl each test was seeded in triplicate into a plate at concentration of cellswell containing rpmi media with fbs nahco3 uml penicillin and µgml streptomycin and each plate was incubated for hours at °c in co2 and humidity atmosphere then μl mtt reagent bio basic inc canada was added over the cells in each well and the plate was incubated in the dark for to hours until a purple precipitate was seen and the absorbance was measured at nm the amount of color produced was directly proportional to the number of viable cells viable cell a samples a blanka control a blank the inhibitory concentration ic50 is the dose of a drug which reduces the viability to and was calculated using nonlinear regression analysisfree radical scavenging assay the antioxidant activity of bromelain was evaluated by 1diphenyl2picrylhydrazyl dpph radical assay and its scavenging power was compared with some antioxidants naringin polyphenolic antioxidant garlic oil and glutathione sulfur containing antioxidants about µl of samples mgml dissolved in dist water was added to µl of a solution of dpph g100 ml dissolved in vv methanol after minutes incubation at room temperature in the dark the absorbance was read at nm against a blank µl dist water µl dpphmethanol solution the experiments were done in triplicate according to the method of braca 27 glutathione mgml was used as a standard antioxidant the scavenging percentage of dpph was calculated according to the followscavengin ing equation where b was the absorbance of the blank and a was the absorbance of samples or standard ec50 is defined as concentration of sample that causes dpph loss there values were calculated using nonlinear regression analysisb ab\uf8ee\uf8ef\uf8f0\uf8f9\uf8fa \uf8fbin vivo studiesradiation processing whole body Îirradiation of mice was carried out using gamma cell40 137cesium manufactured by the atomic energy of canada limited ontario canada installed in the national center for radiation research and technology ncrrt cairo egypt the dose rate was gymin during the experimental period daily correction for humidity barometric pressure and temperature were madeanimals adult female swiss albino mice weighing to g obtained from the breeding unit of ncrrt cairo egypt all animal procedures were performed in accordance with the committee of scientific ethics at faculty of 0cmekkawy table sequences of primers for realtime quantitative pcrgeneparp1 nm0074152nfκb nc0000696pparα nc0000816βactin nc0000716forward primerreverse primer²ccatcgacgtcaactacga3²²caatggctacacaggacca3²²actccacctgcagagcaacca3²²gcgtggggacagccgcatctt3²²gtgcgtggtagcatgagtgt3²²cactgtcacctggaaccaga3²²tagatctcctgcagtagcggg3²²atcggcagaaggggcggaga3²pharmacy alazhar university egypt following the guidelines for animal use the animals were housed in colony cages micecage under proper environmental conditions that is hours darklight cycle good ventilation condition and temperature to humidity at the ncrrt animal house fed with standard diet pellets and provided with water ad libitum animals were left week for acclimatization on the lab environment before starting the experimenttumor transplantation the eac cell line was supplied by serial subculturing at the national cancer institute cairo university egypt it was implanted in each donor female swiss albino mice by ip injection of cells22 g b wt and allowed to multiply28 the ehrlich solid tumor est was obtained by the intramuscular inoculation of ml of viable eac in the right lower limb of each mouse29 mice with a palpable solid tumor diameter mm3 that was maintained within to days after inoculation were used in the studyanimal grouping animals were randomly divided into groups mice each group control not bearing tumor received pbs ip for days group ehrlich solid tumor est bearing mice received pbs ip for days group est Îirradiation gy fractionated doses starting days after tumor appearance mm3 and lasting for days group est bearing mice receiving freshly prepared bl dissolved in pbs mgkg ip daily for days according to pilot study starting once est becomes mm3 bl was purchased from merck kgaa co darmstadt germany group est bearing mice received bl as in group hours before Îirradiation as in group mice were anesthetized days after last irradiation dose using urethane mgkg30 blood samples were collected through cardiac puncture and divided into parts edta coated and plain vials at that time they were euthanized by cervical dislocation liver and tumor tissues were dissected out rinsed with icecold saline dried on a filter paper and weighed then homogenized in icecold pbs ph and stored at °c until used for subsequent biochemical analysisestimation of total body tumor and liver weights animals in each group were checked daily for any adverse clinical symptoms and deaths after to days post inoculation with eac body weights were recorded so body weight change could be estimated tumor and liver weights were measured during sample collection and then the tumor inhibitory ratio was calculated by the following formula inhibition ratio aba where a is the tumor weight average of the control and b is that of the treated group also relative liver weight was calculated as liver weighttotal body weight histopathological examination three tumors of each group were collected and fixed in neutral buffered formalin the specimens were dehydrated in ascending grades of ethyl alcohol cleared in xylene and embedded in paraffin wax four micron thick paraffin sections were mounted on clean slides stained with ehrlichs hematoxylineosin he31 and examined using an olympus microscope bx41 hamburg germany histopathological evaluation was done by assessment of necrosis and calculation of tumor area percentage using image analysis software image j 146a nih usa through the following equation of tumor area area of tumortotal area of the field molecular analyses the mrna levels of poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα genes and of the housekeeping gene βactin were measured by real time polymerase chain reaction rtpcr total rna was isolated from liver tissues using qiagen tissue extraction kit qiagen usa in accordance with the manufacturers instructions the extracted rna μg was used for cdna conversion using high capacity cdna reverse transcription kit fermentas usa and μl reaction volume sybr chemistry in applied biosystems thermal cycler usa to amplify pcr under the following conditions °c for denaturation then °c to °c for annealing using primers mentioned in table and °c for elongationresults were expressed using the comparative ct method for relative mrna quantification of target genes normalized to an endogenous reference βactin and a relevant control equal to ct ct is the difference between the mean ctsample and mean ctcontrol where ctsample is the difference between the mean ctsample and the mean ctβactin and ctcontrol is the difference between the mean ctcontrol and the mean ctβactin 0c integrative cancer therapies estimation of lipid peroxidation lpo reactive oxygen species ros and paraoxonase pon1 in liver homogenate liver lipid peroxidation was estimated by measurement of malondialdehyde mda formation using the thiobarbituric acid method of yoshioka 32 a modified technique of vrablic 33 was used to measure the generation of ros by the intracellular conversion of nitro blue tetrazolium nbt to formazan by the action of superoxide anion paraoxonase activity was estimated by using fluorometric assay enzchek® kit invitrogen uk for the anophosphatase activity of paraoxonase based on the hydrolysis of a fluorogenic anophosphate analog34hematological and biochemical analyses whole blood was immediately analyzed for complete blood count with platelet count using the fully automated analyzer abx cobas micros roche germany estimation of serum alanine aminotransferase alt aspartate aminotransferase ast and albumin alb assays follow the recommendations of the international federation of clinical chemistry ifcc but were optimized for performance and stability using the rochehitachi cobas c 311systemstatistical analysis the statistical analysis was performed using oneway analysis of variance anova and the groups were compared by tukeykramer test viability percentage at different concentrations and body weight change analyzed by twoway anova followed by bonferronis posttest graphs were sketched using graph pad prism isi® software usa version software data were presented as mean ± standard error se and p values considered significantresultsin vitro studieseffect of bromelain and gammairradiation blirr on tumor cell growth and viability the radiosensitizing effect of bl on eac cells was determined by performing mtt assay eac cells exposed to gy Îradiation showed high cell viability percentage reflecting a radioresistance of eac cell line while bl treatment showed in vitro cytotoxic activity with ic50 value of mgml however the maximum cytotoxic effect appeared when the eac cells were subjected to bl then Îradiation gy compared to control or irradiated group with ic50 mgml table effect of bromelain and some natural compounds as free radical scavengers the inhibitory percentage of each compound is shown in figure the ec50 value concentration of sample that causes dpph activity loss is a reliable way for estimation of the radical scavenging activity the ec50 value of glutathione referenced antioxidant is mgml while table cytotoxic activity of blirr against eac cell line bromelain concentration mgmleac bl mgmlic50 mgmlviability non irradiated eac irradiated eac48ab59ab60ab69a787a10ab158ab18ab27ab52ab ± ± each value indicates the mean of records statistical analysis carried out by twoway anova followed by bonferroni posttests a significant versus control ehrlich ascites carcinoma eac group where b significant versus irradiated eac group at p ic50 ± se values were calculated by using nonlinear regression analysisbromelain and garlic oil ec50 are almost equal and mgml respectively however the naringin phenolic antioxidant is the least potent one ec50 mgml in this comparisonin vivo studieseffect of bromelain and gammairradiation blirr on tumor weight and volume table shows a significant decrease in tumor weight in groups treated with bl andor Îirradiation as compared to the est nontreated group the more drastic decrease in the tumor weight ratio observed in the combined therapy group bl irr compared with the estirradiated group as well as est group indicates that combination therapy is more significantly effective than single agent therapy the photograph of est xenografts at the time of sacrifice shows the synergistic effect of bl and irr on tumor volume figure effect of bromelain and gammairradiation blirr on tumor histopathological features of est bearing mice histopathological examination of solid tumor sections revealed typical malignant features including sheets of malignant cells infiltrating adjacent muscular tissue the malignant cells show pleomorphism hyperchromatism and mitotic activity while the necrotic cells appear as nonviable homogenous structureless material with degenerated or karyorrhectic nuclei untreated est bearing group shows a deeply stained tumor cells arrow head and areas of necrosis arrow figure 3a also it displays intact cancer cells arrow and giant cells arrow figure 3b and c the estirradiated group shows muscle fibers invaded by deeply stained tumor cells arrow head and large areas of necrosis arrow figure 3d also displays a notable necrosis of cancer cells n figure 3e the bl treated group shows a 0cmekkawy figure dpph 1diphenyl2picrylhydrazyl reduction curve for glutathione bromelain naringin and garlic oil each value represents mean ± se all experiments were replicated timestable tumor weight and inhibition ratio of ehrlich solid tumor estbearing mice treated with gammairradiation irr gy andor bromelain bl mgkggroupsestest irrest blest bl irrtumor weight g ± ± 004a ± 005a ± 005abtumor inhibitory ratio ± 24a ± 14ab ± 204ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe values shown are mean ± se of data a significant versus est group where b significant versus estirradiated group at p figure a photograph of ehrlich solid tumor est xenografts at the time of scarification showing the effect of bromelain and gammairradation blirr on tumor volume e ehrlich solid tumor e ir ehrlich solid tumor irradiation e br ehrlich solid tumor bromelain e ir br ehrlich solid tumor bromelain irradiation 0c integrative cancer therapies figure photo micrograph of ehrlich solid tumor est xenografts in different animal groups est sections show the degree of tumorogenesis necrosis n regression of tumor by appearance of muscle fibers m and a b c est ehrlich solid tumor d e est irr ehrlich solid tumor irradiation f g est bl ehrlich solid tumor bromelain h i est bl irr ehrlich solid tumor bromelain irradiationwide area of necrosis arrow and n few groups of cancer cells arrow head and muscle fiber m figures 3f and 4g however combined treatment bl irr displays muscle fiber m significant regression of tumor or wide areas of necrotic cancer cells n and few groups of intact cancer cells arrow figure 3h and 3i the tumor area percentage per total tissue area could determine the degree of proliferation as seen in figure there is a great regression of tumor area in the group treated with bl alone or bl and irr compared with untreated est or estirradiated group indicating that combination therapy significantly more effective than single agent therapy 0cmekkawy bl irr shows nonsignificant group change additionally bl irr group significantly upregulated pparα expression compared with est and estirradiated groups indicating that bl might have a hepato as well as radioprotective effect figure effect of bromelain and gammairradiation blirr on the hepatic lipid peroxidation lpo level reactive oxygen species ros content and paraoxonase1 pon1 activity of ehrlich solid tumor est bearing mice lpo in liver tissues significantly increased in all est bearing groups compared to the control group except the combined treated group irr bl succeeded in returning mda lpo measured as mda malondialdehyde level to the normal level however liver ros significantly increased only in untreated and Îirradiated est bearing groups when compared to the control group while a significant decrease in liver ros showed in estirradiated mice treated with bl in comparison with both est untreated and estirradiated groups pon1 activity in liver homogenate was significantly decreased in est untreated and estirradiated groups when compared with the control group bl treated groups revealed significant increases in pon1 when compared with both est untreated and estirradiated groups showing that bl might have a hepato and radioprotective effect figure effect of bromelain and gammairradiation blirr on hematological measurements wbcs and plts were significantly elevated while hgb and hct significantly decreased in the untreated estbearing mice in comparison with control mice however Îirradiation resulted in a significant decrease in wbcs rbcs plt hgb and hct compared with the control mice treatment of the estbearing mice with bl shows a significant amelioration in wbcs plt and hct compared to est untreated mice combined treatment bl irr shows an enhancement in wbcs plt and hct compared to est untreated and gammairradiated est bearing mice table effect of bromelain and gammairradiation blirr on the serum alanine transaminase alt aspartate transaminase ast and albumin alb to investigate the cytoprotective effects of bl against irradiation the levels of serum alt ast and alb were measured figure it was found that alt and ast significantly increased and conversely alb significantly decreased in est untreated and estirradiated groups compared with the control group however estbearing mice treated with bl alone show nearly the same result of alt and alb as control values estbearing mice treated with bl in combination with irradiation initiated a significant decrease in ast and alt as compared with estirradiated group which may reflect a potential hepatic radioprotective effect of blfigure percentage of tumor areatotal tissue area of ehrlich solid tumor est bearing mice treated with gammairradiation irr gy andor bl mgkg the values shown in the plotted area are mean of records from animals ± se significant versus est group where significant versus estirradiated group at p effect of bromelain and gammairradiation blirr on body weight change and relative liver weight regarding the day by day documented recording of body weight bwt illustrated in figure there is almost no change in bwt of bl treated group while it increases significantly in the untreated est group conversely estirradiated groups with or without bl treatment show a significant decrease in bwt when compared with the control group table relative liver weight was compared after normalization to mg body weight untreated estbearing group shows a significant increase in liver weight by hepatomegaly while nonsignificant changes were observed in bl treated groups compared to the normal group table effect of bromelain and gammairradiation blirr on the poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα relative gene expression of ehrlich solid tumor est bearing mice to test the possibility that bl reduces radiation damage to the liver mrna gene expression of parp1 nfκb and pparα was measured in the liver homogenates of est bearing mice and compared to control pbs treated mice the results illustrated in figure show that irr causes significant increases in parp1 and nfκb expression compared to the control group however combined treatment bl irr shows a significant increase in parp1 and nfκb expression compared to control group and a significant attenuation compared to estirradiated groupmoreover all est bearing groups show significant decreases in hepatic pparα relative gene expression compared to the control group except the combined therapy 0c integrative cancer therapies figure effect of bromelain and gammairradiation blirr on body weight during experiment period each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationtable change in body weight and relative liver weight of control mice and ehrlich solid tumor est bearing mice treated with gammairradiation irr gy andor bromelain bl mgkggroupscontrolestest irrest blest bl irrbody weight change ± ± 101a ± 217ab ± 201b ± 341abrelative liver weight ± ± 026a ± 022a ± 026a ± 026ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationeach value represents the mean ± se a significant versus control group where b significant versus est group at p body weight changes percent are related to the initial weight of animalsdiscussionresistance of tumor cells to chemoradiotherapy as well as the damaging effects to nearby normal tissues remains a major obstacle to successful cancer management therefore the current study has been conducted to estimate the effect of bromelain bl as a tumor radiosensetizer and to show to what extent it can protect normal tissue from radiation hazardsradiosensitizers are compounds that when combined with radiation therapy achieve greater cytotoxicity they can be determined in vitro by the mtt assay2635 the present study has found that the radioresistant eac cells could be sensitized when incubated with bl before irradiation it was known previously that in vitro treatment with bl on mouse tumor cell lines resulted in inhibition of cell growth and invasion capacities3637 the anticancer property of bl has been mainly attributed to the protease component through digestion and diffusion in tumor cells38 it may also be due to the bl enhancement of p53 expression as well as another activator of apoptosis eg bax39 in addition it decreases the activity of cell survival regulators such as akt and erk it also deactivates aktdependent proapoptotic regulator foxo3a thus promoting apoptotic cell death in tumors40it is well known that during cancer and radiotherapy excessive energy is used from the host41 ultimately contributing to mechanisms that promote loss of weight as shown in the present study which also showed that bl could return body weight to a normal level by decreasing tumor weight and volume currently the combined therapy bl irr has been shown to be more effective than single agent therapy in reducing tumor volume and weight indicating that bl could possess a radiosensitizing effect in addition the combined therapy has revealed a drastic decrease in tumor area percentage wide areas of necrotic cancer cells and presence of muscle fiber in the histopathological examination compared with the control est and estirradiated groups this seems to be in agreement with other findings of the role of bl in reducing metastasis and local tumor growth2342 in chemically induced mouse skin papillomas topical application of bl reduced tumor formation tumor volume and caused apoptotic cell death39 bl is a hydrolytic enzymatic complex which shows an efficient digestion and diffusion in tumor cells through attacking the glycosidic linkages and hence denatures glycoproteins thus it protects against tumor growth37 another study has demonstrated the use of controlled proteolytic activity on tumor as a successful strategy to increase therapeutic efficacy43 0cmekkawy figure effect of bromelain and gammairradiation blirr on relative gene expression of liver a parp1 b nfκb and c pparα each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe aim of the radiotherapy protocols is to achieve the maximum curative effect on tumor cells with minimal damaging effect on normal cells hence antioxidants and other nutrients which do not interfere with therapeutic modalities for cancer may enhance the killing property decrease side effects and protect normal tissue44for estimation of the antioxidant ability of bl dpph assay was conducted in vitro and the free radical inhibitory action of bl was compared with some antioxidant compounds it was found that bl has a powerful free radical scavenging power bl belongs to thiol proteases in which the catalytic nucleophile is sulfhydryl groups of cysteine residues which in turn accounts for its antioxidant activity45the involvement of ros mda and pon1 are important mechanisms that play a vital role during radiation toxicity the use of antioxidants is an important preventive to decrease the toxic and pathological effects associated with oxidative stress caused by radiation46 the attained results show a hepatic impairment on the third day from exposure to Îradiation elevation of lpo and ros levels and inhibition of pon1 activity compared to normal mice however treatment with bl revealed an amelioration in hepatic damage caused by irradiation these results were in accordance with liu 47 who described the effect of radiation induced ros generation which in turn might attack cell membrane phospholipids and circulating lipids and thus increases production of mda48 lpo acts as a sensitive biomarker for oxidative stress that occurs as part of the pathogenesis of irradiation49 bl has sulfhydryl groups consequently accounting for its antioxidant activity45 thus it could act as ros scavengermeasurement of pon1 postradiotherapy could be an effective clinical biomarker of hepatic and systemic oxidative stress and may be used as an index of the usefulness of radiotherapy50 it has been demonstrated to catalyze hydrolysis of lipid hydroperoxides and lactones51 pon1 protects serum hdl and ldl ps against lipid peroxidation52 in the present study the decreased activity of pon1 upon radiation exposure might be due to its super saturation of lipid hydroperoxides and lactones upon treatment with bl the activity of pon1 was restored near to the normal level hence the pon1 | Colon_Cancer |
dysregulation of lncrnas is frequent in glioma and has emerged as an important mechanism involved in tumorigenesis previous analysis of chinese glioma genome atlas cgga database indicated that lbx2as1 expression is one of differentially expression lncrna between lower grade glioma lgg grade ii and iii and glioblastoma multiforme gbm however the function and mechanism of lbx2as1 in glioma has not been evaluated yetmethods here we analyzed the expression of lbx2as1 in gtex data normal brain tcgalgg and tcgagbm rtpcr was performed to detect lbx2as1 in surgery obtained normal brain and glioma cck8 kit and annexin vfitcpi apoptosis detection kit were used to study the function of lbx2as1 on glioma proliferation and apoptosis bioinformatic analysis rna immunoprecipitation rtpcr western blotting and dual luciferase reporter assay were carried out to investigate the target mirna of lbx2as1 the discovered mechanism was validated by the rescue assayresults following study of gtex and tcga data lbx2as1 was significantly elevated in glioma compared with normal brain and in gbm compared with lgg higher expression of lbx2as1 was associated with poor prognosis of patients with glioma expression of lbx2as1 was positively correlated with pathology classification of glioma knockdown of lbx2as1 inhibited cell proliferation and induced cell apoptosis in glioma lbx2as1 have complimentary binding site for tumor suppressor mir4915p and we showed that lbx2as1 sponged mir4915p to upregulate trim28 expression in glioma cells trim28 overexpression attenuated the effect of lbx2as1 knockdown on glioma cellss in lbx2as1 was an increased lncrna in glioma mechanistically lbx2as1 promoted glioma cell proliferation and resistance to cell apoptosis via sponging mir4915pkeywords lbx2as1 mir4915p cell proliferation cell apoptosis glioma trim28 gliomas are the most common primary cancer types derived from the neural ectoderm accounting for approximately half of all brain malignancy gliomas are histologic classified into several groups including grade ii oligodendrogliomas and astrocytomas and correspondence houruizhejldxjlueducn department of neurosurgery chinajapan union hospital of jilin university changchun jilin chinafull list of author information is available at the end of the grade iii anaplastic oligodendrogliomas anaplastic astrocytomas anaplastic oligoastrocytomas anaplastic ependymomas and grade iv glioblastomas gbm according to world health anization who classification [ ] overall gliomas are lethal cancer type and patients with high grade glioma gbm have a median overall survival less than one year the worse prognosis of patients with glioma is partly due to resistance to radiotherapy and chemotherapy induced cell apoptosis and the strong proliferation ability of cancer cells [ ] the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cchen a0et a0al cancer cell int page of long noncoding rnas are rna molecules longer than nucleotides without protein coding potential as part of competing endogenous rna cerna network the function of many lncrnas rely on their interaction with micrornas mirnas to regulate expression of gene with same mirna response elements mres accumulating evidences suggested that lncrnas are implicated in glioma progression via sponging mirnas [] for example lncrna ccat1 sponged mir181b increased mir181b target fgfr3 and pdgfrα and promoted glioma cell proliferation invasion and resistance to cell apoptosis lbx2as1 is a recently identified cancer associated lncrna in several cancer types overexpression of lbx2as1 has been reported in hepatocellular carcinoma gastric cancer nonsmall cell lung cancer and esophageal squamous cell carcinoma and it promoted cancer cell proliferation with different mechanism in different cell [] in nonsmall cell lung cancer lbx2as1 activated notch pathway to facilitate cancer cell proliferation migration and invasion in esophageal squamous cell carcinoma lbx2as1 stabilized zeb1 and zeb2 to promote epithelialmesenchymal transition of cancer cells lbx2as1 is one of aberrantly expressed lncrnas between lgg and gbm from cgga database the biological role of lbx2as1 has not been examined in gliomathe current study revealed that lbx2as1 was a significantly upregulated lncrna in glioma and its expression associated with prognosis of patients with glioma we aimed to explore the biological role and molecular mechanism of lbx2as1 in gliomamaterials and a0methodspatients and a0samplestissue samples including normal brains and glioma tissues were obtained from the therapeutic surgery of patients in the third hospital of jilin university during june to july samples were confirmed histologically by two neuropathologists following the criteria of who classification guidelines written informed consents were provided and the protocol was approved by the third hospital of jilin university institutional review board the collected samples were stored at until rna extractioncell lines and a0culturehuman glioma cell lines u87mg u251mg and a172 were purchased from atcc manassas va human astrocyte cell line nha was bought from the cell bank of the chinese academy of sciences shanghai china cells were maintained in dulbeccos modified eagle medium invitrogen carlsbad ca supplemented with fbs gibco rockville md all cells were cultured in a humid incubator with co2 at rna immunoprecipitation rip assaya magna rip rnabinding protein immunoprecipitation kit millipore bedford ma was used to perform rip in a brief u87mg cell lysate was incubated with magnetic beads preincubated with mouse igg negative control or human antiago2 antibody cat no millipore billerica ma total rnas were then isolated by trizol reagent and detected by rtpcr to measure the enrichment of mir4915p and lbx2as1bioinformatic analysisthe expression of lbx2as1 in gtex tcgalgg and tcgagbm projects were obtained from gepia software httpgepia cance rpkucn which was also used to study the association between lbx2as1 expression and prognosis of patients with glioma the association between lbx2as1 and trim28 was also analyzed by gepia based on tcgalgg project encori software tarb asesysueducn was used to predict target mirnas of lbx2as1 and the association between lbx2as1 and mir4915p expression in tcgalggquantitative realtime polymerase chain reactiontotal rna was extracted from cells and tissues with trizol reagent invitrogen rna concentration was detected by nanodrop thermo fisher scientific wilmington de firststranded cdna was synthesized from rna with primescript rt reagent kit takara dalian china realtime polymerase chain reaction was performed with sybr prime script rtpcr kit takara on an abi fast realtime pcr system applied biosystems foster city ca mrna and lncrna were normalized to actin mirna was normalized to u6 the relative expression of gene was calculated by δδct method primer sequences were listed in table a0cell transfection²uuc ucc gaa cgu guc acg utt3² sicontrol silbx2as11 ²cca uua auu cag caa aca uuc cut t3 and silbx2as12 ²uga uuu uuu aaa gaa aaa ucc aat t3² were designed and synthesized by genepharma suzhou china for transfection sirna was mixed with lipofectamine rnaimax invitrogen in serumfree dmem and added into cultured ²cag cug guu gaa ggg gac caaa3² mir4915p mimic ²agu ggg gaa ccc uuc cau gagg3² and mir4915p inhibitor ²ccu cau gga agg guu ccc cacu3² was synthesized by ribo bio guangzhou china these mirnc mir4915p mimic and inhibitor was mixed with cells mirnc 0cchen a0et a0al cancer cell int page of table sequence of a0rtqpcr primersprimerlbx2as1trim28actinmir4915pu6 snrnasequence²aat tcg cag gaa ggg gag tg3²tgc caa acc tgg gac aaa ct3²tga gac ctg tgt aga ggc g3²²cgt tca cca tcc cga gac tt3²²cat gta cgt tgc tat cca ggc3²²ctc ctt aat gtc acg cac gat3²²ctc aac tgg tgt cgt gga gtc ggc aat tca gtt gag cct cat ²²tcg gca gga gtg ggg aac ccttc3²²ctc aac tgg tgt cgt gga ²²cgc ttc acg aat ttg cgt gtca3²²gct tcg gca gca cat ata cta aaa t3²²cgc ttc acg aat ttg cgt gtcat3²forwardreverseforwardreverseforwardreversestem loopforwardreversestem loopforwardreverselipofectamine invitrogen in serumfree dmem and added into cultured cells the transfection efficiency was detected a0h after transfectionwestern blottingps6k cat and s6k cat antibodies were obtained from cst beverly ca trim28 antibody cat was product of active motif carlsbad ca actin antibody cat kc5a08 was bought from aksomics shanghai china hrpconjugated mouse cat ab6728 and rabbit cat ab6721 antibodies were purchased from abcam cambridge uk ripa lysis buffer thermo fisher scientific was used to extract proteins from cells proteins were separated by an sdspage gel and transferred to pvdf membranes the membrane was blocked in nonfat milk and incubated with primary and secondary antibody ecl substrate thermo fisher scientific was used to develop blotscell proliferation and a0apoptosis assaythe cck8 kit beyotime shanghai china was used to detect cell proliferative ability briefly µl cck8 was mixed with culture medium and sustained for an additional a0h after that the absorbance at a0nm of each well was detected by a microplate reader biorad hercules ca percentage of apoptotic cells was measured with dead cell apoptosis kit with annexin v fitc and pi kit invitrogen by flow cytometry analysis cells were suspended in annexinv binding buffer provided by the kit and stained with annexinv fitc and pi sequentially the cells were then subjected to flow cytometry analysis cells positive for annexin v with or without pi positive were apoptotic cellsdual luciferase reporter assaylbx2as1 trim28 ²utr or their mutant forms were inserted into pmirglo luciferase vector these vectors were then cotransfected with mirnc or mir4915p into cells by lipofectamine after a0 h the relative luciferase activity of each group was measured with the dual luciferase reporter assay system kit promega corp madison wi firefly luciferase was normalized to renilla luciferasebioinformatic analysisthe data were analyzed with graphpad prism software the difference between the groups were examined with students t test or oneway anova analysis followed by tukey test the association between expression of two genes was studied with pearson correlation analysis p value less than was considered as statistically significantresultslbx2as1 was a0overexpressed in a0glioma and a0its expression was a0associated with a0poor prognosis of a0glioma patientswe firstly studied the expression of lbx2as1 in normal brains from gtex project low grade glioma lgg and glioblastoma gbm from tcga project the results suggested that lbx2as1 was 4fold and 10fold highly expressed in glioma tissues from tcgalgg and tcgagbm compared with normal brains from gtex expression of lbx2as1 was nearly 3fold higher in highgrade glioma gbm compared with lowgrade glioma lgg fig a0 1a we collected normal brains and glioma tissues and analyzed lbx2as1 expression by rtpcr it showed that lbx2as1 was approximately 3fold highly expressed in glioma compared with normal brains fig a01b furthermore lbx2as1 was increased in high grade glioma grade iv vs grade iii grade iii vs grade ii fig a01c retrospective analysis of the clinical outcome from tcgalgg and tcgagbm datasets suggested that lbx2as1 high expression group n have a shorter diseasefree survival compared with lbx2as1 low expression group n fig a0 1d meanwhile patients with higher expression of lbx2as1 showed a shorter overall survival time compared with their counterparts fig a01elbx2as1 was a0negatively associated with a0mir5p in a0gliomato study the mechanism of lbx2as1 in glioma we firstly used rtpcr method to detect lbx2as1 expression in a panel of glioma cell lines expression of lbx2as1 was significantly increased in u87mg u251mg and a172 cells in comparison with normal human astrocyte 0cchen a0et a0al cancer cell int page of fig lbx2as1 was an upregulated lncrna in glioma a analysis of lbx2as1 expression in normal brains from gtex project and glioma tissues from tcgalgg and tcgagbm projects b rtpcr detection of lbx2as1 expression in normal brains and gliomas c comparison of lbx2as1 expression in normal brain grade ii glioma grade iii glioma and grade iv glioma d e kaplan meierplotter analysis of association between lbx2as1 expression and diseasefree survival d overall survival e of patients with glioma from tcgalgg and tcgagbm projects p nha fig a0 2a lbx2as1 functioned as a cerna to exert its function on cell behavior we used the following method to screen for target mirnas of lbx2as1 fig a0 2b firstly encori database prediction showed that lbx2as1 have putative binding sites for mirnas pearson correlation analysis suggested that the expression of three of these mirnas mir19115p mir219a23p and mir4915p were negatively correlated with lbx2as1 expression in tcgalgg dataset in these three mirnas mir4915p was an upregulated mirna in tcga data of glioma as reported by qi et a0 al the strong negative correlation between mir4915p and lbx2as1 expression was showed in fig a0 2c pearson r a0 p expression of mir4915p was decreased by more than in collected glioma compared with normal brains fig a02d in contrast to lbx2as1 mir4915p was lowly expressed in high grade glioma compared with those of low grade grade iv vs grade iii fig a02e the negative correlation between lbx2as1 and mir4915p expression was also observed in these glioma tissues fig a0 2f to study whether lbx2as1 interact with mir4915p we firstly transfected mir4915p mimic into u87mg and u251mg cells to elevate mir4915p expression fig a02g full length of lbx2as1 was inserted into luciferase vector pmirglo luciferase reporter assay showed that mir4915p overexpression did repress luciferase activity of pmirglolbx2as1 by around half in u87mg fig a02h and u251mg cells fig a02i more importantly rip assay showed that ago2 antibody enriched both lbx2as1 and mir4915p in u87mg cells fig a0 2j these data implied that lbx2as1 might regulate mir4915p in glioma cellsa mutual regulatory association between a0mir5p and a0lbx2as1 in a0gliomato study the association between mir4915p and lbx2as1 we transfected increasing concentrations a0 nm and a0 nm of mir4915p mimic into u87mg and u251mg cells expression of mir4915p was increased by and 9fold in u87mg cells transfected with a0nm and a0 nm mir4915p mimic respectively fig a0 3a in u251mg cells mir4915p levels were increased by and 45fold after transfection of a0nm and a0nm mir4915p fig a0 3a overexpression of mir4915p decreased lbx2as1 expression by more than half in u87mg and u251mg cells and lower expression of lbx2as1 was observed in cells with high concentration of mir4915p mimic fig a0 3b two independent sirnas targeting lbx2as1 silbx2as11 and 0cchen a0et a0al cancer cell int page of fig lbx2as1 expression was associated with mir4915p levels in glioma a rtpcr detection of lbx2as1 expression in a panel of glioma cell lines u87mg u251mg a172 and normal human astrocyte nha b flow chart of screen for target mirnas of lbx2as1 in glioma c the association between lbx2as1 and mir4915p expression in tcgalgg project was analyzed by pearson correlation analysis d rtpcr detection of mir4915p expression in normal brains and gliomas e comparison of mir4915p expression in normal brain grade ii glioma grade iii glioma and grade iv glioma f the association between lbx2as1 and mir4915p expression in collected glioma samples was analyzed by pearson correlation analysis g the expression of mir4915p was detected in u87mg and u251mg cells transfected with mirnc or mir4915p mimic in u87mg h and u251mg i cells the luciferase activity of pmirglo empty vector as the control group and pmirglolbx2as1 was detected j rip assay was performed with igg and ago2 antibody followed by rtpcr detection of lbx2as1 and mir4915p levels p p silbx2as12 were transfected into glioma cells these sirnas greatly decreased lbx2as1 expression by more than in u87mg and u251mg cells fig a03c in addition knockdown of lbx2as1 increased mir4915p expression in u87mg and u251mg cells fig a0 3d transfection of silbx2as12 which reduced lbx2as1 expression in a relatively larger extent compared with silbx2as11 was more effective in upregulation of mir4915p expression in the cells fig a03d indicating the negative regulation of mir4915p by lbx2as1 we analyzed the complementary site between lbx2as1 and mir4915p and constructed pmirglo vector with mutant lbx2as1 lbx2as1 mut fig a0 3e overexpression of mir4915p repressed luciferase activity of lbx2as1 wt instead of lbx2as1 mut in u87mg fig a03f and u251mg cells fig a03glbx2as1 upregulated trim28 expression in a0gliomaprevious study suggested that mir4915p targeted trim28 to regulate glioma cell proliferation after investigation of trim28 and lbx2as1 expression in tcgalgg and tcgagbm datasets it was found that trim28 was positively correlated with lbx2as1 expression in glioma pearson r p fig a0 4a additionally trim28 was highly expressed in our collected glioma samples compared with normal brains fig a0 4b pearson correlation analysis showed a strong positive correlation between lbx2as1 and trim28 levels in our collected glioma tissues pearson r p fig a04c knockdown of lbx2as1 decreased approximately trim28 mrna expression in u87mg and u251mg cells fig a0 4d trim28 induced dephosphorylation of s6k to control glioma development lbx2as1 downregulation decreased trim28 protein expression and the downstream phosphorylation of s6k ps6k by half in u87mg cells as measured by western blotting fig a04e similarly lbx2as1 knockdown also decreased trim28 and ps6k in u251mg cells to the same extent as in u87mg fig a04f 0cchen a0et a0al cancer cell int page of fig mir4915p mutually regulated lbx2as1 in glioma mir4915p a and lbx2as1 b expression was detected in u87mg and u251mg cells transfected with mirnc or increasing concentration of mir4915p mimic nm nm c lbx2as1 expression was detected in u87mg and u251mg cells transfected with sicontrol or silbx2as11 or silbx2as12 by rtpcr d mir4915p expression was detected in u87mg and u251mg cells transfected with sicontrol or silbx2as11 or silbx2as12 e sequence alignment of lbx2as1 wt lbx2as1 mut and mir4915p luciferase activity was detected in u87mg f and u251mg g cells transfected with lbx2as1 wt or lbx2as1 mut p p lbx2as1 regulated trim28 via a0sponging mir5pwe first transfected mir4915p inhibitor into u87mg and u251mg cells to decrease mir4915p expression in these cells mir4915p inhibitor decreased mir4915p expression in cells fig a05a we found that mir4915p inhibitor could reverse the downregulation of trim28 mrna expression by silbx2as11 in u87mg and u251mg cells fig a05b western blotting further showed that mir4915p inhibitor could reverse the downregulation of trim28 protein expression by silbx2as11 in u87mg and u251mg cells fig a05c luciferase vectors containing wildtype and mutant trim28 ²utr trim28 ²utr wt trim28 ²utr mut were constructed fig a05d in u87mg cells mir4915p inhibitor could reverse the repression of luciferase activity of trim28 ²utr wt by silbx2as11 fig a05e these data collectively demonstrated a lbx2as1mir4915ptrim28 axis in gliomalbx2as1 promoted cell proliferation and resistance to cell apoptosis by repression of mir4915pit is known that aberrant expression of mir4915p and trim28 mediated glioma cell proliferation and survival to investigate the impact of the lbx2as1mir4915ptrim28 axis on cell proliferation we performed cck8 assay in glioma cells transfected with silbx2as1 with or without mir4915p inhibitor knockdown of lbx2as1 inhibited cell proliferation in u87mg cells and the effect of silbx2as1 was reversed upon cotransfection of mir4915p 0cchen a0et a0al cancer cell int page of fig lbx2as1 regulated trim28 expression in glioma a pearson correlation analysis of lbx2as1 and trim28 expression in tcgalgg and tcgagbm projects b rtpcr detection of trim28 mrna expression in normal brains and gliomas c pearson correlation analysis of lbx2as1 and trim28 expression in collected glioma d trim28 mrna expression was detected in u87mg and u251mg cells transfected with sicontrol or silbx2as11 or silbx2as12 by rtpcr trim28 ps6k protein expression was detected in u87mg e and u251mg f cells transfected with sicontrol or silbx2as11 or silbx2as12 by western blotting actin and s6k were internal controls for trim28 and ps6k p p inhibitor fig a0 6a in consistent with u87mg silbx2as11 and silbx2as12 inhibited u251mg cell proliferation which was attenuated by mir4915p inhibitor fig a0 6b with the flow cytometry we also found that lbx2as1 knockdown induced cell apoptosis in u87mg cells and the effect of silbx2as1 was partially rescued by mir4915p inhibitor fig a06c similar results were found in u251mg cells fig a06d these data manifested that lbx2as1 mainly relied on regulation of mir4915p to mediate glioma cell proliferation and survivaldiscussionlbx2as1 is a most recently identified oncogenic lncrna across several cancer types lbx2as1 was reported as a highly expressed lncrna in nonsmall cell lung cancer especially in tumors of advanced stage high expression of lbx2as1 was also found in stomach adenocarcinoma and hepatocellular carcinoma [ 0cchen a0et a0al cancer cell int page of fig lbx2as1 regulated trim28 expression via sponging mir4915p a the expression of mir4915p was detected in u87mg and u251mg cells transfected with mirnc or mir4915p inhibitor b the expression of trim28 mrna was detected in u87mg and u251mg cells transfected with mirnc or mir4915p inhibitor in combination with sicontrol or silbx2as11 by rtpcr c the expression of trim28 protein was detected in u87mg cells transfected with mirnc or mir4915p inhibitor in combination with sicontrol or silbx2as11 by western blotting d sequence alignment of lbx2as1 trim28 ²utr wt trim28 ²utr mut and mir4915p e luciferase activity was detected in u87mg cells transfected with trim28 ²utr wt or trim28 ²utr mut in combination with sicontrol or silbx2as11 and mirnc or mir4915p inhibitor p p see figure on next pagefig lbx2as1 regulated cell proliferation and apoptosis via sponging mir4915p transfection of silbx2as11 or silbx2as12 inhibited cell proliferation and was reversed by transfection of mir4915p inhibitor in u87mg a and u251mg b cells as indicated by cck8 assay transfection of silbx2as11 or silbx2as12 induced cell apoptosis and was partially reversed by transfection of mir4915p inhibitor in u87mg c and u251mg d cells as indicated by flow cytometry analysis p p 0cchen a0et a0al cancer cell int page of 0cchen a0et a0al cancer cell int page of ] here we analyzed lbx2as1 expression in glioma by using data of tcgalgg and tcgagbm projects in combination with normal brains from gtex project similar to observation in other cancer types it was observed that lbx2as1 was increased in glioma especially highgrade glioma gbm we further revealed that patients with high expression of lbx2as1 have a short diseasefree survival and overall survival indicating lbx2as1 could predict poor prognosis of glioma as the published studies showed that lbx2as1 was associated with poor prognosis of patients with hepatocellular carcinoma and nonsmall cell lung cancer [ ] the findings suggested that lbx2as1 might be a predictive biomark for a variety of cancer typeslbx2as1 exerted its procancer functions via acting as a cerna to sponge tumor suppressive mirnas for example lbx2as1 directly interacted with tumor suppressive mir384 to enhance cell proliferation and resistance to cell apoptosis in hepatocellular carcinoma our bioinformatic analysis indicated that lbx2as1 have a putative binding site for mir4915p mir4915p was a tumor suppressor in several cancer types [] aberrant expression of mir4915p was the consequence of upregulated circrna circ_0001361 and lncrna xist in bladder cancer and nasopharyngeal carcinoma respectively [ ] in glioma mir4915p was decreased in cancer tissues and correlated with good prognosis we confirmed mir4915p as a target mirna of lbx2as1 in glioma upregulation of mir4915p induced cancer cell apoptosis to cease cell proliferation [ ] we showed that lbx2as1 mediated glioma cell proliferation and resistance to cell apoptosis downregulation of mir4915p could partially rescue the impact of lbx2as1 knockdown on glioma cell proliferation and apoptosis thus the current data indicated a novel interaction between lbx2as1 and mir4915p and manifested that lbx2as1 promoted glioma proliferation via sponging mir4915p however as the cell apoptosis induced by lbx2as1 knockdown was not fully rescued by mir4915p downregulation we believe there are several other mechanisms underlying the function of lbx2as1 in glioma for example the notch signaling was regulated by lbx2as1 in nonsmall cell lung cancer and the activity of notch signaling determined the cell apoptosis in glioma [ ] it remains unknown whether lxb2as1 uses the same mechanism to control notch signaling in glioma future study will reveal the complexity of signaling network regulated by lbx2as1 in glioma in addition due to the involvement of mir4915p in cancer cell metastasis further studies will be needed to evaluate the effect of lbx2as1 on glioma metastasistrim28 is a cancerassociated e3 ligase in several cancer types upregulation of trim28 was found in glioma and the proproliferative function of trim28 was supported by in vitro and in vivo data [ ] in glioma trim28 mediated degradation of tumor suppressor ampk activated mtorc1 and regulated cell apoptosis trim28 expression was repressed by several noncoding rna in different cell [ ] in the current study in addition to the known mir4915ptrim28 interaction in glioma we further discovered that lncrna lbx2as1 could regulated trim28 via sponging mir4915p in glioma mechanistically trim28 form complex with mage to regulate mtor activity and the downstream phosphorylation of s6k [ ] we found that lbx2as1 not only upregulated trim28 expression but also increased phosphorylation level of s6k in glioma cells therefore the data revealed a lbx2as1mir4915ptrim28 axis in gliomasour results suggested that lncrna lbx2as1 promoted glioma cell proliferation and resistance to cell apoptosis via sponging mir4915p lbx2as1 could be a novel biomarker for patients with gliomaacknowledgementsnoneauthors contributions qc rh and jg performed the experiments and acquired the data qc and yz analyzed data qc and rh designed the study rh supervised the study manuscript was written by rh all authors read and approved the final manuscript funding none availability of data and materialsall data generated or analyzed during this study are included in this published ethics approval and consent to participateinformed consent was obtained from all patients and the study protocol and consent procedures were approved by the third hospital of jilin university review boardconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details department of neurosurgery chinajapan union hospital of jilin university changchun jilin china department of gynecology and obstetric the second hospital of jilin university changchun jilin china received may revised july accepted july references ostrom qt bauchet l davis fg deltour i fisher jl langer ce pekmezci m schwartzbaum ja turner mc walsh km et al the epidemiology of glioma in adults a state of the science review neuro oncol 0cchen a0et a0al cancer cell int page of chen r smithcohn m cohen al colman h glioma subclassifications pan sj ren j jiang h liu w hu ly pan yx sun b sun qf bian lg and their clinical significance neurotherapeutics wesseling p capper d who classification of gliomas neuropathol appl neurobiol ostrom qt gittleman h stetson l virk sm barnholtzsloan js epidemiology of gliomas cancer treat res xu h chen b xing j wei z liu c qiu y lin y ren l upregulation of lgmnp1 confers radiotherapy resistance in glioblastoma oncol rep krakstad c chekenya m survival signalling and apoptosis resistance in glioblastomas opportunities for targeted therapeutics mol cancer castrooropeza r melendezzajgla j maldonado v vazquezsantillan k the emerging role of lncrnas in the regulation of cancer stem cells cell oncol dordr zhang z qian w wang s ji d wang q li j peng w gu j hu t ji b et al analysis of lncrnaassociated cerna network reveals potential lncrna biomarkers in human colon adenocarcinoma cell physiol biochem liu x zhu q guo y xiao z hu l xu q lncrna linc00689 promotes the growth metastasis and glycolysis of glioma cells by targeting mir3383ppkm2 axis biomed pharmacother magea6 promotes human glioma cell survival via targeting ampkalpha1 cancer lett wang sn luo s liu c piao z gou w wang y guan w li q zou h yang zz et al mir491 inhibits osteosarcoma lung metastasis and chemoresistance by targeting alphabcrystallin mol ther zhang q li q xu t jiang h xu lg mir4915p suppresses cell growth and invasion by targeting notch3 in nasopharyngeal carcinoma oncol rep xu y hou r lu q zhang y chen l zheng y hu b mir4915p negatively regulates cell proliferation and motility by targeting pdgfra in prostate cancer am j cancer res sun r liu z tong d yang y guo b wang x zhao l huang c mir4915p mediated by foxi1 functions as a tumor suppressor by targeting wnt3abetacatenin signaling in the development of gastric cancer cell death dis 201783e2714 liu f zhang h xie f tao d xiao x huang c wang m gu c zhang x jiang g hsa_circ_0001361 promotes bladder cancer invasion and metastasis through mir4915pmmp9 axis oncogene cheng q xu x jiang h xu l li q knockdown of long noncoding rna xist suppresses nasopharyngeal carcinoma progression by activating mir4915p j cell biochem liao y shen l zhao h liu q fu j guo y peng r cheng l lncrna casc2 denoyelle c lambert b meryetfiguiere m vigneron n brotin e lecerf c interacts with mir181a to modulate glioma growth and resistance to tmz through pten pathway j cell biochem ni w luo l zuo p li rp xu xb wen f hu d lncrna ghet1 downregulation suppresses the cell activities of glioma cancer biomark cui b li b liu q cui y lncrna ccat1 promotes glioma tumorigenesis by sponging mir181b j cell biochem tang lx su sf wan q he p xhang y cheng xm novel long noncoding rna lbx2as1 indicat | Colon_Cancer |
introduction postoperative ileus poi a common complication after surgery severely affects postoperative recovery it is unclear whether pretreatment with transcutaneous electrical acupoint stimulation teas can improve recovery from poi this trial will evaluate the effects of pretreatment with teas on poimethods and analysis this will be a prospective randomised controlled trial american society of anesthesiologists asa physical status classification iiii level patients aged years and scheduled for laparoscopic colon surgery will be included in the study it is planned that subjects will be randomised to the teas and sham teas steas groups the groups will undergo two sessions of teassteas daily for days before surgery with a final teassteas treatment min before anaesthesia the primary endpoint of the study will be time to first defaecation secondary endpoints will include time to first flatus time to tolerance of oral diet gi2 composite outcome of time to first defaecation and time to tolerance of oral diet time to independent walking length of hospital stay postoperative pain visual analogue scale score on the first days after surgery analgesic requirements complications and plasma concentrations of interferon ifn ifnÎ interleukin6 il6 and il1 multiple linear regression will be used to identify independent predictors of outcome measuresethics and dissemination this study has been approved by the chinese registered clinical trial ethics review committee no chiecrct20170084 the results of the trial will be published in an international peer reviewed trial registration number this study has been registered with the chinese clinical trial registry no chictr inr17013184trial status the study was in the recruitment phase at the time of manuscript submissionintroductionpostoperative dysfunction ileus poi of gastrointestinal is a transient gi strengths and limitations of this study º this study aims to evaluate whether pretreatment with transcutaneous electrical acupoint stimulation teas can prevent postoperative ileus poi º teas is a safe non invasive and easily accepted adjunctive intervention º this study will provide deeper insights into the mechanism by which teas pretreatment reduces the inflammatory response º this is a single centre study which is a potential limitationpropulsion that often occurs after abdominal surgery and may also occur after surgery at other sites1 the main symptoms of poi include abdominal pain and distention nausea vomiting difficult defaecation and intolerance to solid food poi is usually temporary but if prolonged may lead to surgical incision dehiscence intestinal anastomotic fistula abdominal cavity infection intestinal ischaemia aspiration pneumonia and other serious complications2 a retrospective cohort study involving nearly hospitals in the usa showed that poi is a key reason for prolonged hospitalisation and increased medical costs for patients undergoing abdominal surgery1 the usa spends more than billion treating poi every year5 at present the most common methods used to treat poi include rational perioperative use of narcotic drugs and opioids eating as soon as possible after surgery avoidance of nasogastric tubes after the operation early ambulation postoperative epidural analgesia restriction of fluid intake the use wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0copen access of minimally invasive surgery such as laparoscopic drug therapy and the use of chewing gum despite the numerous treatment strategies poi remains a difficult clinical challenge that compromises the rapid recovery of postoperative patients it is therefore necessary to find more effective convenient and economical treatment methods6the main mechanism underlying poi may be activation of macrophages in the external muscular layer during the surgical procedure11 intestinal manipulation during surgery can activate macrophages in the outer muscle layer of the small intestine leading to release of inflammatory factors interleukin6 il6 il1 and the chemokine mip1α together with increased expression of the adhesion molecule icam1 on endothelial cells and induction of neutrophils and monocytes in the circulation into the small intestine muscle layer these cells and activated macrophages can release a large amount of inducible nitric oxide synthase and prostaglandin which inhibit the movement and contraction of the gi tract12 transport of these inflammatory mediators in the bloodstream causes activation of macrophages in the distal gi tract leading to poi over the entire intestinal tract14 it has been confirmed by a large number of animal experiments that reducing the inflammatory response is an effective way to treat poi15there is a long history in traditional chinese medicine tcm of using acupuncture to treat functional gi diseases and in recent years there has been significant global interest in the beneficial effects of acupuncture on poi the positive effect of electroacupuncture ea on poi has been clearly demonstrated ng used ea to treat poi in patients undergoing laparoscopic colon surgery18 defaecation time and length of hospital stay were significantly shortened in patients who received ea compared with those who did not receive the treatment in patients undergoing hepatic resection you found a significant reduction in the incidence of poi in patients treated with a combination of acupuncture and chinese herbal medicine the length of hospitalisation was also significantly shortened in the treated group ± days vs ± days p001419in the previous studies we proved that pretreatment with acupuncture could reduce excessive activation of the innate immune system and inhibit the inflammatory response this effect may be achieved by activation of the vagal nervous system20 other studies have shown that transcutaneous electrical acupoint stimulation teas and ea have similar effects in the treatment of pain and alleviating the inflammatory response22 tcm holds that the best treatment for disease is prevention based on all of the above studies we hypothesise that the use of teas as a preoperative treatment may reduce the incidence of poi there have so far not been any studies that address this questionwe have therefore designed a randomised controlled trial to investigate whether pretreatment with teas can reduce the incidence of poi in patients undergoing laparoscopic colon resection the study is also designed to verify that the anti inflammatory effect is associated with the immunomodulatory function of teasmethods and analysisstudy objectivethe primary objective is to assess the effect of teas on clinical recovery of bowel function after laparoscopic colon surgery the secondary objective is to verify that suppression of overactivation of the innate immune system and reduction of the inflammatory response are the mechanisms underlying the ability of pretreatment of percutaneous acupuncture to prevent poistudy locationa prospective single centre double blinded randomised controlled trial will be conducted at shuguang hospital which is affiliated to the shanghai university of traditional chinese medicine chinastudy populationparticipants will be recruited according to the inclusion and exclusion criteriainclusion criteria male and female patients aged years patients undergoing elective laparoscopic colonic surgery and upper rectal resection such as left collect right colectomy and anterior resection of the upper part of the rectum and lower part of the sigmoid body mass index kgm2 asa classification iiii patients provide signed informed consent the consent form can be viewed in online supplementary appendix exclusion criteria middle and lower rectal resection totalproctocolectomy or the need for complex endoscopic surgery need for abdominal wall fistula gi fistula fistula surgery or stoma creation history of abdominalpelvic operations or complications patients receiving epidural anaesthesia or epidural analgesia patients with skin infections surgical incision or scar at the point of application of acupuncture patients have a history of limb surgery spinal surgery or nerve injury patients who participated in other clinical trials or received other acupuncture therapy in the previous weeks patients with cardiac pacemakers patients have one of the following conditions before surgery chronic pain drug addiction or alcohol dependence patients with preoperative combination of severe central nervous system disease and severe mental illnesswang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0cendpointsprimary endpointfirst defaecation time h that is time to first anal defaecation after laparoscopic surgerysecondary endpointstime to first flatus h time to tolerance of solid oral diet h gi2 composite outcome of time to first defaecation and time to tolerance of oral diet time to walk independently h length of hospital stay defined as the number of days from operation to discharge d criteria for hospital discharge include stability of vital signs with no fever achievement of flatus or defaecation ability to tolerate solid food without vomiting control of postoperative pain absence of other postoperative complications and ability to function at home independently or with home care provided pain will be assessed using the visual analogue scale vas on postoperative days and scale of to where represents complete absence of pain and represents the worst pain intensity postoperative requirements for analgesia will also be assessed inflammatory mediators interferon ifn ifnÎ interleukin6 il6 and il1 in blood will be measured before teassteas intervention and on days and after the operation postoperative complications will be recorded using the clavien dindo classification for complication assessment24 the follow up period will be at least monthswe add gi2 as a secondary outcome to the original protocol after recruitment of the study had already begun gi2 is a time indicator which will be calculated from two existing outcomes time to first defaecation and time to tolerance of oral diet there will be no harm to subjects no additional cost and no more workopen accessrandomisation and blindingpatients will be randomised to receive either teas or steas by stratified randomisation according to sex in a ratio figure using a computer generated random sequence a sealed envelope will be opened to determine to which group the patient has been assigned the acupuncturist will be aware of the treatment group patients as well as the outcome investigator nurse anaesthetist will be blinded to the treatment allocationcurrent sample size justificationaccording to wang jian and song jiangangs preliminary study of teas pretreatment for prevention of poi in patients undergoing laparoscopic colon surgery in shuguang hospital the mean time to first defaecation following laparoscopic colon surgery was ± hours m±sd working on the assumption that a clinically meaningful difference in mean time to first defaecation between the teas and steas groups is day or hours patients would be needed in each group to reach a power of and a type i error rate if the dropout rate is a total sample size of patients for the two groups is needed for this studystatistical analysisdata for continuous variables ie first defaecation time first passage of flatus time to tolerance of oral diet time to walking independently length of hospital stay will be reported using the mean and sd m±sd for normally distributed data or median range for skewed data data for categorical variables will be expressed as a number percentage intergroup differences will be assessed using the students t test or mann whitney u test intergroup differences in inflammatory mediators at time points of pre teassteas treatment and on figure flowchart of the study protocolwang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0copen access figure acupoints selected in this trial a hegu il4 and neiguan p6 b zusanli st36 and shangjuxu st37 c hans acupoint nerve stimulatorpostoperative days and were assessed by two way repeated measures analysis of variance with bonferroni post hoc test the significance level will be set at all data will be analysed using spss v170 software or other appropriate statistical software packagespretreatmentpatients randomised to the teas and steas groups will undergo two treatment sessions daily for three consecutive days before surgery the patients will then be treated for a final time min before anaesthesiafor patients in the teas group the zusanli st36 shangjuxu st37 hegu li4 and neiguan p6 acupoints will be identified before electrical stimulation with surface electrodes figure selection of these acupoints is based on a consensus between the acupuncturists carrying out the study the acupuncturist will stimulate these acupoints using a hans acupoint nerve stimulator hans200a nanjing jisheng medical technology nanjing china at a frequency of hz the intensity will be adjusted for each individual to maintain a slight twitching of the regional muscle and achieve de qi sensations such as soreness numbness distention and heaviness the steas group will receive a strong but comfortable current for s and the current will then gradually vanish over the next s25 the participants of both groups will be told that they are receiving current stimulation each session of acupoints treatment will last for min during the application of teas patients will be required not to change the current settings themselves a prompt beep at the end of teas will indicate the end of treatmentall surgery will be carried out under general anaesthesia using standardised anaesthetic procedures patients will be fasted for hours before surgery right upper extremity venous access will be established before the patients entering the operating theatre ringers lactate solution mlkg will be administered by intravenous infusion for compensatory expansion before induction of anaesthesia patients will then receive midazolam mgkg fentanyl µgkg vecuronium bromide mgkg and propofol mgkg intravenously for induction of anaesthesia anaesthesia will be maintained using a cp600 anaesthesia delivery system slgo medical technology beijing china the dose wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0cof propofol will be adjusted to maintain the bispectral index in the range of after surgery all patients will remain in the post anaesthesia care unit and then return to the ward for recovery until dischargethe perioperative management of all patients will be standardised early ambulation will be encouraged and oral feeding will be resumed as early as possible all patients will be followed up for at least months after discharge from the hospitaladverse eventsall adverse reactions will be closely monitored through spontaneous reports by patients or direct observation by clinicians or by asking the patients about adverse events using open questions all adverse reactions will be recorded and appropriate treatment will be provided if necessary serious adverse events will be reported to the ethics committeedata collection and managementdemographic variables and clinical data will be collected from all patients during the study blood pressure heart rate and oxygen saturation will also be monitored any adverse events will be recorded data will be collected throughout the study and will be securely managed under conditions of confidentiality data collection will be performed by a nurse anaesthetist the participants will be referred to by their participant number rather than by their name throughout the study unless otherwise specified all relevant documents and files will be archived for years the data will be accessible only by investigators who sign the confidential disclosure agreement and by institutional or governmental auditors during the study data without patient identifiers will be publicly accessible after the study data collection and management will be monitored by the institutional ethics committee for clinical research of shuguang hospitalpatient and public involvementthis study is currently in the recruitment phase patients andor the public were not involved in study design or conduct of the study the participants will be able to access the study results through social mediadiscussionpoi continues to represent an important cause of morbidity after colon surgery the prevention of poi is thus of great importance in reducing perioperative complications and reducing hospitalisation costs although it has been shown that ea can shorten the duration of poi18 the effectiveness of teas which is a similar technique in preventing poi has not been investigated it is therefore important to assess the effectiveness of teas in preventing poi through a clinical studythis study has several strengths first the intervention strategy of the protocol will be pretreatment with teas previous studies have shown that pretreatment open accesshas a prophylactic effect for example pretreatment with teas has been shown to improve pain treatment26 and to improve resuscitation after anaesthesia with reduction of postoperative nausea and vomiting28 it is however unclear whether preoperative teas can prevent poi studies suggest that early preoperative intervention may be more beneficial in regulating physiological functions and preventing poi29 in an extension to these findings the present study will help to determine whether teas pretreatment could improvement poisecond the effectiveness of teas will be evaluated by assessing clinical function and by serological examination in this randomised controlled trial of patients undergoing laparoscopic colorectal surgery our aim is to assess the effects of preoperative teas on poi using relevant clinical parameters associated with bowel function these include time to first defaecation time to first flatus time to tolerance of oral diet and gi2 importantly we will also measure serum concentrations of inflammatory mediators associated with poi such as ifn ifnÎ il6 and il1 our findings may thus provide deeper insights into the mechanisms by which teas improves poithere are also limitations to this protocol various clinical indicators have been used in studies for the diagnosis of poi but there is no consensus on which clinical parameter is the best for assessment of gi transit9 two indicators that are widely used to assess bowel movement will be used in this study time to first defaecation will be the primary outcome and time to first flatus will be one of the secondary outcomes there is a possibility that we may observe conflicting results ie significant improvement in time to flatus but not defaecation because flatus can vary considerably between patients clinical trials support the time to tolerance of oral diet and gi2 defined as the later of the following two events time to first tolerance of solid food and time to first bowel movement as supplementary secondary outcomes to measure the recovery time of gi function and these will be used in this study32 other limitations of these indicators are that they require objective measurement of motility and are time consuming to measure34 recently this situation has been improved by the use of in vivo monitoring techniques to assess the function of gi movements innovative devices such as sitz markers have been used to evaluate postoperative recovery of small bowel movement by counting the number of sitz markers that did not pass through the ileocecal valve but remained in the small intestine using radiography36 the smartpill is a swallowable device that record parameters within the gi tract indicators such as ph temperature and intracavitary pressure can be collected to analyse gi transit times in vivo37 these devices acquire objective parameters to evaluate bowel movement and could save time research into the satisfaction of both doctors and patients with these device needs to be carried out furthermore this study is a single centre trial and because the therapeutic effect of teas may be affected by ethnicity and region it will wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0copen access be necessary to conduct multicentre and large sample studies in the futurenotwithstanding its limitations this study can clearly indicate the overall effects of teas on postoperative recovery we hypothesise that pretreatment with teas could improve recovery of gi function in patients undergoing laparoscopic surgery if this study provides positive results it will be possible to recommend this pretreatment strategy for patients undergoing abdominal surgery relevant cost effectiveness studies are also worthy of considerationauthor affiliations1anesthesiology shuguang hospital affiliated to shanghai university of traditional chinese medicine shanghai china2anesthesiology wenzhou medical university the sixth affiliated hospital lishui china3research institute of acupuncture anesthesia shuguang hospital affiliated to shanghai university of traditional chinese medicine shanghai chinaacknowledgements we thank dr stanley tao from shanghai ruihui biotech for his valuable assistance in the statistical design of this studycontributors jw conceived the study dl wt jg and gf participated in its design and coordination wc yy ws and jg collected references and developed the protocol gy and ly will perform statistical analyses rf will follow up with patients and record data jw lf and js drafted the manuscript all authors have read and approved the final manuscriptfunding the present study is supported by the project of the national natural science foundation of china nos and and the commercial sponsorship of sinch pharmaceuticals techcompeting interests none declaredpatient consent for publication obtainedprovenance and peer review not commissioned externally peer reviewedopen access this is an open access article distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial see a0http creativecommons org licenses by nc orcid idlihua a0fan http orcid org references iyer s saunders wb stemkowski s economic burden of postoperative ileus associated with colectomy in the united states j manag care pharm boelens pg heesakkers ffbm luyer mdp et a0al reduction of postoperative ileus by early enteral nutrition in patients undergoing major rectal surgery prospective randomized controlled trial ann surg melis m fichera a ferguson mk bowel necrosis associated with early jejunal tube feeding a complication of postoperative enteral nutrition arch surg moghadamyeghaneh z hwang gs hanna mh et a0al risk factors for prolonged ileus following colon surgery surg endosc goldstein jl matuszewski ka delaney cp et a0al inpatient economic burden of postoperative ileus associated with abdominal surgery in the united states p and t bragg d el sharkawy am psaltis e et a0al postoperative ileus recent developments in pathophysiology and management clin nutr wolthuis am bislenghi g fieuws s et a0al incidence of prolonged postoperative ileus after colorectal surgery a systematic review and meta analysis colorectal dis 201618o1 nguyen dl maithel s nguyen et et a0al does alvimopan enhance return of bowel function in laparoscopic gastrointestinal surgery a meta analysis ann gastroenterol studies and clinical aspects of gadolinium salts and chelates cardiovasc drug rev koscielny a kalff jc t helper cell type memory cells and postoperative ileus in the entire gut curr opin gastroenterol mikkelsen hb thuneberg l opop mice defective in production of functional colony stimulating factor1 lack macrophages in muscularis externa of the small intestine cell tissue res van bree shw nemethova a cailotto c et a0al new therapeutic strategies for postoperative ileus nat rev gastroenterol hepatol hilton wm lotan y parekh dj et a0al alvimopan for prevention of postoperative paralytic ileus in radical cystectomy patients a cost effectiveness analysis bju int wehner s behrendt ff lyutenski bn et a0al inhibition of macrophage function prevents intestinal inflammation and postoperative ileus in rodents gut wehner s straesser s vilz to et a0al inhibition of p38 mitogen activated protein kinase pathway as prophylaxis of postoperative ileus in mice gastroenterology schwarz nt kalff jc türler a et a0al prostanoid production via cox2 as a causative mechanism of rodent postoperative ileus gastroenterology engel dr koscielny a wehner s et a0al t helper type memory cells disseminate postoperative ileus over the entire intestinal tract nat med adding lc bannenberg gl gustafsson le basic experimental ng ssm leung ww mak twc et a0al electroacupuncture reduces duration of postoperative ileus after laparoscopic surgery for colorectal cancer gastroenterology you x m mo x s ma l et a0al randomized clinical trial comparing efficacy of simo decoction and acupuncture or chewing gum alone on postoperative ileus in patients with hepatocellular carcinoma after hepatectomy medicine 201594e1968 song jg li hh cao yf et a0al electroacupuncture improves survival in rats with lethal endotoxemia via the autonomic nervous system anesthesiology zhang j yong y li x et a0al vagal modulation of high mobility group box1 protein mediates electroacupuncture induced cardioprotection in ischemia reperfusion injury sci rep balogun ja biasci s han l the effects of acupuncture electroneedling and transcutaneous electrical stimulation therapies on peripheral haemodynamic functioning disabil rehabil jiang y wang h liu z et a0al manipulation of and sustained effects on the human brain induced by different modalities of acupuncture an fmri study plos one 20138e66815 dindo d demartines n clavien p a classification of surgical complications a new proposal with evaluation in a cohort of patients and results of a survey ann surg rakel b cooper n adams hj et a0al a new transient sham tens device allows for investigator blinding while delivering a true placebo treatment j pain huang l pan y chen s et a0al prevention of propofol injection related pain using pretreatment transcutaneous electrical acupoint stimulation turk j med sci zhang q gao z wang h et a0al the effect of pre treatment with transcutaneous electrical acupoint stimulation on the quality of recovery after ambulatory breast surgery a prospective randomised controlled trial anaesthesia zheng lh sun h wang gn et a0al effect of transcutaneous electrical acupoint stimulation on nausea and vomiting induced by patient controlled intravenous analgesia with tramadol chin j integr med stakenborg n labeeuw e gomez pinilla pj et a0al preoperative administration of the ht4 receptor agonist prucalopride reduces intestinal inflammation and shortens postoperative ileus via cholinergic enteric neurons gut vather r trivedi s bissett i defining postoperative ileus results of a systematic review and global survey j gastrointest surg wu z boersema gsa dereci a et a0al clinical endpoint early detection and differential diagnosis of postoperative ileus a systematic review of the literature eur surg res deng g wong wd guillem j et a0al a phase ii randomized controlled trial of acupuncture for reduction of postcolectomy ileus ann surg oncol van bree shw bemelman wa hollmann mw et a0al identification of clinical outcome measures for recovery of gastrointestinal motility in postoperative ileus ann surg wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0c maffezzini m campodonico f canepa g et a0al current perioperative management of radical cystectomy with intestinal urinary reconstruction for muscle invasive bladder cancer and reduction of the incidence of postoperative ileus surg oncol bungard tj kale pradhan pb prokinetic agents for the treatment of postoperative ileus in adults a review of the literature pharmacotherapy open access chae h d kwak m a kim i h effect of acupuncture on reducing duration of postoperative ileus after gastrectomy in patients with gastric cancer a pilot study using sitz marker j altern complement med vilz to pantelis d lingohr p et a0al smartpill® as an objective parameter for determination of severity and duration of postoperative ileus study protocol of a prospective two arm open label trial the pidusa study bmj open 20166e011014wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0c' | Colon_Cancer |
" it is estimated that around of patients with early stage colon cancer benefit from adjuvantchemotherapy we are currently not capable of upfront selection of patients who benefit from chemotherapywhich indicates the need for additional predictive markers for response to chemotherapyit has been shown that the consensus molecular subtypes cmss defined by rnaprofiling have prognostic andorpredictive value due to postoperative timing of chemotherapy in current guidelines tumor response tochemotherapy per cms is not known which makes the differentiation between the prognostic and predictive valueimpossible therefore we propose to assess the tumor response per cms in the neoadjuvant chemotherapy settingthis will provide us with clear data on the predictive value for chemotherapy response of the cmsscontinued on next page correspondence jpmedemaamsterdamumcnljhjm van krieken jnm ijzermans jp medema and m koopman havejoint last authorship3laboratory for experimental oncology and radiobiology center forexperimental and molecular medicine cancer center amsterdamamsterdam umc university of amsterdam meibergdreef azamsterdam the netherlands5oncode institute amsterdam umc university of amsterdam amsterdamthe netherlandsfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cberg bmc cancer page of continued from previous pagemethods in this prospective single arm multicenter intervention study patients with resectable microsatellite stablect34nxm0 colon cancer will be treated with two courses of neoadjuvant and two courses of adjuvant capecitabine andoxaliplatin the primary endpoint is the pathological tumor response to neoadjuvant chemotherapy per cms secondaryendpoints are radiological tumor response the prognostic value of these responses for recurrence free survival andoverall survival and the differences in cms classification of the same tumor before and after neoadjuvant chemotherapythe study is scheduled to be performed in dutch hospitals the first patient was included in february discussion patient selection for adjuvant chemotherapy in early stage colon cancer is far from optimal the cmsclassification is a promising new biomarker but a solid chemotherapy response assessment per subtype is lacking in thisstudy we will investigate whether cms classification can be of added value in clinical decision making by analyzing thepredictive value for chemotherapy response this study can provide the results necessary to proceed to future studies inwhich neo adjuvant chemotherapy may be withhold in patients with a specific cms subtype who show no benefitfrom chemotherapy and for whom possible new treatments can be investigatedtrial registration this study has been registered in the netherlands trial register nl8177 at wwwtrialregisternltrial8177 the study has been approved by the medical ethics committee utrecht mec18712keywords colon cancer consensus molecular subtypes neoadjuvant chemotherapy surgery colon cancer is one of the most common types of cancer in the netherlands with an incidence of around patients in approximately of patients present with local disease stage iiii curativesurgery followed by adjuvant systemic chemotherapy isstandard of care in patients with microsatellite stablemss highrisk stage ii and stage iii colon cancer despite this intensive treatment of the patients develop metastatic disease these patients do not benefitenough from the current adjuvant systemic therapymoreover it is estimated that will not develop metastases after surgery alone and are therefore overtreated with adjuvant chemotherapy identifying the patients at risk of developing metastases as well as thoseresponding to therapy is a clear unmet need in coloncancer care the development of new prognostic andpredictive markers for chemotherapy response is therefore of utmost importancemany efforts have been undertaken to stratify crc patients into biologically and clinically distinct subtypesone of these led to the development of the consensusmolecular subtypes cmss which is based on rna expression profiling of tumor tissue and which is currentlyconsidered to be the most robust molecular stratificationin crc cms1 is characterized by hypermutationmicrosatellite instability msi and strong immune infiltration cms2 the canonical subtype has marked wntand myc signaling activation cms3 is enriched forkrasmutations and shows evident metabolic deregulation cms4 the mesenchymal subtype is characterizedby prominent tgf activation stromalinvasion andangiogenesis activation subtyping in a large heterogeneous patient cohort n with stage i to iv colorectal cancer showed significant differences in prognosiswith cms4 as the poorprognosis subtype confirmingthe clinical relevance of the intrinsic processes implicated in each cms these results support the idea that the cmss mighthave predictive value for response to chemotherapy dueto the postoperative timing of chemotherapy in currentguidelines the tumor response to chemotherapy is notassessable which makes a distinction between the prognostic value and predictive value of the subtypes impossible only a randomized controlled trialin whichpatients would be randomized in either surgery plus adjuvant chemotherapy or surgery alone would make thisdistinction possible howeverthis causes ethical dilemmas because chemotherapy would be withhold in patients who might actually benefit yet a solid responseassessment per subtype is necessary for implementationin clinical decisionmaking we therefore propose totreat patients with two neoadjuvant and two adjuvantcourses of capox and determine the response in tumorresected specimensapplying neoadjuvant chemotherapy may have severaladvantages the possibility of response monitoring earlyeradication of micrometastases and more complete resections neoadjuvant treatment is already standard ofcare for different gi malignancies including esophagealgastric and rectal cancers [] the foxtrot collaborative group was the first to set up a neoadjuvant trialin patients with locally advanced resectablecolon cancer and concluded that preoperative chemotherapy is feasible with acceptable toxicity and perioperative morbidity after this pilot studytheyconducted a randomized phase trial investigating theeffect of neoadjuvant chemotherapy in patients with act3 n02 m0 colon cancer patients were randomized between weeks of neoadjuvant combined with 0cberg bmc cancer page of weeks of adjuvant folfoxcapox or weeks of adjuvant folfoxcapox neoadjuvant chemotherapywas safe with less major surgical complications significant downstaging and a reduced risk of incomplete resection although the primary endpoint of the studyfreedom from recurrent or persistent disease after years was not met the risk of a recurrence after yearswas reduced to with perioperative chemotherapycompared to with adjuvant chemotherapy onlyhr p in the proposed study we will investigate the predictivevalue of the cms classification on chemotherapy response in a neoadjuvant setting including pathologicalresponse and radiological response and their correlationwith rfs and os this allows us to determine therapyefficacy in individual patients and per subtypeobjectivethe primary aim of this study is the evaluation of thepathological tumor response to neoadjuvant systemicchemotherapy per cms in patients with mss high riskstage ii and stage iii colon cancermethodsstudy designconnection ii is a prospective multicenter interventional cohort study that will be performed as a substudyofthe prospective dutch colorectal cancer cohortplcrc plcrc is a nationwide cohort study of thedutch colorectal cancer group dccg facilitating scientific research to improve the outcome and quality oflife of patients with colorectal cancer we aim to include patients in dutch hospitals that participatein plcrcin connection ii patients with a mss ct34nxm0 colon tumor will be treated with two courses ofneoadjuvant and two courses of adjuvant capecitabineand oxaliplatin capox fig and table the cmsclassification will be determined on both the pretreatment biopsies and the resection specimen at least multiregion biopsies will be taken pretreatment toensure a sample with vital tumor and sufficient rnaquality tumor response will be assessed on the resection specimen using the tumor response grading trgsystem as proposed by dworak radiologicalresponse evaluation will be centrally performed by dedicated radiologists on sequential ct scans made at baseline and after two courses of neoadjuvant chemotherapybut before resection pathologists and radiologists will beblinded for the cms classificationoptionally blood samples are taken for circulatingtumor dna ctdna analysis and plasma storage atfour time points at baseline after neoadjuvant treatment after surgery and after completion of the adjuvantchemotherapy followup will be performed until years postsurgery data on local recurrences metastasesand survival will be documentedstudy populationpatients diagnosed with resectable ct34nxm0 coloncancer are eligible for the connection ii trial baselinectscans of all patients will be reviewed by dedicated radiologists in the treating hospitals with special focus ontumor staging msi status will be determined on biopsymaterial to exclude patients with an msi tumor patients are eligible when they meet the followingconsent for the connection ii studycriteria 0f able and willing to provide written informed 0f informed consent signed for plcrc components 0f mss based on pretreatment biopsy by immunohis 0f fit to undergo neoadjuvant chemotherapy withclinical data and future studiestochemistry ihccapecitabine oxaliplatin and subsequent surgeryjudged by the primary treating physician 0f adequate bone marrow liver and renal functionpatients will be excluded if any of the following criteriaare met 0f any other malignant disease within the preceding years apart from nonmelanotic skin cancerfig flow diagram of clinical study patients with an msi tumor will be excluded from this study at time points blood samples will becollected for ctdna analyses and future biomarker studies 0cweekcheck in and exclusionsign informed consentblood withdrawal for ctdna plasmactscansurgerycapoxrecord medical history xxx axxxcxbx fxgc1d1c2d1xxc3d1exxdc4dxxberg bmc cancer page of table study flowchart of clinical studystudy proceduresinclusion neoadjuvantsurgeryadjuvant chemotherapyfollow upchemotherapy xxxdocument concomitant medicationtherapiesa blood withdrawal may be done at screening or immediately before cycle day b blood withdrawal to be performed after cycle week and before surgeryc blood withdrawal to be performed before cycle day d blood withdrawal to be performed approximately weeks after surgerye cycle day should ideally start within weeks after surgery at the latest weeks after surgeryf ct should be performed after completion of cycle and before surgeryg surgery should ideally be performed weeks after cycle day but has to be performed weeks after cycle day xcarcinoma in situ and early stage disease with a recurrence risk of less than 0f colonic obstruction that cannot be defunctioned by 0f pregnant or lactating womena stomamain study parameterendpointthe primary endpoint is the pathological tumor responseto neoadjuvant chemotherapy per cms the pathologicalresponse will be centrally scored on hestained slidesfrom the resection specimen using the tumor responsegrading system according to dworak [ ] based on results from the foxtrot study a good response will bedefined as trg2 trg3 or trg4 poor response as trg1or trg0 the cms classification will be determined onthe pretreatment biopsies and on the resection specimens rna will be isolated from ffpe material and analyzed on the ncounter sprint profiler a reliable androbust platform for samples with degraded rna such asffpe samples [ ]secondary study parametersendpoints 0f additional pathological markers to assess the tumorresponse the modified ryan scheme trs andexpression of ki67 and caspase3 and morphological cytostaticcytotoxic effects on hestained tissue slides 0f pathological response per trg and trs categoryseparately for the different cms subtypes 0f radiological tumor response to neoadjuvantchemotherapy 0f recurrence free survival rfs at two and threeyears rfs is defined as the time elapsed betweenthe diagnosis of the primary tumour and either thedate of any recurrence of disease time of death orthe date of the last followup visit at which a patientwas considered to have no recurrence 0f overall survival os at five and ten years 0f therapyinduced cms differences 0f prognostic and predictive value of cytotoxiclymphocytes cytolym and cancerassociated fibroblasts caf infiltration scores 0f diagnostic accuracy of ctdna measurements formonitoring treatment response to neoadjuvanttreatment and detection of residual disease 0f exploration of proteome profiles for monitoringtreatment response to neoadjuvant treatment anddetection of residual disease 0f percentages of pathological complete r0pathological microscopic incomplete r1 andpathologically macroscopic incomplete r2resections 0f surgical complication rate ie wound infections andanastomotic leakstatistical analysisprimary study endpointthe primary study endpoint is the pathological tumorresponse per cms using the trg system according todworak pathologic tumor regression rates withcorresponding confidence intervals will be analyzedper cms subgroup using the wilson method 0cberg bmc cancer page of secondary study endpointscategorical data pathological tumor response accordingto the modified ryan scheme are compared using chisquare analysis or fishers exact test and are shown asnumbers relative and absolute rates continuous datacytolym and caf infiltration scoresradiologicaltumor response pathological response by percentage ofki67 and caspase3 positive neoplastic cells are compared using nonparametric ttest or mannwhitney utest where appropriate and are shown as mean andstandard deviation or median and interquartile range pvalues are twotailed and results areconsidered significantthe os at and years and rfs at and years willbe calculated and depicted by means of the kaplanmeier technique and will be compared using the stratified logrank test hazard ratios and confidence intervals will be calculated with a stratified coxproportional hazard analysis the rfs will be analyzedper cms subgroup per trg and radiological responseall estimates will be accompanied by confidenceintervalssample size calculationwe based our sample size calculation on the desiredprecision with which we will be able to estimate thepathological response rates to neoadjuvant chemotherapy within each cms subtype this precision is quantified by the margin of error the radius of the confidence interval which we set at a maximum of this margin of error is achieved with patients inthe least prevalent cms subgroup namely cms3 andan anticipated pathologic responses yielding a response rate of with a 95ci of based onthe currently observed ratios of subtypes derived fromthe large consensus dataset after exclusion of the msitumors which holds cms1 tumors for most part wewill need a total of mss patients cms2 cms3 cms4 with this sample size we anticipatemaximum margins of error of and forcms2 cms3 and cms4 respectively and overallthe above depends on the assumption that the response rates will not be higher than within eachcms subgroup if response rates will actually be closerto the maximum margin of error will increasethe sample size hence indicates that for the analysis patients will be needed for whom followup andsubtype is known we expect a loss in patients dueto loss of followupinsufficient quality of the biopsymaterial or failure to faithfully assign patients to a subgroup based on the rna expression profiles resulting ina total of patients needed to have sufficient data forboth the primary and secondary outcomesdata collection and data managementdata collection and data management will be performedby the netherlands comprehensive cancer anizationiknl they have broad experience with continuousdata collection based on high quality electronic case reportforms ecrfs which guarantees complete andtimely recording handling and storage of data and documents all local and central data managers are registeredand the electronic database trias is iso certifieddata will be documented in line with good clinicalpractice gcp and dutch legal requirements major violations of the protocol will be recordedmonitoringno data and safety monitoring board dsmb will beassigned since patients are subjected to an interventionwith a low postoperative morbidity that is already beingperformed in routine clinical practice no interim analyses will be performedauditingindependent monitoring of the study is performed by aqualified monitor of iknl the monitor plan is basedon the judgement of the irb that study participation isof low to moderate risk monitoring will be performedby investigating the electronic trial database and performing site visits each participating site will be visitedat least once with repeat visits to sites where performance is a concern the quality assessment will focus onthe safety wellbeing and rights of the patients the quality of the documented data in the ecrf and their traceability to source documents and the completeness of theregulatory binder after each monitor visit the trialmonitor reports feedback to the project leader study coordinator and local investigatoradverse eventsthe treatment with capox in this study is standard ofcare therefore ae and sae are not expected to be different as both the treatment with capox and the surgery are part of the standard of care only two specificsaes are defined which are possibly related to the adjusted study schedule information will be collected onpatients who prematurely stop chemotherapy treatmentand of patients who are not able to undergo plannedsurgery due to progressive diseaseobstructionthe following two saes will be reported 0f if the surgery has to be postponed for more than 0f if patients can not complete all the neoadjuvantweeks after the start of cycle of capoxchemotherapy courses 0cberg bmc cancer page of the study coordinator will report these saes to thethat apaccredited institutional review board irbproved the study protocoldiscussioncolon cancer is one of the most common types of cancer in the netherlands the standard of care for patientswith mss high risk stage ii and stage iii colon cancercurrently consists ofsurgery followed by systemicchemotherapy patient selection for adjuvant chemotherapy is still far from optimal approximately wouldnever develop metastases after surgery alone and istherefore overtreated with adjuvant chemotherapymoreover still develop metastatic disease despite this intensive treatment leaving merely thatin fact benefit from adjuvant chemotherapy this illustrates the evident need for additional predictive markersfor chemotherapy benefitone potential marker is the cms classification whichis based on the integration of six different molecularclassification systems based on rna expression profiling the cms classification divides crc patients intofour subtypes with distinctive biological features guinney showed a clear relapse free survival and overallsurvival advantage for cms1 compared to cms4 in aheterogeneous patient cohort with stage iiv crc withdivergent treatment schemes besides the prognostic value literature provides somesupport for a predictive value of cms for response tosystemic treatment in a retrospective analysis of thensabp c07 trial on patients n with stage iiicolon cancer only cms2 was associated with benefitfrom oxaliplatin treatment patients with cms4 tumorsdid not benefit from addition of oxaliplatin treatment the mesenchymal subtype showed no benefit from5fu monotherapy compared to no systemic therapy ina nonrandomized retrospective analysis of stage iiicrc patients although being a promising molecular marker a solidchemotherapy response assessment per subtype has notbeen performed and it remains unknown whether thedifference in longterm outcome between cms1 andcms4 originates from differences in prognosis or response to therapythis makes it impossible to know whether patientswith the poorprognosis subtype cms4 have an impaired survival due to the aggressive nature of the tumoror due to a limited response to chemotherapy therefore it is unknown whether these patients should receivechemotherapy or not this also holds true for the othersubtypes although cms1 show superior outcomes tocms4 it is unknown whether this is due to a favorabletumor biology or due to a substantial response tochemotherapy wesolidtherefore believethatachemotherapy response assessment per subtype is animportant and essential step to distinguish betweenprognosis and prediction and to incorporate the cmssin clinical decisionmakingadministering neoadjuvant chemotherapy in the suggested study population was proven safe and feasible inthe foxtrot study [ ] importantly the pathological tumor response was evidently associated with recurrence free survival patients with a complete responsetrg4 developed no recurrences after years of followup compared to of patients that showed no regression at all trg0 this illustrates that the responseto chemotherapy of the primary tumor may indeed be areliable measurement for chemotherapy efficiencythe primary endpoint of the proposed study is thepathological tumor response which will be centrallyscored using the trg by dworak a highly reproduciblescoring system which is often used and clinically meaningful evidently tumor response monitoring usinghistology requires invasive procedures as a secondaryendpoint radiological response will be scored by a central board of radiologists and compared to the pathologicaltumor response to evaluate this noninvasivetechnique as a response modality both the histologicaland radiological response will be correlated to rfs andos to assess their prognostic valueinvolvementthe proposed neoadjuvant approach requires reliableclinical tnm staging to minimize the risk of overtreating patients with stage i or low risk stage ii colon cancer a metaanalysis analyzing the accuracy of t and nstaging on ct imaging showed that t1 can be reliablydistinguished from t3 sensitivity and specificity while nodalis unreliable with apooled sensitivity and specificity of and respectively therefore only t stage will be used to selectpatients second only patients with an mss status willbe included which will be determined on the biopsiesfollowing the latest recommendations of the update ofthe esmo guideline to refrain from adjuvant chemotherapy in highrisk stage ii msi colon cancer patientsas the possible clinical benefit is too low this wasalso seen in the foxtrot trial where msi status wasassociated with a significantly higher rate of poorno response vs p using the proposed selection of patients with a mss ct34nxm0colon tumor up to of patients is estimated to beovertreated [ ]results from this study in which we analyze both thepathological and radiological tumor response per cmswill lead to improved patient stratification and clearerinsight into which patients benefit from chemotherapythis will allow us to identify the group of patients thatreceives chemotherapy appropriately and the group ofpatients that may not benefit from the current treatment 0cberg bmc cancer page of regimen future studiesshould focus on whetherchemotherapy can be withheld in this patient group oron the development of new therapies to improve patientoutcomeabbreviationscaf cancer associated fibroblast capox capecitabin and oxaliplatincms consensus molecular subtype ctdna circulating tumor dnacytolym cytotoxic lymphocytes dccg dutch colorectal cancer groupesmo european society of medical oncology mec medical ethicscommittee msi microsatellite instable mss microsatellite stable os overallsurvival plcrc prospective dutch colorectal cancer cohort rfs recurrencefree survival trg tumor response gradingacknowledgementsnot applicableauthors contributionsib sw jr gv rb1 cj se dv mk1 eh wg mo rb2 jk ji jm mk2 authors make substantial contributions toconception and design andor acquisition of data andor analysis andinterpretation of data authors participate in drafting the orrevising it critically for important intellectual content authors give finalapproval of the version to be published served as scientific advisorfundingthe connection ii trial is funded by the dutch cancer society alpedhuzes the dutch cancer society is a nonprofit society that funds cancerresearch and has had no direct influence in the structuring of the trial andwill also not benefit financially from the outcomeavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the principal investigator on reasonable request results will becommunicated via plcrc presentations at international conferences and viapublications in peer reviewed journalsethics approval and consent to participatethe study has been approved by the medical ethics committee utrechtmec18712 the medical ethics committee utrecht belongs to the umcutrecht and the prinses máxima center reference number slrc19009541the study will be conducted according to the principles of the declarationof helsinki 10th version fortaleza and in concordance with the dutchmedical research improving human subjects act wmo and otherapplicable guidelines regulations and actsauthorships will be defined following the international committee ofmedical journal editors guidelines the patients treating physicians local investigator or research nurse of theparticipating hospitals will follow ichgcp and other applicable regulationsin informing the patient and obtaining consent this includes explaining theconnectionii study to the patient providing himher with informationsuch as the expected efficacy and possible side effects and that refusal toparticipate will not influence further options for therapy before informedconsent may be obtained the investigator should provide the patient ampletime and opportunity to inquire about details of the trial and to decidewhether or not to participate in the trial all questions about the trial shouldbe answered to the satisfaction of the patient only after written informedconsent the patient will be included in this study the inclusion has to takeplace shortly after diagnosis to prevent delay in treatmentpatients are well informed that participation in voluntary and that they maywithdraw at any point during the studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests nielsen t wallden b schaper c ferree s liu s gao d analyticalvalidation of the pam50based prosigna breast cancer prognostic geneauthor details1department of surgery erasmus mc university medical center rotterdamrotterdam the netherlands 2department of medical oncology universitymedical center utrecht utrecht university utrecht the netherlands3laboratory for experimental oncology and radiobiology center forexperimental and molecular medicine cancer center amsterdamamsterdam umc university of amsterdam meibergdreef azamsterdam the netherlands 4department of pathology radboud universitymedical centre nijmegen the netherlands 5oncode institute amsterdamumc university of amsterdam amsterdam the netherlands 6netherlandscomprehensive cancer anisation department of research utrecht thenetherlands 7department of medical oncology amsterdam umc locationvumc amsterdam the netherlands 8julius center for health sciences andprimary care university medical center utrecht utrecht university utrechtthe netherlands 9department of radiology the netherlands cancer instituteamsterdam the netherlands 10department of surgery university medicalcenter utrecht utrecht university utrecht the netherlandsreceived may accepted july referencesnederlandse kankerregistratie nkr iknl retrieved from wwwiknlnlnkrcijfers in may guinney j dienstmann r wang x de reynies a schlicker a soneson c the consensus molecular subtypes of colorectal cancer nat medvan hagen p hulshof mc van lanschot jj steyerberg ew van bergehenegouwen mi wijnhoven bp preoperative chemoradiotherapy foresophageal or junctional cancer n engl j med bosset jf mercier m triboulet jp conroy t seitz jf surgical resection withand without chemotherapy in oesophageal cancer lancet author reply cunningham d allum wh stenning sp thompson jn van de velde cjnicolson m perioperative chemotherapy versus surgery alone forresectable gastroesophageal cancer n engl j med sebagmontefiore d stephens rj steele r monson j grieve r khanna s preoperative radiotherapy versus selective postoperativechemoradiotherapy in patients with rectal cancer mrc cr07 and ncicctgc016 a multicentre randomised trial lancet roh ms yothers ga connell mjo beart rw pitot hc shields af theimpact of capecitabine and oxaliplatin in the preoperative multimodalitytreatment in patients with carcinoma of the rectum nsabp r04 j clinoncol foxtrot collaborative g feasibility of preoperative chemotherapy for locallyadvanced operable colon cancer the pilot phase of a randomisedcontrolled trial lancet oncol matthew t seymour dm and on behalf of the international foxtrot trialinvestigators foxtrot an international randomised controlled trial in patients pts evaluating neoadjuvant chemotherapy nac for colon cancerj clin oncol 20193715_suppl3504 coebergh van den braak rrj van rijssen lb van kleef jj vink gr berbeem van berge henegouwen mi nationwide comprehensive gastrointestinal cancer cohorts the 3p initiative acta oncol dworak o keilholz l hoffmann a pathological features of rectal cancerafter preoperative radiochemotherapy int j colorectal dis veldmanjones mh brant r rooney c geh c emery h harbron cg evaluating robustness and sensitivity of the nanostring technologiesncounter platform to enable multiplexed gene expression analysis ofclinical samples cancer res weissenberg e tnm staging of colorectal carcinoma ajcc 8th ed song n poguegeile kl gavin pg yothers g kim sr johnson nl clinical outcome from oxaliplatin treatment in stage iiiii colon canceraccording to intrinsic subtypes secondary analysis of nsabp c07nrgoncology randomized clinical trial jama oncol lee j sohn i do ig kim km park sh park jo nanostringbasedmultigene assay to predict recurrence for gastric cancer patients aftersurgery plos one 20149e90133 0cberg bmc cancer page of signature assay and ncounter analysis system using formalinfixed paraffinembedded breast tumor specimens bmc cancer roepman p schlicker a tabernero j majewski i tian s moreno v colorectal cancer intrinsic subtypes predict chemotherapy benefit deficientmismatch repair and epithelialtomesenchymal transition int j cancer nerad e lahaye mj maas m nelemans p bakers fc beets gl diagnostic accuracy of ct for local staging of colon cancer a systematicreview and metaanalysis ajr am j roentgenol committee eg eupdate early colon cancer treatment recommendations murakami k west n westwood a hemmings g bottomley d davis j the relationship between dna mismatch repair and response to folfoxbased preoperative chemotherapy in the international phase iii foxtrottrial journal of patholog | 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 |
in china esophageal cancer ec is the fourth most frequently diagnosed form of malignant tumor inmales and the fifth most commonly diagnosed form in females approximately and casesoccurred in respectively the incidence of ec in eastern asia is in the top five worldwide including china esophageal squamous cell carcinoma escc is a major histological subtype accountingfor of all ec cases the complex interaction of economical and environmental conditions with individuals hereditary factors may lead to ec development the etiology and development of ec is notfully understood despite many investigations have payed close attention to the importance of immunity recently it was hypothesized that some important variants in immunerelated genes may influencethe susceptibility of esccreceived november revised july accepted july accepted manuscript online august version of record published august the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895interleukin4 il4 coded by the il4 gene is an important regulator of the inflammation pathways il4 apleiotropic cytokine may be correlated with survival and growth of lymphocytes il4 is produced by mast cellprecursors and by the tcell thymocyte populations it is important for bcell activation proliferation and differentiation it is reported that il4 is necessary for producing immunoglobulin e and implicated in immune diseasesin the process of innate immune responses il4 may activate m2 macrophage and then play a specific role it hasantiinflammatory effect which is relevant to the development of escc recently a number of studies have focusedon the relationship of il4 with cancer development il4 single nucleotide polymorphisms snps have alsobeen explored for an association with susceptibility to cancer [] the rs2070874 tc located in the 5cid3utrregion of the il4 gene is an important snp in cancer development some metaanalyses have indicated that il4rs2070874 may be associated with cancer development in asian populations [] kim reported that il4rs2070874 might affect the role of aspirin in regulating il4 expression rs2243263 gc polymorphism is anintron snp of il4 gene this intron snp might play a role in splicing although the exact role of this intron snpis unknown the associations of il4 rs2243263 gc snp with the human disease have been explored a previousstudy suggested that il4 rs2243263 was associated with the reverse seroconversion of hepatitis b virus hbv this snp was also studied for the relationship of the susceptibility to cancer a previous report investigated the correlation of the il4 rs2243263 locus with colorectal cancer although in this study a null association was identifiedhowever lan in a large simple size study found that the il4 rs2243263 gc snp might increase the susceptibility to nonhodgkin lymphoma currently the associations of il4 the rs2070874 tc and rs2243263 gcpolymorphisms with escc development are unknownthe il10 gene is located in chromosome 1q322 il10 another immune regulator serves as an inhibitor of dendritic cells and macrophages and inhibits the production of many inflammatory cytokines eg tumor necrosis factorα il1 il6 il12 and others il10 is a vital antiinflammatory regulator after il10 combineswith its receptor il10r signal transducer and activator of transcription is triggered which plays a vital role inantiapoptosis and proliferation an investigation found that the upregulated mrna expression of the il10gene and higher serum levels of il10 were found among subjects who carried the rs1800896 gallele thers1800872 snp a promotor variant could influence the level of il10 protein some investigations have suggested that the il10 rs1800896 ag and rs1800872 ac variants may influence thesusceptibility to escc of late a metaanalysis indicated that these il10 snps increased the risk of ec however in this earlier metaanalysis the sample size was very limited ec patients and controls includedthe association of the il10 rs1800896 ag and rs1800872 ac polymorphisms with ec development should befurther studiedherpesvirus entry mediator hvem also known as tnfrsf14 plays a major role in the immune response[] hvem has been found to be expressed in lymphoid cells as well as in other cells a previous study suggestedthat the hvemb and tlymphocyte attenuatorlymphotoxincd160 network in immune reaction to infection andinflammation could play a bidirectional regulatory role several investigations have focused on the role of hvemin cancer survival [] zhu reported that higher expression of hvem may promote apoptosis and herald agood prognosis for bladder cancer patients additionally a previous study has indicated that hvem is implicatedin the development of breast cancer bc a snp in the hvem gene the g to a of rs2234167 in the exon regionwas found to influence the development of bc however the association of hvem rs2234167 ga snp withthe expression of hvem is unknown recently migita found that hvem is critical for both tumor survival andthe escape of the host immune system in escc cases thus it could be a useful target for escc therapy to dateinvestigation has not been performed to identify a relationship of the hvem rs2234167 ga polymorphism withescc susceptibilitytherefore in this investigation the hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 andrs1800872 polymorphisms were selected and investigated for their effect on escc development in a chinese hanpopulationmaterials and methodssubjectsour casecontrol study was performed in fujian union hospital fuzhou china and the no1 peoples hospital of zhenjiang city zhenjiang china this investigation was approved by jiangsu university registration idk20160036y and fujian medical university registration id 2016zqn25 participants were recruited betweenfebruary and april our study included escc cases and controls these escc patients werehistopathologically confirmed and were from to years old controls were cancerfree individuals from to 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 bsr20193895101042bsr20193895 years old the controls were not related to any escc case using a prestructured questionnaire we collectedepidemiological data from participants the escc patients and normal controls signed consent formsdna extraction and genotyping of hvem rs2234167 il4 rs2070874 andrs2243263 and il10 rs1800896 and rs1800872 lociwe collected a blood sample ml from each participant dna was extracted carefully as described in a previousstudy using an snpscan¢ assay genesky biotechologies inc shanghai china we determined the genotypesof hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 polymorphisms to confirm the accuracy of genotyping samples were selected and retested the genotypes of hvem rs2234167 il4rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 loci were reanalyzed by another technician thegenotypes of hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 snps wereunchangedstatistical analysisthe difference in alcohol consumption body mass index bmi gender cigarette use and age were tested by using Ï2 test mean age was calculated by using a students t test we used a chisquare test Ï2 or fishers exact testto determine whether the frequencies of hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896and rs1800872 variants in escc cases and controls were different a multivariate logistic regression analysis methodwas used to calculate the crude and adjusted odds ratios ors and confidence intervals cis sas softwarepackage sas institute inc cary nc usa the relationship of hvem rs2234167 il4 rs2070874 and rs2243263and il10 rs1800896 and rs1800872 polymorphisms with escc development was determined by ors and cisthe statistical significance of all analyses was p005 twosided an internetbased hardyweinberg equilibriumhwe test httpihggsfdecgibinhwhwa1pl was also harnessed to assess whether the distribution of hvemrs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 genotypes could represent the included populationresultsbaseline characteristicsin total escc cases and controls were recruited table of these escc cases were females and were males average age was years in the control group there were females and maleswith an average age of years there was no difference in terms of mean age p0413 the categoricalvariables age and gender were wellmatched p005 however the distribution of other categorical variables egtobacco use bmi and drinking status were significantly different all p0001 among escc cases there were with lymphatic metastasis the ajcc version criteria was used to determine the escc stage and escc cases were stage iii and were stage iiiiv after genotyping the participants the association ofhvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 genotypes with escc riskwas assessedthe minor allele frequencies mafs of hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896and rs1800872 loci are shown in table they are similar to the data of chinese population as presented in table the hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 genotypes in controlsaccorded with hwerelationship of hvem rs2234167 il4 rs2070874 and rs2243263 andil10 rs1800896 and rs1800872 loci with escctable shows the hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 genotypesthe frequencies of il4 rs2070874 tt tc and cc genotypes were and inescc cases and and in controls when the reference was il4 rs2070874 ttgenotype we found the il4 rs2070874 cc genotype significantly decreased the risk of escc p0023 when thereference was il4 rs2070874 tttc genotype the il4 rs2070874 cc genotype also significantly decreased the riskof escc p0028 adjustment for bmi smoking drinking age and gender the decreased susceptibility was alsoidentified cc vs tt p0008 cc vs tttc p0010hvem rs2234167 il4 rs2243263 and il10 rs1800896 and rs1800872 genotypes are shown in table both crudeand adjusted comparisons indicated that hvem rs2234167 il4 rs2243263 and il10 rs1800896 and rs1800872 lociwere not associated with the risk of escc 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 0ctable distribution of selected demographic variables and risk factors in escc cases and controlsbioscience reports bsr20193895101042bsr20193895p1variableage yearsage yearssex¥malefemaletobacco usenevereveralcohol usenevereverbmi kgm2¥lymph node statuspositivenegativetmn stageiiiiiiivgradeg1g2g3cases n721n controls n1208n bold values are statistically signiï¬cant p005 abbreviation tmn tumorlymph nodemetastasis1twosided Ï2 test and students t testtable primary information for the included snpsgenotyped polymorphismschromosomeposition regionmaf1 in database 1000g chinese hanpopulatonsmaf in our controls n1208pvalue for hwe2 test in our controls genotyping value1maf2hwehvemrs2234167 gail4 rs2070874tcil4 rs2243263gcil10 rs1800872tgil10 rs1800896tc3cid3utr5cid3utrintron variant5cid3ï¬anking5cid3ï¬ankingadditionally a subgroup analysis was conducted by escc stage we identified an association between il4rs2070874 tc snp and the decreased susceptibility of escc in stage iii subgroup cc vs tt p0022 cc vstttc p0025 table however this association could not been identified for other snps 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 bsr20193895101042bsr20193895table the frequencies of hvem rs2234167 il4 rs2070874 rs2243263 and il10 rs1800896 and rs1800872polymorphisms in different escc subgroupsstage iii patientsstage iiiiv patientsn328n393controls n1208ngenotypehvem rs2234167 gagggaaaa alleleil4 rs2070874 tctttcccc alleleil4 rs2243263 gcgggcccc alleleil10 rs1800872 tgtttgggg alleleil10 rs1800896 tctttcccc alleleoverall casesn721nnnrelationship of hvem rs2234167 il4 rs2070874 and rs2243263 andil10 rs1800896 and rs1800872 loci with escc in stratiï¬ed analysesin a stratified analysis the il4 rs2070874 genotypes are listed in table after an adjustment we suggested that il4rs2070874 c allele was a protective factor for escc in five subgroups male subgroup cc vs tt p0028 cc vstttc p0031 ¥ years old subgroup cc vs tt p0026 cc vs tttc p0029 never smoking subgroupcc vs tt p0041 cctc vs tt p0013 and tc vs tt p0042 drinking subgroup cc vs tt p0025cc vs tttc p0024 and bmi kgm2 subgroup cc vs tt p0010 cc vs tttc p0012 in othersubgroups no association of l4 rs2070874 with escc risk was found table the il4 rs2243263 gc genotypes in the stratified analysis are listed in table after adjustment we identifiedthat il4 rs2243263 gc polymorphism was a risk factor for escc development in the bmi ¥ kgm2 subgroupgc vs gg p0030 and gccc vs gg p0018 table in other stratified analyses adjustment comparisons suggested that hvem rs2234167 and il10 rs1800872 andrs1800896 loci did not confer a risk of escc data not shownassociation of hvem rs2234167 il4 rs2070874 and rs2243263 andil10 rs1800896 and rs1800872 loci with lymphatic metastasis in escccasesamong the escc patients patients had lymphatic metastasis as presented in table we found a null association of hvem rs2234167 il4 rs2070874 rs2243263 and il10 rs1800896 and rs1800872 snps with differentlymph node status 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 0cilcenseccbytheauthorsthisisanopenaccessarticelpublishedbyportlandpressilmitedonbehalfofithebochemcailsocetyianddistributedunderthecreativecommonsattributiontable logistic regression analyses of association of hvem rs2234167 il4 rs2070874 rs2243263 and il10 rs1800896 and rs1800872polymorphisms with risk of esccgenotypehvem rs2234167 gaga vs ggaa vs gggaaa vs ggaa vs gggail4 rs2070874 tctc vs ttcc vs tttccc vs ttcc vs tttcil4 rs2243263 gcgc vs cccc vs gggccc vs ggcc vs gggcil10 rs1800872 tgtg vs ttgg vs ttggtg vs ttgg vs tttgil10 rs1800896 tctc vs ttcc vs tttccc vs ttcc vs tttcpatientsn721vscontrolsoveralln1208crude or cipadjustedor1 ci pstage iii patients n328 vs controlsn1208crude or ciadjustedor1 ci ppstage iiiiv patients n393 vs controlsn1208crude or ciadjustedor1 ci pp 1adjusted for age sex smoking status alcohol use and bmi status bold values are statistically signiï¬cant p005iiboscencereportsbsrbsr 0cbioscience reports bsr20193895101042bsr20193895table stratiï¬ed analyses between il4 rs2070874 tc polymorphism and crc risk by sex age bmi smokingstatus and alcohol consumptionil4 rs2070874 tccasecontrol1tttccctttcadjusted or2 ci pcctc cccc vs tcttvariablesexmalefemaleage years¥smoking statusnevereveralcohol consumptionnevereverbmi kgm2¥ p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1for il4 rs2070874 tc the genotyping was successful in crc cases and controls2adjusted for multiple comparisons [age sex bmi smoking status and alcohol consumption besides stratiï¬ed factors accordingly] in a logisticregression modelbold values are statistically signiï¬cant p005association of hvem rs2234167 il4 rs2070874 and rs2243263 andil10 rs1800896 and rs1800872 loci with tumor grade of escc casesas presented in table patients had welldifferentiated tumors had moderately differentiated tumors and has poorly differentiated tumors we found an association of the il10 rs1800872 tg snp with a worse differentiation tg vs tt p0048 and ggtg vs tt p0032 table discussionimmunotherapy is altering how we comprehend malignancies and offers new methods to treat them ec is a representative model of immune and inflammationrelated cancer recently some studies indicated that the snps ininflammation and immunerelated genes might influence the risk of ec in this study we explored the role ofimmunerelated gene snps hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872to escc development we observed that il4 rs2070874 tc could decrease a risk to escc even in the stage iiisubgroup however in bmi ¥ kgm2 subgroup il4 rs2243263 gc might increase the risk of escc we alsofound an association of the il10 rs1800872 tg snp with a worse differentiationil4 is an important regulator of immune and inflammation pathways some reports have suggested that il4 levelsare higher in untreated escc patients than in controls [] it is considered that il4 levels may be implicated inthe development of escc the il4 rs2070874 tc polymorphism is a 5cid3utr snp in a highrisk gastric cancergc region a previous study suggested that rs2070874 c allele in the il4 gene might decrease the susceptibilityto gc in a chinese population lu reported that the rs2070874 c allele increased the risk of hcc in amale subgroup however chang and wang found that the il4 rs2070874 polymorphism might notinfluence the susceptibility of cancer in chinese population in this study we included subjects andinvestigated the correlation of this snp to escc susceptibility we found that il4 rs2070874 tc polymorphism 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 stratiï¬ed analyses between il4 rs2243263 gc polymorphism and crc risk by sex age bmi smokingstatus and alcohol consumptionbioscience reports bsr20193895101042bsr20193895il4 rs2243263 gccasecontrol1gggcccgggcadjusted or2 ci pccgccccc vs gcggvariablesexmalefemaleage¥smoking statusnevereveralcohol consumptionnevereverbmi kgm2¥ p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1for il4 rs2243263 gc the genotyping was successful in crc cases and controls2adjusted for multiple comparisons [age sex bmi smoking status and alcohol consumption besides stratiï¬ed factors accordingly] in a logisticregression modelbold values are statistically signiï¬cant p005seemed to be a protective factor for escc development our findings were similar to a previous metaanalysis thatsuggested that the il4 rs2070874 c allele could be associated with a decreased susceptibility of gastrointestinal cancer a functional study indicated that the il4 rs2070874 allele c could promote a higher level of il4 in plasma il4 has an antiinflammatory effect and may decrease the risk of escc by inhibiting the inflammation fitzgerald reported that the il4 rs2070874 allele c could decrease the risk of prostate cancer specific mortality consistent with that report we identified an association between the il4 rs2070874 tc snp and a decreasedsusceptibility to escc in the stage iii subgroup however we did not find an association of il4 rs2070874 tcpolymorphism with lymphatic metastasis this might be due to the limited sample sizes in the future the relationshipof the rs2070874 snp in il4 gene with progress and prognosis should be further exploredrs2243263 gc an intron snp in the il4 gene was studied for the relationship of this snp to some diseasesthis snp might decrease the risk of asthma in the african american children while this relationship was not identified in caucasians hsiao reported that the il4 rs2243263 c allele was a protective factor for hbv surfaceantigen reverse seroconversion in nonhodgkin lymphoma cases undergoing rituximab treatment a previous studyinvestigated the relationship of il4 rs2243263 gc with colon and rectal cancer risk but no association wasfound however in a large simple size study lan found that the il4 rs2243263 gc snp increased the susceptibility to nonhodgkin lymphoma in this study we found that the il4 rs2243263 gc might increase therisk of escc in obese and overweight subjects table it was reported that the il4 level in mothers was inverselylinked to overweight in early childhood and might influence the metabolic profile of childhood in addition thelevel of il4 decreased with antipsychoticinduced weight gain it is suggested that the level of il4 could influence obesity and overweight introns are regulatory sequences that can affect the expression of genes here we foundthat the rs2243263 gc polymorphism a snp in il4 intron region might alter the risk of escc it is presumed 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 bsr20193895101042bsr20193895table logistic regression analyses of association between hvem rs2234167 il4 rs2070874 rs2243263 andil10 rs1800896 and rs1800872 polymorphisms and lymph node status in escc patientsgenotypepositive n405negative n316crude or cipadjusted or1 ciphvem rs2234167 gagggaaaga aagggaaaa alleleil4 rs2070874 tctttccccctctttcccc alleleil4 rs2243263 gcgggccccgccgggcccc alleleil10 rs1800872 tgtttgggggtgtttgggg alleleil10 rs1800896 tctttccccctctttcccc allelenn1adjusted for age sex smoking alcohol use and bmi status that the rs2243263 gc polymorphism influences the level of il4 by regulating gene transcription in the future afunctional study should be considered to explore the potential mechanismthe il10 rs1800872 tg is a promotor snp torrespoveda reported that the expression of il10 mrnaand the level of serum il10 were significantly higher in subjects with the il10 rs1800872 t allele a recent studyfound that il10 rs1800872 tg snp promoted the risk of ec a metaanalysis also confirmed this association in our casecontrol study we did not find the association of il10 rs1800872 tg snp with the developmentof ec even in stratified analyses and reviewing different lymph node status additionally liu reported thatil10 rs1800872 gg genotypes predicted the worse survival of diffuse large bcell lymphoma patients treated withrituximabchop cyclophosphamide doxorubicin vincristine and prednisone in this study we found that 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 bsr20193895101042bsr20193895table logistic regression analyses of association between hvem rs2234167 il4 rs2070874 rs2243263 andil10 rs1800896 and rs1800872 polymorphisms and grades of esccgenotypeg2g3 n579hvem rs2234167 gagggaaaga aagggaaaa alleleil4 rs2070874 tctttccccctctttcccc alleleil4 rs2243263 gcgggccccgccgggcccc alleleil10 rs1800872 tgtttgggggtgtttgggg alleleil10 rs1800896 tctttccccctctttcccc allelenng1 n142crude or cipadjusted or1 cip 1adjusted for age sex smoking alcohol use and bmi statusbold values are statistically signiï¬cant p005the il10 rs1800872 g allele was associated with poorly differentiated tumor thus in the future the association ofthe il10 rs1800872 tg snp and the survival of escc cases should be further studiedlimitations in the present study should be acknowledged first in the present study we only included five functional snps and explored the association of the risk to escc second there were other environmental risk factorseg vegetable and fruit intake aspirin and nsaids use and physical exercise which we did not consider for theirinfluence to the development of escc third the number of escc patients was limited and our study may be 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 bsr20193895101042bsr20193895underpowered in some subgroups fourth in this investigation the protein expression levels of the suspect factors were not measured finally considering the low penetrance of snp the other functional polymorphisms in thehvem il4 and il10 genes should not be ignoredin summary the present study suggests that the il4 rs2070874 tc polymorphism is a protective factor for esccdevelopment while the il4 rs2243263 gc increases a risk to escc in obese and overweight subjects additionallyit is highlighted that the il10 rs1800872 g allele is associated with poorly differentiated tumorcompeting intereststhe authors declare that there are no competing interests associated with the manuscriptfundingthis work was supported in part by the young and middleaged talent training project of health development planning commission in fujian province [grant number 2016zqn25] the program for new century excellent talents in fujian province university[grant number ncetfj2017b015] and the joint funds for the innovation of science and technology fujian province [grantnumber 2017y9099]author contributionall authors contributed significantly to the present study conceived and designed the experiments wt and mk performed theexperiments sc rc and cl analyzed the data wt and mk contributed reagentsmaterialsanalysis tools mk wrote themanuscript sc and rc other please specify noneacknowledgementswe appreciate all subjects who participated in the present study we wish to thank dr yan liu genesky biotechnologies incshanghai china for technical supportabbreviationsajcc american joint committee on cancer bc breast cancer bmi body mass index ci confidence interval ecesophageal cancer escc esophageal squamous cell carcinoma gc gastric cancer hbv hepatitis b virus hvem herpesvirus entry mediator hwe hardyweinberg equilibrium il interleukin nsaid nonsteroidal antiinflammatory drug or odds ratio snp single nucleotide polymorphismreferences bray f ferlay j soerjomataram i global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin 103322caac21492 matejcic m and iqbal parker m geneenvironment interactions in esophageal cancer crit rev clin lab sci 1031091040836320151020358 qin jm yang l chen b interaction of methylenetetrahydrofolate reductase c677t cytochrome p4502e1 polymorphism andenvironment factors in esophageal cancer in kazakh population world j gastroenterol pmcid pmc2773864103748wjg146986 lin ew karakasheva ta hicks pd the tumor microenvironment in esophageal cancer oncogene pmcidpmc5003768 101038onc201634 park r williamson s kasi a immune therapeutics in the treatment of advanced gastric and esophageal cancer anticancer res 1021873anticanres12891 nelms k keegan ad zamorano j the il4 receptor signaling mechanisms and biologic functions annu rev immunol 101146annurevimmunol171701 rush js and hodgkin pd b cells activated via cd40 and il4 undergo a division burst but require continued stimulation to maintain divisionsurvival and differentiation eur j immunol 101002152141412001043143c1150aidimmu11503e30co2v suzuki a leland p joshi bh targeting of il4 and il13 receptors for cancer therapy cytokine 101016jcyto201505026 francipane mg alea mp lombardo y crucial role of interleukin4 in the survival of colon cancer stem cells cancer res 10115800085472can076874 tan n song j yan m association between il4 tagging single nucleotide polymorphisms and the risk of lung cancer in china molgene genom med e00585 pmcid pmc6465665 101002mgg3585 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc by 0cbioscience reports bsr20193895101042bsr20193895 cardenas dm sanchez ac rosas da preliminary analysis of singlenucleotide polymorphisms in il10 il4 and il4ralpha genesand proï¬le of circulating cytokines in patients with gastric cancer bmc gastroenterol pmcid pmc6288868101186s1287601809139 shamoun l skarstedt m andersson re association study on il4 il4ralpha and il13 genetic polymorphisms in swedish patientswith colorectal cancer clin chim acta 101016jcca201809024 jia y xie x shi x associations of common il4 gene polymorphisms with cancer risk a metaanalysis mol med rep pmcid pmc5561993 103892mmr20176822 cho ya and kim j association of il4 il13 and il4r polymorphisms with gastrointestinal cancer risk a metaanalysis j epidemiol pmcid pmc5394226 101016jje201606002 zhenzhen l xianghua l qingwei w three common polymorphisms in the il4 gene and cancer risk a metaanalysis involving cases and controls tumour biol 101007s1327701307618 kim bs park sm uhm tg effect of single nucleotide polymorphisms within the interleukin4 promoter on aspirin intolerance inasthmatics and interleukin4 promoter activity pharmacogenet genomics hsiao lt wang hy yang cf human cytokine genetic variants associated with hbsag reverse seroconversion in rituximabtreatednonhodgkin lymphoma patients medicine baltimore e3064 pmcid pmc4839912 101097md0000000000003064 bondurant kl lundgreen a herrick js interleukin genes and associations with colon and rectal cancer risk and overall survival intj cancer pmcid pmc3470814 101002ijc27660 lan q wang ss menashe i genetic variation in th1th2 pathway genes and risk of nonhodgkin lymphoma a pooled analysis ofthree populationbased casecontrol studies br j haematol pmcid pmc3075370101111j13652141201008424x kwasniak k czarnikkwasniak j maziarz a scientiï¬c reports concerning the impact of interleukin interleukin and transforminggrowth factor beta on cancer cells central eur j immunol pmcid pmc6745546 105114ceji201876273 dandrea a asteamezaga m valiante nm interleukin il10 inhibits human lymphocyte interferon gammaproduction bysuppressing natural killer cell stimulatory factoril12 synthesis in accessory cells j exp med pmcid pmc2191152101084jem17831041 miteva ld stanilov ns deliysky ts signiï¬cance of 1082ag polymorphism of il10 gene for progression of colorectal cancer andil10 expression tumour biol 101007s1327701425892 pereira apl trugilo kp okuyama ncm il10 c592ca rs1800872 polymorphism is associated with cervical cancer j cancerres clin oncol 101007s00432020032560 yang y and fa x role of il10 gene polymorphisms on the susceptibility for esophageal cancer and its association with environmental factorsint j clin exp pathol pmcid pmc4583954 sun jm li q gu hy interleukin rs1800872 tg polymorphism was associated with an increased risk of esophageal cancer in achinese population asian pac j cancer prev zhao x lu c chu w microrna214 governs lung cancer growth and metastasis by targeting carboxypeptidased dna cell biol 101089dna20163398 breloer m hartmann w blankenhaus b cutting edge the btlahvem regulatory pathway interferes with protective immunity tointestinal helmin | Colon_Cancer |
" the diagnostic gold standard of hirschsprungs disease hd is based on the histopathological assessmentof colorectal biopsies although data on cholinergic innervation and ganglion cell gc distribution exist only few studieshave examined these two key features together we assessed the pattern of cholinergic innervation and the amount ofgcs in colorectal specimens of hd patientsmethods we established a semiquantitative score for cholinergic innervation using acetylcholinesterase ache enzymehistochemistry and quantitatively analyzed the number of gcs via nadh tetrazolium reductase nadh enzymehistochemistry we examined both the entire length of the resected specimens as well as defined areas of the transitionzone of both pathological and healthy appearing segmentresults high ache score values were associated with absence of gcs and ache scores were inversely correlated withthe number of gcs nevertheless we observed several cases in which one of the two features revealed a normaldistribution pattern whereas the other still displayed pathological featuress our data support the need for transmural colon biopsies to enable the best evaluation of both cholinergicinnervation and gcs for a reliable assessment of hdkeywords hirschsprungs disease colon rectosigmoid ache aganglionosis hirschsprungs disease hd is a rare cause of constipation in infants that is microscopically characterized bycongenital absence of gcs in a variable extension mainlyaffecting the rectum and sigmoid colon hd has a prevalence of approximately in newborns male babiesare more frequently affected with a ratio of malefemale a registerbased study has described a slightincrease in incidence over the recent years the clinicalsymptoms of hd comprise severe chronic constipation correspondence michelmittelbronnlnsetatlu anne k braczynski and stefan gfroerer contributed equally to this work4institute of neurology edinger institute goethe university frankfurtgermany13department of oncology donc luxembourg institute of health lihstrassen luxembourgfull list of author information is available at the end of the ongoing defecation problems and occasionally bowel obstruction most newborns with hd present with a delayedmeconium passage in infancy a postnatal passagetime of longer than h combined with defecationproblems is clinically suggestive for hd the treatmentof choice for hd is the surgical resection of the affectedaganglionic segment the transition zone which is onlypartially populated by gcs should also be resected theoperation technique should preserve the anorectal function which is usually achieved by connecting the distalfunctional bowel with the sphincter muscle at the area ofthe dentate line despite proper surgical treatmentsthe quality of life may remain negatively affected duringchildhood most cases of hd occur sporadicallynevertheless genetic causes of this disorder have beenidentified [ ] the most frequent mutations affect 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 0cbraczynski bmc pediatrics page of ret gene that codes for a receptor tyrosine kinase approximately different ret mutations have been reported in hd patients accounting for of familialcases of hd and to of sporadic cases of hd the diagnosis of hd is histopathologically confirmed bythe absence of gcs in the enteric plexus of the distal rectum usually several biopsies ranging between and cmabove the dentate line [] via either superficial suction or a fullthickness transmural specimen intraoperative diagnostics to detect gcs is most frequentlybased on the hematoxylin and eosin he staining ofcryosections this method can be combined with enzymehistochemistry for i the assessment of cholinergic nerve fibers using acetylcholinesterase ache and ii the detection of ganglion cells via lactate dehydrogenase ldhsuccinate dehydrogenase sdh and nadh tetrazoliumreductase reactions nadh [ ] although averagevalues exist concerning the number and distribution of ganglion cells in normal intestines and intestines affected byhd these values are highly dependent on the stainingmethod histopathologically the diagnosis consists ofan absence of gcs and the presence of hypertrophicachepositive nerve fibers however little is known aboutthe correlation of the gc distribution pattern and the cholinergic innervation hd cases therefore in the currentstudy we aimed at analyzing both the extent and distribution pattern of cholinergic innervation in the mucosal layerin relation to the density of gcs in the myenteric plexus ina cohort of hd patientsmethodspatient datahd patients who were included in this retrospectivemonocentric study underwent transanal pull throughtapt surgeries at the department of pediatric surgery university hospital frankfurtmain germany inall cases the diagnosis of hd was confirmed by transanal rectal biopsies prior to tapt surgeries the specimens for detailed information please see table wereindependently reviewed by at least two experienced neuropathologists akb pnh mm from the institute ofneurology according to the diagnostic criteria that wereproposed by knowles patients with longsegmentdiseases were excluded from this study the ages at thetime of the surgeries ranged from days to monthsmean age months the study cohort consisted of male m and female f patientsethical statementthe parents of the patients gave written informed consent for the surgery the use of patient material wasapproved by the ethical committee ofthe goetheuniversity frankfurt germany gn samplingpartial colectomy specimens were examined and measured n additionally each resected bowel segment was macroscopically analyzed with regard to thechanges in diameter in the zones of bowel obstructionthe length between the proximal resection margin andthe beginning of the dilated colon was documentedfig 1a swiss roll specimens were prepared accordingto the following guidelines first from each of the entirerectosigmoidal specimens a longitudinal cm widestrip was excised in paramedian orientation to the antimesocolic tenia afterwards the strip of the intestinalwall was coiled in caudocranial orientation in cryogel tissue tek oct sakura alphen aan den rijntable sample dataidlength of aganglionic segment total length cmswiss rollxtz cmxtz cmxtz cmxtz cmna125abbreviations x specimen available na not availablexxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx 0cbraczynski bmc pediatrics page of fig sampling and study design a intraoperative aspect of rectosigmoid colon during tapt procedure there was a notable change ofdiameter at the beginning of tz tz cm white lines mark the biopsy sites at and cm proximal to the beginning of the transition zonetz cm asterisk indicating aboraldistal end of colon arrowhead the oralproximal resection region b swiss roll specimens stained for heache nadh and ldh asterisk and arrowhead as described in a c first we analyzed the swiss rolls of the fulllength specimens and locatedareas with each of the four grades of the achescore in the same region we counted gcs d second we analyzed the specimen tz and cm for their respective achescore ache and counted the gcmm in the corresponding area nadhnetherlands attached to a cork plate and snap frozensee below swiss rolls were then used to analyze theoverall innervation pattern the serial biopsies referring to the specimens from the transition zone tz tz cm cm cm and cm were prepared as followsthe beginning of the change in the diameter was definedas the beginning of the transition zone and referred toas tz cm a horizontal incisional antimesocolic biopsyof approximately cm à cm was excised at cm further rostral biopsies were performed at cm cm and cm tz cm tz cm tz cm fig 1a the biopsies were fixed in cryogel on the cryostat specimenholder all specimens were frozen at °c in isopentane which was precooled in liquid nitrogen then μmthick sections were cut with a manual cryostat system leica cm wetzlar germany 0cbraczynski bmc pediatrics page of staining and enzyme histochemistrycryosections were stained with he nadh reactionldh reaction and ache reaction according to standardprotocols fig 1b the incubations were performed at °c for min nadh min ldh and minache stained sections were mounted in entellanmerck millipore darmstadt germanyscoringbefore performing semiquantitative and quantitativeanalyses a joint training was done to reduce interobserver variability akb pnh mm in case of doubtduring quantification a joint analysis was performeduntil agreement was reached a semiquantitative scoring system was applied to evaluate the pathological cholinergic innervation in the mucosal layer fig 1c d thefour grades of the ache score were defined as followsscore no achepositive nerve fibers in the mucosallayer fig 2a e score mild positivity fiber percrypt within the lamina propria mucosae lp fig 2bf score moderate positivity up to fibers per cryptfig 2c g and score strong positivity with or morefibers per crypt fig 2d h in the lp gcs were countedwithin the myenteric plexus using nadh enzyme histochemistry fig based on the ache scoring two different analyseswere performed first we determined the number ofgcsmm in the nadh stainings in areas correspondingto each distinct ache score in the swiss roll fig 1cfor raw values see supplementary table in mostspecimens areas meeting each grade of the ache scorewere present for the second analysis transition zonesamples were analyzed for ache scores as well as forthe number of gcsmm in the corresponding area fig1d for raw values see supplementary table thelength of the myenteric plexus segment was determinedby an open polygon line using image analysis softwareanalysis docu olympus hamburg germany depending on the quality of the specimens the amount gcs ina segment of mm were counted absolute gccount was then normalized to the length of the respective myenteric plexus segment to compare gc density ingcmm the imaging and evaluation were performedusing a light microscope olympus bx41 at a magnification of 40x length or 400x cell countstatistical analysisfor statistical analyses we used single case overlay diagrams fig 4b significance levels were calculated byusing an anova and tukeys test fig 4a c d a significance level of α was determined for all testsstatistical analyses were performed by using the jmp software sas cary nc usaresultsache score negatively correlated with ganglion cell countin areas with no achepositive nerve fibers in the mucosal layer score fig 5ac a median of gcsmm min max was observed in areas withmild ache positivity score a median of gcsmmwas counted min max in areas with moderate ache positivity score a median of gcsmmmin max was counted in areas with severeache positivity score fig 5df a median of gcsfig ache scores of cholinergic innervation in the lamina propria mucosae representative areas of the mucosal layer with cholinergicinnervation score absence of achepositive fibers a e score mild cholinergic innervation b f score moderate cholinergic innervationc g and score severe cholinergic innervation d h scale bar ad μm eh μm 0cbraczynski bmc pediatrics page of fig ganglion cell count in the myenteric plexus a and b healthy bowel areas with normal gc density dashed lines indicating gcs c and dclassic hd pathology with complete absence of gc nadh scale bar a c μm b d μmmm min max was observed there were significant differences in the gc counts when comparingscores vs and vs each p as well asscores vs and vs each p fig 4alow ache score values were associated with a highnumber of ganglion cells but the absence of pathologicalcholinergic innervation was not a reliable indicator for aregular ganglion cell countmoderate or severe pathological cholinergic innervationin the lp was associated with complete absence of ganglion cells in the myenteric plexus fig 4b and fig 5gihowever the absence of or presence of only very fewache positive fibers in the mucosa did not assure aregular gc distribution in all cases in out of a total of resection specimens there were no areas detectedthat could be assigned to an ache score furthermore there were two cases with clear aganglionosis atthe aboral resection margin however showing no sign ofpathological cholinergic innervation ache score inthe respective segment exemplarily case id fig 5githe colonic areas with mild cholinergic innervation corresponding to an ache score of still exhibited a considerable variation in the gc counts with variationsbetween gcsmm case id and gcsmmcase id fig ache scores did not continuously decrease in thetransition zoneserial biopsies from the defined areas within the transition zone were collected fig 1a and d depending onthe available material and the lengths of the resectedspecimens the sample counts varied tz cm n tz cm n tz cm n and tz cm n the evaluation of the pathological cholinergic innervation in the transition zone specimens revealed a medianache score of at tz cm min max a medianscore of at tz cm min max a median scoreof at tz cm min max and a median scoreof at tz cm min max the decrease between tz cm and tz cm and the increase betweentz cm and tz cm were both significant p fig 4cganglion cell counts increased in the proximal region ofthe transition zone and remained stable rostrallythe gcsmm were counted in the transition zone biopsies at the most distal point of the transition zone tz cm a median of gcsmm min max wasobserved at tz cm a median of gcsmm min max was counted at tz cm a median of gcsmm min max was observed finally at tz cm a median of gcsmm min 0cbraczynski bmc pediatrics page of fig see legend on next page 0cbraczynski bmc pediatrics page of see figure on previous pagefig analysis of the swiss role and ache score and gc distribution in hd transition zone a gcsmm in areas assigned to ache score to inthe full length swiss roll preparations b single case overlay diagram gcsmm per ache score are grouped according to achescore to in ain cases no areas with score were detectable c cholinergic innervation as measured by the ache score and d gcsmm in the tz samples tz cm and cm p p p max was counted there was a significant increase in the the number of gcsmm between cm and cm p and between cm and cm p fig 4d regarding the differences between the othersamples tz cm to tz cm and tz cm to tz cm there was a statistically nonsignificant trend of increasing gc counts with the difference being the smallest between tz cm and tz cm however onlyn for tz cmdiscussionin our cohort of hd patients we assessed the extentof cholinergic innervation in the lamina propria mucosaeby ache scoring and the number of gcs in colonic biopsies over a maximal length of cm extending fromthe proximal area to the beginning of the macroscopically defined transition zonethe amount of gcs was in a similar range to thoseamounts reported by meierruge and brunner byusing ldh stainings gcsmm in dysor hypoganglionic colon segments as well as gcsmm inunaffected colon tissues were reported our slightlyhigher cell numbers may be related by the differentstaining protocol nadh instead of ldh dependingon the staining method as well as the interobservervariability gc counts can vary considerably [ ]fig discrepancy of ganglion cell count in areas with normal appearing ache distribution in general an ache score a corresponded to gcpopulation counts as described in b and c while an ache score d corresponded to the absence of gcs e and f however case id showed no cholinergic innervation ache score g associated with absence of gcs h and i scale bar a d g μm b e h μm c fi μm 0cbraczynski bmc pediatrics page of fig variation of ganglion cell distribution in cases with similarly pathological ache reaction three cases with ache score are shown accase id df case id gi case id while the amount of pathologic cholinergic innervation is comparable gc counts per mm varies from14mm hi case id to 472mm bc case id scale bar a d g μm b e h μm c f i μmswaminathan summarizes the variation of cellcounts depending on the staining method ranging from to gcscm even with laborious counting ofthe entire circumference patienttopatient variationremained very high suggesting that either significant variation in myenteric gc density is normal or variation in technical factors eg stretch might be relevant the question whether the dilatation of the transition zone and the oral part of healthy gut leads to neuronal death or whether a reduced cell count is the resultof dilatation and stretching the same amount of cgsdistributed in a wider circumference remains unsolvedduring tapt surgery longitudinal stretching seems tobe more prominentthan intravital circumferentialstretching which is difficult to assess another factor istissue shrinking during the embedding process which iswell known from ffpe tissue this can be neglected incryoconserved material as in our case current literaturereflectson the disrupted craniocaudal migratorythe gcs and a locoregional partialbehaviour ofphenotype which leads to the reduced amount of gcs inthe transition zone rather than technical factors additionally the work by white and langer nicely corroborates the findings on circumferential gc count differences swaminathan and kapur examined thisissue in nonhd cadaveric specimens and found that almost the entire circumference of transverse sectionsfrom a single sample had to be counted to get an accurate estimate of gc density nevertheless all published staining and counting techniques were reportedto have high sensitivity and specificity in an experiencedlaboratory with accuracy rates as high as conventional staining techniques do however not discriminate between gcs and glial bystander cells in the gangliawhich may result in further variation that is caused byinadequate gc identificationour surgical specimens were sampled in a longitudinalorientation from aboral to oral and the beginning of 0cbraczynski bmc pediatrics page of the tz was macroscopically defined based on the end ofthe constricted gut two phenomena may affect gccounts a concerning the circumference some authorshave observed a varying uneven distribution of the gcsin the gut wall see discussion above and b the beginning of the tz cm tz biopsies cm tz in ourstudy was macroscopically defined thereby still mainlyshowing aganglionic bowel segments in contrast definingthe beginning of the transition zone based on microscopiccriteria a cm tz biopsy might be randomly located at cm from the aganglionic segment boundary to reduce the impact of these uncertainties further researchstudies should consider an evaluation of intervals that areshorter than cm as expected from normal embryonicdevelopment our samples showed a longitudinal increaseof gcs in oral direction we found significant changes inthe transition zone between tz cm and tz cmfollowed by a trend of increasing gcs in more proximalzones kapur analyzed the relationship between the lengthof the partially aganglionic transition zone compared tothe length of the aganglionic segment in patients andstated that excluding the very long aganglionic segmentsthe hypoganglionic transition zone was consistently lessthan cm in length nevertheless there were significant methodological differences in the study design thecriteria used for the diagnosis of myenteric hypoganglionosis on hestained paraffin sections were descriptiveand designed to detected only moderatetosevere hypoganglionosis despite these formal differences andlimitations regarding to low number of specimens in ourstudy our results suggest a relatively longer transitionzone with still slightly increasing gc counts in areas tz and tz in our study high ache score values score wereonly observed in aganglionic bowel absent or low achescore values score were mostly associated with a highnumber of gcs but the absence of cholinergic innervation did not prove a normal innervation mild cholinergicinnervation has been described in rectal biopsies from patients who did not suffer from hd possibly because mildcholinergic innervation is part of the normal variation little is known about mild persistent cholinergic innervationin the lp in hd patients it is possible that rare achepositive neurites also resolve with the maturation of thechild or persist as a normal variant in older children oradults finding rare achepositive neurites protrudinginto the lp is described to be a relatively common findingin suction biopsies from pediatric patients who do not suffer from hirschsprung disease [] our findings question that a low amount of remaining ache positive fibersache score has to be considered as a reliable pathological feature in hd patients no significant correlationwas observed between ganglion cell density and absentache score versus ache score which extended upto cm proximally to the aganglionic segment in fourcases we did not identify an area with an ache score of in these cases some degree of cholinergic innervationremained despite a resection margin of up to cm proximal from the tz the lack of correlation between ganglion cell density and low ache score may be based onthe variables discussed above variability in ganglion cellcounts different staining protocolsinterobserver variability tissue stretching and others or alternatively because ache score might be part of the normal variationin our study we exclusively evaluated transmural biopsiesfor both initial diagnosis as well as during definite surgerywhile in other centers mucosal or submucosal biopsiesmay be performed thus resulting in the risk of insufficienttissue being collected for definite diagnostics becauseno definite abnormal cholinergic innervation patternache score was found in the hypoganglionic bowelof most hd patients mucosal ache activity alone shouldnot be used to define the proximal transition zonenevertheless the definition of the end of the transitionzone and the beginning of the normal colon by the use ofgc count cutoff values may not lead to improved surgical patient outcomes with respect to the interobservervariability and methodological differences based on ourresults both mucosal and submucosal tissues should beevaluated however the choice of the resection margins requires further interdisciplinary discussionsin our study we examined the relationship between theamount of cholinergic mucosal innervation and the density of myenteric ganglion cells in resected intestinal specimens from hd patients although we identified anassociation between absent gc counts aganglionosis andmoderate or high ache activity no statistically significantcorrelation between ache activity and ganglion cellcounts in proximal ganglionic intestinal segments couldbe established thus the presence of sparse cholinergicnerves in the mucosa is not a reliable marker of myenterichypoganglionosis and transmural biopsies are essential toquantify ganglion cells to exclude nonacherelated features of transition zone our data support the high validityof transmural colon biopsies for hd diagnostics especiallyfor cases lacking one of the classic features and highlightsthe necessity of a close collaboration between pediatricsurgeons and neuropathologistssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s1288702002299zadditional file table s1 gc count and length of corresponding mpsegment according to ache score swiss role specimen table s2 0cbraczynski bmc pediatrics page of ache score gc count and length of the corresponding mp segment inthe transition zonereceived september accepted august abbreviationsache acetylcholinesterase f female gc ganglion cell he hematoxylinand eosin hd hirschsprungs disease ldh lactate dehydrogenaselp lamina propria mucosae m male mp myenteric plexus nadh nadhtetrazolium reductase reactions tz transition zone tapt transanal pullthroughacknowledgementswe thank maika dunst tatjana starzetz and cornelia penski for theirexcellent technical assistance this manuscript was language edited byamerican journal experts 475ecb814533a9066fd4authors contributionsthe paper is the result of the intellectual collaboration of all listed authors inparticular akb sg pnh ur and mm participated in the conception anddesign of the study drafted the manuscript and performed the neuropathologicalanalysis the statistical methods of this study were reviewed by mm pb followedup the patients rb cosupervised the study and gave critical advice concerningthe stainings sg and ur treated the patients performed the surgery supervisedthe study and helped interpreting the findings all authors read and approved thefinal manuscriptfundingakb received funding through the start and the rotation program of themedical faculty of rwth aachen university the start rotation fund is apersonal grant to akb allowing physicians to do research during thespecialization training mm would like to thank the luxembourg nationalresearch fond fnr for the support fnr pearl p16bm11192868 grantthe pearl fund is a personal grant to mm to develop a research program ofexcellence in the field of clinicaltranslational neuropathologyavailability of data and materialsthe dataset supporting the s of this is included within the and its additional filesethics approval and consent to participatethe use of patient material was approved by the ethical committee of thegoethe university frankfurt germany gn the parents of thepatients gave written informed consent for the surgeryconsent for publicationnot applicablecompeting intereststhe authors declare no conflicts of interestauthor details1department of neurology rwth aachen university hospital aachengermany 2department of physical biology heinrichheine universitydüsseldorf germany 3institute of biological information processing ibi7structural biochemistry forschungszentrum jülich jülich germany 4instituteof neurology edinger institute goethe university frankfurt germany5department of pediatric surgery helios hospital berlinbuch berlingermany 6institute of pathology and neuropathology eberhardkarlsuniversity tuebingen germany 7german cancer consortium dktkheidelberg germany 8german cancer research center dkfz heidelberggermany 9frankfurt cancer institute fci frankfurt am main germany10department of neurosurgery goethe university frankfurt germany11department of pediatric surgery university of frankfurt am main frankfurtgermany 12university childrens hospital goethe university frankfurtgermany 13department of oncology donc luxembourg institute ofhealth lih strassen luxembourg 14luxembourg centre for systemsbiomedicine lcsb university of luxembourg luxembourg cityluxembourg 15national center of pathology ncp laboratoire national desanté lns rue louis rech l3555 dudelange luxembourg16luxembourg center of neuropathology lcnp rue louis rech l3555dudelange luxembourgreferencesborrego s ruizferrer m fernandez rm antinolo g hirschsprung's diseaseas a model of complex genetic etiology histol histopathol best ke addor mc arriola l balku e barisic i bianchi f hirschsprung'sdisease prevalence in europe a register based study birth defects res aclin mol teratol friedmacher f puri p classification and diagnostic criteria of variants ofhirschsprung's disease pediatr surg int gfroerer s rolle u pediatric intestinal motility disorders world jgastroenterol das k mohanty s hirschsprung disease current diagnosis andmanagement indian j pediatr collins l collis b trajanovska m khanal r hutson jm teague wj quality of life outcomes in children with hirschsprung disease j pediatrsurg brooks as oostra ba hofstra rm studying the genetics of hirschsprung'sdisease unraveling an oligogenic disorder clin genet de lorijn f boeckxstaens ge benninga ma symptomatologypathophysiology diagnostic workup and treatment of hirschsprungdisease in infancy and childhood curr gastroenterol rep edery p lyonnet s mulligan lm pelet a dow e abel l mutations ofthe ret protooncogene in hirschsprung's disease nature meierruge wa bronnimann pb gambazzi f schmid pc schmidt cp stossf histopathological criteria for intestinal neuronal dysplasia of thesubmucosal plexus type b virchows arch meierruge w ultrashort segment hirschsprung disease an objective pictureof the disease substantiated by biopsy z kinderchir holschneider am puri p hirschsprung's disease and allied disorders berlinheidelberg springer friedmacher f puri p rectal suction biopsy for the diagnosis ofhirschsprung's disease a systematic review of diagnostic accuracy andcomplications pediatr surg int schappi mg staiano a milla pj smith vv dias ja heuschkel r apractical guide for the diagnosis of primary enteric nervous systemdisorders j pediatr gastroenterol nutr meierruge w lutterbeck pm herzog b mer r moser r scharli aacetylcholinesterase activity in suction biopsies of the rectum in thediagnosis of hirschsprung's disease j pediatr surg meierruge wa bruder e pathology of chronic constipation in pediatricand adult coloproctology pathobiology meierruge wa brunner la morphometric assessment of hirschsprung'sdisease associated hypoganglionosis of the colonic myenteric plexuspediatr dev pathol knowles ch veress b kapur rp wedel t farrugia g vanderwinden jm quantitation of cellular components of the enteric nervous system in thenormal human gastrointestinal tractreport on behalf of the gastro international working group neurogastroenterol motil knowles ch de giio r kapur rp bruder e farrugia g geboes k gastrointestinal neuromuscular pathology guidelines for histologicaltechniques and reporting on behalf of the gastro internationalworking group acta neuropathol meierruge wa brunner la engert j heminghaus m holschneider amjordan p a correlative morphometric and clinical investigation ofhypoganglionosis of the colon in children eur j pediatr surg swaminathan m kapur rp counting myenteric ganglion cells in histologicsections an empirical approach hum pathol white fv langer jc circumferential distribution of ganglion cells in thetransition zone of children with hirschsprung disease pediatr dev pathol mckeown sj stamp l hao mm young hm hirschsprung disease adevelopmental disorder of the enteric nervous system wiley interdiscip revdev biol kapur rp histology of the transition zone in hirschsprung disease am jsurg pathol 0cbraczynski bmc pediatrics page of schofield de devine w yunis ej acetylcholinesterasestained suction rectalbiopsies in the diagnosis of hirschsprung's disease j pediatr gastroenterolnutr moore sw johnson g acetylcholinesterase in hirschsprung's diseasepediatr surg int pacheco mc bove ke variability of acetylcholinesterase hyperinnervationpatterns in distal rectal suction biopsy specimens in hirschsprung diseasepediatr dev pathol chow cw chan wc yue pc histochemical criteria for the diagnosis ofhirschsprung's disease in rectal suction biopsies by acetylcholinesteraseactivity j pediatr surg publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Colon_Cancer |
" in the current research we have developed silver and iron nanops of isolated proanthocynidinpac from grape seed by green synthesis and evaluated for antimicrobial antioxidant activity and in vitrocytotoxicity against colon cancer cell linesresults one percent solution of isolated proanthocynidin in water was vigorously mixed with silver nitrate and ferric chloride solution and kept for h to yield pacagnp and pacfenp the synthesized nanops werecharacterized by uv ftir xrd and sem analysis and evaluated for antimicrobial potential against selectedmicrobes moreover the synthesized nanops were studied for dpph assay and in vitro cytotoxicity usingcolon cancer cell lines colo320dm and ht29 mtt srb and trypan blue assay uv spectroscopy confirmed thedevelopment of nanops sem analysis showed that the ps were aggregated in the size range of to nm antimicrobial potential was found to be less against staphylococcus aureus pseudomonas aeruginosa andescherichia coli whereas cytotoxicity of pacagnp and pacfenp against colo320dm and ht29 exhibited promisingresults as compared to the pure pac pacagnp and pacfenp exhibited ± and ± inhibition respectively against dpph radical whereas pure pac showed ± inhibition and standardascorbic acid exhibited ± inhibition of dpph radical the silver and iron nanops were successfully developed by green synthesis method usingisolated proanthocynidin which is economical and ecofriendly the use of metal nanops may open up a newopportunity for anticancer therapies to minimize the toxic effects of available anticancer drugs specifically intargeting specific sitekeywords proanthocynidin silver and iron nanops antioxidant activity cytotoxicity colorectal cancercolo320dm ht29 correspondence randivedheerajgmailcom1department of pharmaceutics rajarambapu college of pharmacy kasegaonwalwa sangli maharashtra indiafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 0cshejawal of genetic engineering and biotechnology page of highlights 0f the present research work is focused on greensynthesis of silver and iron nanops of isolatedproanthocynidin from grape seed extract 0f this is the first attempt to assess scientifically theanticancer efficacy of the developed metalnanops on in vitro colon cell lines ht29 andcolo320dm 0f the results of the study revealed that developednanops are having significant cytotoxic activityagainst colon cancer as compared with pureproanthocynidin phytoconstituents 0f this is an ecofriendly economical easy and rapidmethod of development of metal nanops withminimum usage of hazardous chemicals 0f use of isolated phytoconstituents which are devoidof toxic effects of all other anticancer drugs andbetter absorption as well as cellular uptake due tonanosize for targeting specific site will be a newerresearch area a foremost reason of cancer death in men and womenis colorectal cancer crc and it affects nearly millionpeople throughout the world every year it is thethird most frequently diagnosed cancer in both men andwomen about new cases and around deaths have been estimated to have occurred in inthe usa the occurrence of crc in china is lower thanthat of western countries but it has increased in thecurrent years and has become a substantial cancer burden in china mainly in the more developed areas epidemiologicalthe dailyconsumption of fresh fruits and vegetables is connectedwith a reduced risk of cancer the special effects may bemoderately attributed to the presence of several polyphenolic compounds which are known to exhibit antioxidant and free radicalscavenging properties literature hasshown thatit is very essential to deliver anticancer drug at the siteof action and the dosage form must exhibit high cellpenetration or permeability and drug solubility nanops may enter the human body via several routesthe probability of penetration depends on the size andsurface properties of ps and on the anatomicalstructure of the specific sites of the exposure routes nanotechnology can be termed as the exploitation ofmatter through specific chemical andor physical processes to produce materials with specific propertieswhich can be utilized in particular applications [ ] ananop can be a microscopic p or materialthat has at least one dimension less than nm in size[] different bulk materials exhibit sole optical electrical thermal physical and chemical properties andhence they find a range of applications in the areas ofenvironment medicine chemistry energy agricultureinformationandheavy industry [ ] recently there has been a keeninterest in the green synthesis of nanops consumer goodscommunicationowing to the characteristic catalytic and optical properties of the metal nanops as compared to the bulkmaterial they have fetched greater attention to date inmultidisciplinaryin thepharmaceutical and cosmetic which position onwardgrowth in commercial interest and calling for effectualsynthesis procedures to match the growing demand ofsilver iron and gold nanops especiallyscientificareasayurvedic and herbalformulations available in themarket diverge in quality and therapeutic efficiencyowing to the differences in composition of the plantphytoconstituents [] advancement in the ayurvedic herbal medicine has been revolutionized from theshowing of phytochemicals and pharmacological activities to elucidating their mechanisms of action and sitesof action also currently there is an increased interest in the herbal drugs and remedies for the treatmentof chronic diseases proanthocyanidins are the naturally available polyphenolic compounds which varied in chemical structure pharmacological action and characteristics andextensively available in fruits seeds vegetables nutsflowers and bark grape seed is an abundant source of proanthocyanidinpacs which is known to be a powerful inhibitor ofaromatase activity it is an enzyme expressed in higherlevels in cancerous than in usual breast tissues pacs belong to a bigger class of abundant plantderivedcompounds flavonoids which offers numerous valuablehealth effects largely because of its antioxidant properties [ ] it has been shown to defend against oxidative stress and tobaccoinduced dna damage andexhibited selective prominent cytotoxicity against somehuman cancers including colon lung breast prostateand gastric carcinomas []they are secondary plant metabolites available inmany diverse kinds of fruits vegetables and plantbasedbeverages and also available in cocoa apple grapes teaand red wine pac classes of condensed tannins areoligomers and polymers of catechin and epicatechin and other related flavonoids chiefly linked by eitherbtype c4 c6 or c8 or atype linkages c2 o7grape seed extract gse falls into the btype categoryand grape seed extractrich diets have been connectedwith a reduced risk of chronic cardiac diseases andalso a variety of common cancers including colorectalcancer []proanthocyanins are strong free radical scavengers andare supposed to be contributors to the health benefits of 0cshejawal of genetic engineering and biotechnology page of fruits and vegetables [ ] proanthocyanidins obtained from grape seeds have been proven to protectagainst uv lightinduced carcinogenesis stop immunesuppression enhance interleukin il12 and decreaseil10 apple pacs have established synergistic effects with lysosomotropic compounds in increasing theanticancer properties targeting human colon cancerderived metastatic cells [ ]an environmentally friendly option is to prepare nanops dependent on three important parametersnamely solvent medium reducing and stabilizing or capping agent for nps therefore the present study isimportant with respect to the development of metalnanops of phytoconstituent specifically for targeting the cancer it has many advantages like being ecofriendly and costeffective and mainly the isolated phytoconstituents have prominent activity as compared withextract of plants and their parts moreover the side effects of the synthetic drugs can be avoided furtherin vivo animal study of the metal nanopbased formulation is the new area of researchmethodschemicalsproanthocynidin pac was obtained as a gift samplefrom influx healthcare mumbai maharashtra all thechemicals used in the study were of analytical grade silver nitrate agno3 and ferric chloride fecl3 were purchased from loba chem kolhapur ciprofloxacin wasobtained from okasa pharmaceutical satara celllinecolo320dm and ht29 were procured from nccspune maharashtramicroanism usedthe test anisms used in this study were staphylococcus aureusatcc pseudomonas aeruginosaatcc and escherichia coli atcc theculture was obtained from yashawantrao chavan college of science saidapur karad ms indiasynthesis of metallic nanopssynthesis of proanthocyanidin silver nanopspacagnpspacagnps were synthesized using proanthocyanidinsolution and agno3 solution in accordance with theprocedure mentioned by phull with minor modification equal volumes of proanthocyanidinaqueous solution and silver nitrate solution were incubated at ambient temperature for h to obtainpacagnps synthesis of pacagnps was detected bynaked eye with a change of color from dark red to faintbrown which was confirmed by uv spectroscopy thecollected pacagnps were centrifuged for min at rpm and dried in vacuum chamber at °c shownin fig synthesis of proanthocyanidin iron nanopspacfenpsfor the synthesis of profenps proanthocyanidin aqueous solution and ferric chloride solution were used inaccordance with the procedure mentioned by raju with minor modification ferric chloride andproanthocyanidin solution were vigorously combined in a ratio of the mixture was then placed onan orbital shaker for h at an ambient temperature toobtain pacfenps the synthesis of pacfenps was observed with a color change from dark black to darkbrown which was also confirmed by uv spectroscopythe collected pacfenps were centrifuged for min at rpm and dried in vacuum chamber at °cshown in fig characterization of nanopsuvvis absorbance of pac pacagnp and pacfenpthe development of metal nanops of pacagnpand pacfenp by the proanthrocyanidin was recordedperiodically using a uv spectrophotometer shimadzuthe samples were diluted with ml of deionized waterand measured for uvvis spectrum after the formationof nanops that change in color deionized waterwas used as a blank for correction all samples were scanned from to nmsem and histogram analysis of pacagnp and pacfenpscanning electron microscopy sem is a normally usedmethod of evaluation and morphological analysis ofnanops at the nanometer to micrometer scale developed pacagnp and pacfenp were characterizedusing a highresolution scanning electron microscopeschottky field emission scanning microscope su5000the samples were prepared by a simple drop coating ofsuspended gold solution on to an electric clean glass andallowed the solvent to evaporate and the samples weredried atand analyzed in amicroscoperoom temperatureftir spectroscopy analysis of pac pacagnp and pacfenpto recognize the different biomolecules present in theproanthocynidin and the phytocompounds in ag and fenanops after synthesis were analyzed by ftirbruker alpha echo atr spectrum was recorded inthe range of cmxrd analysis of pacagnp and pacfenpthe green synthesized silver and iron nanops ofpac were evaluated by xrd analysis using an xrd xray diffractometer bruker d8 discover operated 0cshejawal of genetic engineering and biotechnology page of fig uv and synthesis npat a voltage of kv and ma with cu kα radiation inθ2θ configurations the crystalline size was determinedfrom the width of the xrd peaks by assuming that theywere free from nonuniform strainsantimicrobial activity of pac pacagnp and pacfenpin vitro antimicrobial activity was performed using theagar well diffusion technique the sterile agar wasinoculated with the bacterial culture s aureus p aeruginosa and e coli for h at °c antimicrobial activities were tested on nutrient medium against s aureusp aeruginosa and e coli which are representative typesof grampositive and gramnegative anisms wellswere bored by using a sterile borer standard solutionciprofloxacin and test samples pac pacagnp andpacfenp mgml was prepared by dissolving the testsample in dmso were placed into the wells μlplates were then kept for h in the refrigerator to enableprediffusion of the extracts into the agar finally theplates were incubated overnight h at °c theantimicrobial activity was determined by measuring thediameter of zone of inhibition [ ]in vitro cytotoxicity studies of pac pacagnp andpacfenp by using mtt assayhuman ht29 cell and colo320dm were obtainedfrom the national center for cell sciences pune msindia the cell cultures were maintained indmem supplemented with fetal bovine serum thecells were plated at a density of à cells per well in a96well plate and cultured for h at °c the cellswere subsequently exposed the plates were incubatedfor h and cell proliferation was measured by adding μl of mtt thiazolyl blue tetrazolium bromide dye mgml in phosphatebuffered saline per well theplates were incubated for a further h at °c in a humidified chamber containing co2 formazan crystalsformed due to reduction of dye by viable cells in eachwell were dissolved in μl dmso and absorbancewas read at nmfinally the percent cytotoxicity of the compounds wascalculated by using the following formulapercent cytotoxicity ¼ reading of controlreading of treated cellsreading of control 02 since the absorbance was directly associated with thenumber of viable cells the percent viability was determined from the absorbancein vitro cytotoxicity studies of pac pacagnp andpacfenp by using srb assayhuman ht29 cells and colo320dm were maintainedin dmem supplemented with fetal bovine serumthe cells were plated at a density of à cells perwell in a 96well plate and cultured for h at °cthe cells were subsequently exposed to μgml compound after drug incubation μl tca waskept for h at °c then the plate was washed with 0cshejawal of genetic engineering and biotechnology page of tdw triple distilled water and air dried thereafter μl srb dye was added in each well and kept for min at room temperature again the plate was washedthree times with acetic acid and air dried finally μl tris buffer was added and the absorbance wasread at nmthe percent cytotoxicity of the compounds was calculated by using following formulapercent cytotoxicity ¼ reading of controlreading of treated cellsreading of control 02 in vitro cytotoxicity studies of pac pacagnp andpacfenp by using trypan blue assaythe dye exclusion test is used to find out the number ofviable cells present in a cell suspension it is based onthe principle thatlive cells possess undamaged cellmembranes that exclude certain dyes such as trypanblue eosin or propidium whereas dead cells do not exclude in this test a cell suspension is simply mixed withtrypan blue dye and then visually examined to determine whether cells take up or exclude dye in the studypresented here a viable cell will have undamaged a clearcytoplasm whereas a nonviable cell will have a bluecytoplasmfifty microliters of celllines of human ht29 cellsand colo d was taken in microcentrifuge tubethey were incubated for min and then added μlof all samples of nanops in concentration of μg ml which were prepared by dissolving inphosphate buffer ph and dmso they were incubated in co2 incubator for min and thereafter trypan blue μl was added in each tube theywere further incubated for min in co2 incubatorand analyzed for total viable cells and nonviable cellsby using nubars slide antioxidant activity of pac pacagnp and pacfenp bydpph 22diphenyl2picryl hydrazyl hydrate assaythe scavenging ability of pac pacagnp and pacfenp on the stable free radical was calculated with themethod expressed by mensor twenty microliters of pac pacagnp and pacfenp solutions wasseparately added in three labeled testtubes subsequently ml of methanolic solution of dpph and ml of methanol were added to each test tube afterwhich all tubes were allowed to react at an ambienttemperature for min the control was prepared as described above without any extract and nanopsmethanol was used to correct the baseline after minof incubation the discoloration of the purple color wasmeasured under a uvvisible spectrophotometer theradicalscavenging activity was determined by the following formula scavenging activity ððþ ¼ aabs controlðþaabs sampleðaabs controlþþ 02 where a control is the absorbance of control samplepacagnp and pacfenp measured at nm and nm respectively and a control is the absorbance ofpac measured at nmstatistical analysisstatistical data ofthe cytotoxicity were assessed ongraphpad prism for windows bit with version results were analyzed by oneway anova withdunnetts posttest analysis of variance the meanstandard error mean sem of all calculated values wasshown in each group a value of p or was considered statistically significantresultsuvvis spectroscopy of pac pacagnp and pacfenpwhen the aqueous pac was mixed with aqueousagno3 and fecl3 solution the color of the solutionchanged which indicated the formation of silver and ironnanops this change in color was due to the collective coherent oscillation of conduction electrons atthe surface of the nanops that interact with the oscillatingaphenomenon called surface plasmon resonance sprthis change in color indicated the reduction of ag andfe ions which was traced with uvvis spectroscopythe pac showed max at nm and silver nanop pacagnp possesses specific wavelength that canabsorb atmax nm whereas iron nanopspacfenp exhibit max at nm as shown in fig incidentelectriclightfieldoftheftir spectrum of pac pacagnp and pacfenpin the ftir of proanthocynidin fairly sharp peaks at and cm were observed which indicate thepresence of the functional group present in the compound however the aromatic at ccvalencecm the oh phenolic at ohvalence and chvalence arene alkane och3 the methoxylic at cm and co stretching at appeared in the ir spectrum of complex as shown infig 2apacagnpthese characteristic vibrations after reduction of agions were shifted to new peaks at and cmwhich indicated the presence of the functional grouppresent in the compound however the aromatic at ccvalencethe oh phenolic atohvalencechvalencecmand 0cshejawal of genetic engineering and biotechnology page of fig a ftir b sem histogram c xrdarene alkane och3 the methoxylic at cmand co stretching at appeared in the irspectrum of complex as shown in fig 2ain addition bioreduction showed that the and cm bands were suppressed in theagnp proanthocynidin and nps showed similar absorption bands indicating that nps might be stabilized byproanthocynidin on the basis of the orange yellow andbrownish green color of the biomass and the groups suggested by ftir analysisit was hypothesized thatproanthocynidin may be involved in silver nanopsynthesispacfenpthese characteristic vibrations after reduction of fe3ions were shifted to new peaks at and cmwhich indicated the presence of the functional grouppresent in the compound however the aromatic at ccvalencethe oh phenolic atohvalencechvalencecmand arene alkane och3 the methoxylic at cmand co stretching at appearin the irspectrum of complex as shown in fig 2a in additionbioreduction showed that the and cm bands were suppressed in the fenpproanthocynidin and nps showed similar absorptionbandsindicating that nps might be stabilized byproanthocynidin on the basis of the orange yellow andblackish color of the biomass and the groups suggestedby ftir analysis it was hypothesized that proanthocynidin may be involved in iron nanop synthesissem and histogram analysis of pacagnp and pacfenpa scanning electron microscope was used to analyze thestructure of pacagnp and pacfenp nanops thatare developed and represented in fig 2b the nanops formed were aggregated having a size range of to nm this aggregation of the nanops can beminimized or prohibited by increasing the concentrationof the proanthocynidin extract histograms of both thenanops have been represented in fig 2b exhibiting 0cshejawal of genetic engineering and biotechnology page of average p size pacfenp μmand pacagnp μmxrd spectrum of pacagnp and pacfenpthe xrd pattern of the synthesized silver and ironnanops formed using proanthocynidin is shown infig 2c the diffraction peak at 2θ ° and subsequenthigher order reflections can be indexed to the ag andother facets of silver nanops by comparing jcpdsfile no in case of pacfenp the peak shown at2θ ° corresponds to the iron compared with standard xrd for iron jcpds data pdf no the xrd spectrum also revealed a weak peak around2θ ° which can be attributed to the phytochemicalcomponents it thus confirmed that the nanopsformed on the membrane consisted of crystalline xrdindicated possible multicomponent product formation athigher energyantibacterial activity of pac pacagnp and pacfenpthe zones of inhibition revealed that there is very little antimicrobial potential of pac pacagnp andpacfenp against pseudomonas aeruginosa staphylococcus aureus and e coli the results are highlightedin table and the zone of inhibition is depicted infig results of cytotoxicity of pac pacagnp and pacfenpthe results of cytotoxicity assay by mtt and srb assayhave been presented in table with respect to two different colon cancer cell lines namely colo320dm andht29 a variation in the results of different assay wasobserved howeverthe silver nanops exhibitedbetter activity than pure pac in both the methods theresults of mtt assay against ht29 demonstrated thatpacagnp showed maximum ± inhibitionrepresented in fig 4x a in case of colo320dm silver nanops exhibited ± inhibition asrepresented in fig 4x b whereas the srb assay ofpac pacagnp and pacfenp against ht29 revealed ± inhibition by pacagnp as shown in fig4x c in case of colo320dm srb assay a maximumof ± inhibition was exhibited by silver nanops as depicted in fig 4x dthe results of trypan blue assay revealed that silvernanopaticles pacagnp showed ± nonviability of colo320dm cell line when observed on amotic microscope whereas iron nanops pacfenpexhibited ± inhibition of ht29 cell lines asrepresented in table results of dpph 22diphenyl2picryl hydrazyl hydrateassay of pac pacaunp and pacfenpthe dpph activity results demonstrated effective freeradical percent scavenging potential of pac pacaunpand pacfenp as depicted in fig 4y as compared to ascorbic acid standardthe concentration responsecurves of dpph radicalscavenging activity of pacpacaunp and pacfenp are shown in table it wasobserved that pacagnps were more effective than pacextract and pacfenps at a concentration of μgml the scavenging activity of pacagnps was observed to be ± while at similar concentration for the pacfenps it was reported to be ± the outcome of antioxidants on dpph is thoughtto be due to their hydrogen donating abilitydiscussionengineered nanomaterials showed imperative benefitsdue to their unique nanostructure along with their significant properties for the designed applications metallic nps have been synthesized using several different methods such as chemical reduction electrochemical microbiological reduction ultrasonication methodand microwave radiation the present researchwork is focused on green synthesis of silver and ironnanops ofisolated proanthocynidin from grapeseed extract it is an ecofriendly economical easy andrapid method of development of metal nanopswith minimum usage of hazardous chemicals the developed silver and iron nanops were characterized byuv spectroscopy which showed the change in absorbance after development of nanops ftir spectrumprovides the information about the chemical change ofthe functional groups involved in bioreduction [ ]the ftir spectra of developed nanops confirmedthe formation of silver and iron nanops as characteristic vibrations after reduction of ag ions weretable results of antibacterial activity of pac pacagnp and pacfenp against selected microbial strainssrnosample namepac purepacagnppacfenpciprofloxacin stdvalues are expressed in triplicate mean sdzone of inhibition diameter mm against the selected microanismspseudomonas aeruginosa ± staphylococcus aureus ± ± ± ± ± ± e coli ± ± 0cshejawal of genetic engineering and biotechnology page of fig antimicrobial activityshifted to new peaks at and cm and characteristic vibrations after reduction of fe3 ions wereshifted to new peaks at and cm alsocharacteristic color change can be attributed to the surface plasmon resonance of deposited agnps and itclearly indicated the development of nanops the size of nanops and its morphology wereclearly observed in the sem images the average size fortable results of cytotoxicity of pac pacagnps and pacfenp by mtt and srb assay using colo320dm and ht29 cell linescompoundmtt assay against ht29 controlpercent inhibitionmean odpercent viabilityproanthocynidin plainproanthocynidin agnpproanthocynidin fenpmtt assay against colo320dm controlproanthocynidin plainproanthocynidin agnpproanthocynidin fenpsrb assay against ht29 controlproanthocynidin plainproanthocynidin agnpproanthocynidin fenpproanthocynidin plainsrb assay against colo320dm controlproanthocynidin plainproanthocynidin agnpproanthocynidin fenpvalues are expressed in triplicate mean sd ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0cshejawal of genetic engineering and biotechnology page of fig graph of x cytotoxicity and y antioxidant dpphpacfenp was observed to be μm and μm for pacagnp the xrd resultsrevealed weak peak at 2θ ° for phytoconstituentwhereas diffraction peak at 2θ ° was observed for silver nanops and iron nanops showed thepeak at 2θ °in case of antimicrobial activity the developed silver andiron nanops exhibited little antimicrobial potential ascompared to pure proanthocynidin metal nps increase theantibacterial potential due to the formation of reactive oxygen species ros developed from different types of ironoxide nanops like feo fe2o3 and fe3o4 the freeradical produced in the reaction causes intracellular stressesthat can damage the dna of the cell the in vitro cytotoxicity results against ht29 andcolo320dm showed that pacagnp exhibited ± inhibition mtt assay and ± inhibitionofcolo320dm pacagnp demonstrated ± inhibition mtt assay and ± inhibition srbassayagainst ht29assaysrbincasein dpph assay pacfenp and pacagnp exhibited ± and ± inhibition respectivelythe obtained parameters of the characterization andevaluation of the nanops clearly revealed that ascompared to proanthocynidin the silver and iron nanops possess better antioxidant and anticancer potential against colorectal cancer thusthe use ofisolated phytoconstituents which are devoid of side effects and promoting better absorption and cellular uptake owing to their nanosize will certainly achieveeffective targeting and has to be provided greater attention as a newer research area also several review papershave been published about the synthesis of silver nanops using natural polymers like kcarrageenan andsynthetic polymers like poly vinyl pyrrolidone and polyethylene glycol to improve the strength and stability ofnanops as per the study of moustafa the use ofnatural and synthetic polymer for the development ofsilver nanops opened up new research area in medicinal and biological field along with food industry we have successfully synthesized pacagnps and pacfenps using proanthocynidin isolated from grape seedextract by employing a green synthesis method which istable results of cytotoxicity of pac pacagnps and pacfenp by using trypan blue assaysrnodrugcolo320dmpercent viability ± percent nonviable ± proanthocynidin plainproanthocynidin agnpproanthocynidin fenpvalues are expressed in triplicate mean sd ± ± ± ± ht29percent viability ± ± ± percent nonviable ± ± ± 0cshejawal of genetic engineering and biotechnology page of table antioxidant activity of dpph radicalscavenging activityconcentrations μg mlstandard percent inhibition ± pac percent inhibition ± pacagnps percent inhibition ± pacfenps percent inhibition ± ic50 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± values are expressed in triplicate mean sdconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of pharmaceutics rajarambapu college of pharmacy kasegaonwalwa sangli maharashtra india 2department of pharmaceuticalchemistry rajarambapu college of pharmacy kasegaon walwa sanglimaharashtra india 3department of pharmaceutics bharatividyapeeth college of pharmacy kolhapur maharashtra indiareceived may accepted august availability of data and materialsall the data required for the processing of the s are presented inthe results section supporting data was included separatelyethics approval and consent to participatenot applicable biao l tan s zhang x gao j liu z fu y synthesis andcharacterization of proanthocyanidinsfunctionalized ag nanopscolloids surf b biointerfaces gurushankar k gohulkumar m prasad nr rishnakumar n synthesischaracterization and in vitro anticancer evaluation of hesperetinloadednanops in human oral carcinoma kb cells adv nat sci nanoscinanotechnol nanops j nanomater1andsimpleenvironmentallybenignrelativelypacagnps and pacfenps were obtained with ps between and nm in size it is easy and costeffective and does not involve any harmful and poisonous chemicals all the other characterization like uvftir xrd and sem confirmed the development ofnanops we have observed significant antioxidantactivitycapacitiespacagnps and pacfenps exhibited a little antibacterial activity against the selected strains of microbes in anutshell the study showed that the developed nps fromthe isolated pac exhibit beneficial antioxidant and anticancer potential when assessed by three different in vitroassay methods with specifically colon cancer cell linesthus it may open up a new opportunity for anticancertherapies that need further researchradicalscavengingfreeandabbreviationspac proanthocynidin pacagnp proanthocynidin silver nanoppacfenp proanthocynidin iron nanop uv ultraviolet visiblespectroscopy ftir fourier transform infrared spectroscopy xrd xraydiffraction sem scanning electron microscope dmem dulbeccos modifiedeagles medium dpph 22diphenyl2picryl hydrazyl hydrate mtt dimethylthiazol2yl25diphenyltetrazolium bromide srb sulforhodaminebacknowledgementsthe authors are thankful to the secretary of kes society kasegaon andprincipal of rajarambapu college of pharmacy kasegaon sanglimaharashtra for providing facilities we are also thankful to the managingdirector influx healthcare mumbai for providing pure phytoconstituentmarkers dr sandip patil md biocyte institute of research and developmentbird sangli mhauthors contributionsthe work was c | 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 |
"tea is the second most popular beverage consumed in theworld next to water green tea is a kind of nonfermentedtea produced from the plant camellia sinensis it is favoredby people for its fresh ï¬avor and health beneï¬ts and consumed worldwide especially in east asian countriesgreen tea contains caï¬eine and polyphenolic compoundsknown as catechins catechins are the primary bioactivesubstances and present significant biological propertiestea leaves drycatechins constitute up to ofweight among that egcg is the main and the most abundant catechin [ ] egcg has been traditionally regardedas beneï¬cial mainly ascribed to its antioxidant action the antioxidant eï¬ects of egcg are manifested in scavenging free radicals in the body and inhibiting the formation ofros the results of earlier studies suggested that egcgcould decrease the risk of several human disorders associatedwith oxidative stress on the other hand egcg also displays significantprooxidant eï¬ects usually under highdose conditions theprooxidant actions of egcg play a dual role being both beneï¬cial and harmful while achieving desired outcomes inchronic disease prevention and treatment reports about thetoxicity of egcg are also emerging a growing body ofevidence continues to demonstrate a variety of harmfuleï¬ects from excessive consumption of green tea or oraladministration of highdose egcg supplement highdoses of egcg not only cause cytotoxicity in vitro but alsoresult in living body hepatotoxicity nephrotoxicity andgastrointestinal disorders vomiting and diarrhea the oral bioavailability of egcg is not so profound inhealthy humans as it was only of the total ingestion most of the ingested egcg is absorbed in the smallintestine and substantially degraded in the large intestine bymicrobiota action the eï¬ective dosage of egcg mightbe close to or higher than the toxic dosage in practical applications considering its low bioavailability therefore it is 0coxidative medicine and cellular longevitynecessary to understand the potential toxicity doses andusage of egcg in this review the prooxidant eï¬ects ofegcg in health beneï¬ts and adverse eï¬ects were discussedespecially concerning their underlying mechanisms involvedand doses used this review is aimed at harnessing the prooxidant eï¬ects of egcg for human health maintenance whileavoiding toxicity thereby better guiding the safety consumption of green tea and egcg chemical structure andautooxidation of egcgbasic catechins contain two or more aromatic ringshydroxyl group on carbon three position andor the higherdegree of hydroxylation of the b ring would be primarilyresponsible for the potent antioxidant activities of catechinsfigure 1a previous structureactivity relationshipstudies of catechins have demonstrated the importance ofthe number and location of the phenolic hydroxyl groupson antioxidative capacity egcg has the remarkablepotential to scavenge radicals and chelate metal ion theseabilities could be ascribed to the presence of dihydroxyand trihydroxy groups in a ring b ring and d ringfigure 1b the catechol structure of egcg makes it susceptible todegradation via autooxidation figure under normal°physiological conditions ph c egcg is autooxidized and converted to oquinone through nonenzymaticaldehydrogenation of phenolic hydroxyl groups at b ring when the cell culture medium is exposed in the airegcg could be oxidized by oxygen and yields superoxide andanion radicals o2
egcg are essential intermediate products in egcg autoox and oxygen could function as oxidants for furidation o2ther oxidation of egcg ï¬nally resulting in the formationof oquinone accompanying the generation of hydrogen could also form substantial amountsperoxide h2o2 o2of h2o2 via disproportionation reaction one egcgmolecule could produce more than two h2o2 molecules inphosphate buï¬er at neutral ph and egcg radicals
egcg o2autooxidation of egcg generates substantial ros theros comprises singlet oxygen hydroxyl radicals superoxideperoxides and h2o2 h2o2 is in a dominant position andusually is regarded as a toxic agent when the ros levelexceeds cellular antioxidant capacity oxidative stress willoccur in other words this is the result of an imbalancebetween prooxidant and antioxidant eï¬ects inclusion ofantioxidant defense enzymes such as catalase cat andsuperoxide dismutase sod could minimize h2o2 levelwhich is essential to maintain the redox balancethe concentration of egcg in the cell environmentseems to be a primary factor in explaining its prooxidanteï¬ects for example egcg treatment alone diminisheddna strand breakage of human blood lymphocytes at lowconcentrations μm while it induced dna strandbreakage at higher concentration μm thusegcg acts as an eï¬ective antioxidant at low doses withinthe range of high nanomolar and low micromolar levelswhile egcg represents a prooxidant at high doses howeverthis blurred boundary might vary depending on the type ofradical stimulants cellular environment and duration ofexposure to egcg health benefitsuntil now egcg has been a major research subject withinthe ï¬eld of healthpromoting eï¬ects the potential role ofthe prooxidant eï¬ects of egcg in cancer and obesity prevention and treatment as well as the antibacterial actionsachieved demonstrable results in previous studies prooxidant eï¬ects and anticancer activity of egcgcancer is one of the most common and lifethreateningdiseases occurring among humankind egcg as a naturalproduct has drawn a great deal of attention from boththe scientiï¬c community and the general public indeedegcg has shown both prophylactic and therapeutic eï¬cacy in multiple human cancers several mechanisms havebeen proposed to accountfor the inhibitory action ofegcg against cancer formation and growth the prooxidant eï¬ects of egcg were thought to be potential mechanisms for anticancer action the anticancer mechanismsvaried depending on the cell type dose andor time oftreatment table []apoptosis is the bestdescribed form of programmed celldeath the induction of apoptosis represents a universal andideal therapeutic strategy for cancer control cell apoptosiscould be triggered by either the intrinsic mitochondrial pathway or the external death receptor pathway the mitochondrial pathway could be induced by intracellularstresses such as oxidative stressthe apoptosistriggering eï¬ects of ros have beennoted in vitro table egcg inhibited cell growth ina dosedependent manner and the decrease in the numberof viable cells was mainly due to apoptosis caused by theegcginduced intracellular ros as early as the last century scientists found that egcg induced h2o2 formationin human lung cancer celllines h661 and 21bes andexogenously added cat completely prevented egcginduced cell apoptosis which suggested that h2o2 isinvolved in the apoptosis process provoked by egcg similar actions were also found in various cancersand tumor cells table thioredoxin trx and thioredoxin reductase trxr are pivotal regulators of cellularredox homeostasis decreased trxtrxr activity mightcontribute to the increased ros level high concentrationof egcg inactivated trxtrxr via the formation of egcgtrx1 and egcgtrxr conjugates which was linked to theelevation of ros level in hela cells and further promotedcancer cell death moreover one of the biochemical hallmarks of apoptosis is genomic dna fragmentation chen performed the dna fragmentation assay in theskov3 cells indicating that egcg induced apoptosis bycausing dna damage this result was consistent withother studies in ovarian and cervical cancer cells [ ]in terms of molecular mechanisms intrinsic apoptosisleads to the release of mitochondrial cytochrome c afterbeing released into the cytoplasm cytochrome c stimulates 0coxidative medicine and cellular longevityohbohohohhoohbohohohohdohocoaohobhoocaohafigure a basic structure of catechins b chemical structure of egcgohbegcgohohautooxidationph75 °cohboautooxidation·egcgoho2h2o2ohboooquinonefigure superoxidemediated chain reaction the formation of oquinoneapoptosome formation followed by activation of caspasecascades egcgmediated mitochondrial ros couldpromote cytochrome c release to the cytosol the antiproliferative action of egcg on prostate cancer and breast cancer is mediated through apoptosis as evident from caspase9[ ] the cells susceptible to egcginduced apoptosisalso showed activation of caspase3 moreover theincreased ros level was observed to result in the stimulationof mitogenactivated protein kinase mapk themapk signaling pathwayincluding extracellular signalregulated kinase erk jun nterminal kinase jnks andp38 plays a vital contribution in cell proliferation diï¬erentiation apoptosis and stress response egcg induced oxidative stress via generation of ros and thereafter activatedthe jnk pathway leading to changes in mitochondrial membrane potential and release of cytochrome c in ht29 humancolon adenocarcinoma cells and mia paca2 pancreaticcancer cells [ ] together these results suggest thategcginduced apoptosis is mediated through ros generation and might subsequently activate the cell intrinsic pathway in the presence of transition metals such as copper andiron h2o2 could convert to a potent oxidant hydroxyl radical via the fenton reaction nakagawa found that egcg μm produced h2o2 and triggered fenton reactionto form highly toxic hydroxyl radicals which resulted in lymphoblastic leukemia jurkat cell death in the presence offeiii and cuii egcg μm induced dna damagein hl60 cells as 8oxo78dihydro2²deoxyguanosine oxodg content increased which was a characteristic ofoxidative dna damage nevertheless no significantincrease in 8oxodg was observed in h2o2resistant colonhp100 cells suggesting that h2o2 was involved in cellulardna damage egcg could inhibit cell proliferation andinduce apoptosis through cellular dna breakage in diï¬erentcancer cell lines such dna breakage involved the mobilization of endogenous copper ions and the generation ofros moreover the observation of site speciï¬city of dnadamage by egcg is valuable cuiimediated dna damageby egcg occurred most frequently at t and g residues egcg was able to mobilize endogenous copper ions andgenerate hydroxyl radicals in situ hydroxyl radicalsserved as the proximal dna cleaving agentleading todna breakage in the nuclei this result was possibly due tothe interaction of egcg with chromatinbound copper ionsand then the nondiï¬usible hydroxyl radicals were formed atthe binding site hence hydroxyl radical generated nearbydna was well established as the cause of strand scissionbecause the concentration of copper is significantly very highin various malignancies egcg could induce cancer celldeath through the metal iondependent pathway thispathway was independent of mitochondriamediated programmed cell deaths such action involved in metal ionmediated dna cleavage would be an important mechanismof anticancer properties of egcgin addition to being transported into the cell egcgcould also function on the cell membrane fraction to regulatethe surface growth factor receptor earlier studies foundthat autooxidation of egcg led to epidermal growth factorreceptor egfr inactivation in human esophageal cancer 0ccell linesbladder cancernbtiibreast cancermcf7mcf7cervical cancerhelahelacolon canceroxidative medicine and cellular longevitytable role of prooxidant eï¬ects in the anticancer activity of egcg based on cell culture studiesegcgconcentrationtimebiological eï¬ectsreferences μm hinduced early apoptosis through dna damage μgml hinduced cell growth inhibition and apoptosis by downregulating survivinexpression via suppressing the akt pathway and activating caspase9 μm hinduced apoptosis at low doses via activation of jnk caspase9 and caspase3while inducing necrosis at high doses which is related to diï¬erences in rosgeneration and atp levels μm μm and h hincreased cell death through dna damageinduced cell death through generation of ros and inactivation of trxtrxrhct116 μm hinduced apoptosis through induction of ros and epigenetic modulation ofapoptosisrelated gene expressionht29 μm hendometrial carcinomaishikawa μm hinduced apoptotic cell death via activating the jnk pathway accompanyingmitochondrial transmembrane potential transition and cytochrome c releaseic50 was μminduced apoptosis via ros generation and p38 map kinase activationic50 was μmesophageal cancerkyse lung cancer μm hinactivated egfr by superoxide generated from autooxidation of egcg μm μm h h h μm hdisplayed strong growth inhibitory eï¬ects against lung tumor cell linesinhibited cell growth through induction of ros ic50 was μmic50 was μminduced apoptosis via h2o2 production and hydroxyl radical formationinduced apoptosis by modifying the redox systemh661 and h1299 μmh1299lymphoblastic leukemiajurkatmyelomaim9 rpmi8226and u266oral cancerscc25 andscc9ovarian cancer μm hreduced cell viability by inducing mitochondrialocalized ros and decreasingsirt3 expressionskov3 μgml dpancreatic cancerpanc1 μm hmia paca2 μm hinhibited cell proliferation and induced apoptosis by inhibiting cell cycle arrest andinducing dna damageinduced apoptosis through generation of ros as well as caspase3 andcaspase9 activationinduced stress signals by damaging mitochondria and rosmediatedjnk activationprimary eï¬usionlymphomabcbl1 and bcprostate cancer μgml hinduced apoptosis and autophagy through ros generationpc3 and μm hreduced cell survival and increased apoptosis caused a significant alteration incaspase9 alternative splicing 0coxidative medicine and cellular longevitycell line kyse one possible explanation is thath2o2 produced from egcg autooxidation in the cell culturemedium could attack and inactivate egfr leading to theinhibition of egfr phosphorylationand preferentialit is worth considering whether high amounts of egcgcould cause damage to normal cells egcgmediated rosproduction was particularly observed in cancer cells compared with normal cells the selectivity of egcginducedapoptosis in cancer cells might be due to the diï¬erentialinducibility of rosexpression ofapoptosisrelated genes moreover tao found thategcg induced diï¬erential mitochondrial dysfunction andoxidative stress in normal and oral cancer cells these eï¬ectswere related to the diï¬erential modulation of sirtuin sirt3 and its downstream targets including glutathionegsh and sod considering the cytotoxicity of egcgin normal cells the ic50 value in normal cells was checkedand showed to be more than μm while that for thecorresponding cancer cells was μm these resultssuggested that cancer cells are more sensitive to egcg thannormal cells and ros might be selectively toxic to cancercellsin addition to being used as preventive agents individually egcg could also be used as adjuvant therapies generally cooperative interaction of two or more agents couldtarget more signaling pathways thus eï¬ectively improvingagent chemosensitivity reducing untoward eï¬ects of treatment expanding the scope of action and showing highertherapeutic outcomes drug resistance is a dauntingchallenge in cancers prooxidant activities of egcg wereproposed to contribute to overcoming drug resistancehighlighted by the fact that h2o2 production induced byegcg increased the potency of cisplatin in ovarian cancercells by three to sixfold in contrast cisplatin alone washighly resistant to the treatment in some cancer cell linescopper transporter ctr1 is a critical determinant toincrease cisplatin uptake egcg could upregulate ctr1expression through the stimulation of ros simultaneous treatment of arsenic trioxide ato with egcgshowed oxidativemediated induction of apoptosis in leukemia cancer cells egcg acted as a prooxidant andincreased intracellular h2o2 and atoinduced hemeoxygenase1 ho1 provided ferrous iron to increase thefenton reaction in both cases cellular oxidative damageeventually occurredin general under typical cell culture conditions egcghas been known to generate i extracellular ros via autooxidative reaction in the cell culture medium ii ros in cellular mitochondria and iii intracellular ros through thefenton reaction upon cell entry figure these three pathways contribute diï¬erently to cancer cells but eventuallycause cell damage and death cancer initiation and progression are generally divided into several stages when egcgacts as an antioxidant it might more eï¬ectively enhance antioxidant capacity at the cancer prevention stage whereaswhen egcg acts as a prooxidant it might be more criticalat suppressing tumor growth stage one possible suppositionis that tumor cells may be more susceptible to oxidativestress because their increased growth rate and metabolismcause a heightened basal ros level the degree of cell proliferation and diï¬erentiation seems to be one factor aï¬ectingthe ros production ability of egcg future research willbe required to determine if egcg is a much more potentros inducer in diï¬erentiated than in undiï¬erentiated cancercells although a limited amount of data has shown that theseprooxidant eï¬ects can occur in vivo it is essential to understand when and to what extent the antioxidant or prooxidanteï¬ects of egcg are working in diï¬erent stages of cancers inanimal models prooxidant and antiobesity eï¬ects of egcg obesity is ametabolic disease characterized by abnormal or excessive fataccumulation it is generally associated with an increased riskof chronic diseases including diabetes hypertension anddyslipidemia a large and growing body of studies hasinvestigated the antiobesity eï¬ects of egcg in cellular andanimal experiments and the underlying mechanismsthe clinical manifestations of obesity are adipocytehyperplasia and hypertrophy in vitro studies have well demonstrated that egcg could inhibit adipocyte growth andinduce adipocyte death through its prooxidant eï¬ects hung reported that high concentrations of egcg μm reduced the cell viability of preadipocytes by induced the appearance of dna fragmentation andincreased the activity of the apoptotic enzyme caspase3 egcg was demonstrated to raise ros level anddescend gsh level in preadipocytes and adipocytes whichinduced oxidative stress thus resulting in decreased cell number ²ampregulated protein kinase ampk represents ametabolitesensing protein kinase hwang foundthat the release of ros by egcg stimulation could furtheractivate ampk rapidly in 3t3l1 adipocytes a recent studyalso proved that ampk was activated by exogenous h2o2and this activation was not through direct redox signalingto ampk but was a secondary consequence of redox eï¬ectson other processes egcg activates ampk via the generation of ros subsequently blocks anabolic pathways and promotes the catabolicpathway and suppresses gluconeogenesis and adipogenesisconsequently leading to body weight reduction and metabolic syndrome alleviation figure the activation ofampk modulates the expression of genes and proteinsinvolved in lipid metabolism downregulates the expressionof fat synthesis proteins and upregulates lipid catabolismproteins it was shown that egcg inhibited the expressions of glucose 6phosphatase g6pase for gluconeogenesis phosphoenolpyruvate carboxykinaseforgluconeogenesis fatty acid synthase fas for fatty acid synthesis acetylcoa carboxylase acc for fatty acid synthesis hydroxymethylglutarylcoa reductase hmgrforregulatory elementbinding proteinscholesterolsrebpsfor sterol synthesis peroxisome proliferatoractivated receptor gamma pparγ for lipid synthesis andstorage and ccaatenhancerbinding protein alphacebpα for adipogenesis as well as enhanced the expression of acylcoa oxidase aco for fatty acid oxidationperoxisome proliferatoractivated receptor alpha pparαpepcksterol 0coxidative medicine and cellular longevityautooxidationrosegcgcellrosfe2cu2fentonreaction·ohegfrcytochrome ccell damagecell deathcaspase9caspase3cell culture mediumfigure prooxidant eï¬ects of egcg in cell cultureegcggeneraterosactivateampkmodulateg6pase pepck fasacc hmgr srebpspparð¾ cebpð¼aco pparð¼ cpt1acad pgc1ð¼ucps atglfat synthesislipid catabolismantiobesityfigure eï¬ects of egcg on lipid metabolism via ros and ampkfor fatty acid oxidation carnitine palmitoyltransferase1cpt1 for fatty acid oxidation acylcoa dehydrogenaseacad for fatty acid oxidation peroxisome proliferatoractivated receptor gamma coactivator1α pgc1α for fattyacid oxidation uncoupling proteins ucps for thermogenesis and adipose triglyceride lipase atgl for triglyceridehydrolysis []accordingly the prooxidant eï¬ects of egcg play avital role in preventing the initiation and progression ofobesity egcg could cause oxidative stress thus damagingadipocyte directly and activating ampk and then aï¬ectingrelative genes and protein expression and signal transduction in various tissues indirectly however the increase ofoxidative stress in fat accumulation might be an importantpathogenic mechanism of obesityrelated metabolic syndrome such as diabetes firm conclusions as to whetherprooxidant eï¬ects of egcg could perform on body weightbody fat and adipose weight in humans will require morethorough clinical studies prooxidant and antibacterial eï¬ects of egcg egcgexhibits a broad spectrum of bactericidal activity against various bacteria its bactericidal eï¬ects include damage to thebacterial cell membrane and inhibition of fatty acid synthesisand enzymatic activity h2o2 which is generated byegcg appears to play an indispensable role in the bactericidal actions of egcg the bactericidal action of egcgwas related to h2o2 level as bactericidal action was inhibitedby the increase of cat concentration egcg was foundto have bactericidal activity at higher concentrations in thesalmonella assay highly correlated with h2o2 production egcg showed a dosedependent μm inhibition on escherichia coli e coli op50 strain growth this inhibitory action was associated with a profoundincrease in intracellular oxidative stress caused by egcghence the use of egcg as a prooxidant is well supportedby these studiesegcg was shown to have broad antibacterial spectrumeï¬ects on both grampositive and gramnegative bacterianevertheless egcg might function through diï¬erent mechanisms against grampositive and gramnegative bacteriaintracellular ros level was determined by ï¬ow cytometrythe results indicated that damage on gramnegative e colicell walls was induced by egcg depending on h2o2 release 0coxidative medicine and cellular longevity in contrast the damages on grampositive staphylococcus aureus resulted from a combination between egcg andpeptidoglycan layer because the outer membrane ofgramnegative bacteria was mainly composed of negativelycharged lipopolysaccharides which could resist the destruction of egcg they are less susceptible to egcg thangrampositive bacteria bacterial cell membrane damage not only prevents thebinding of bacteria to host cells but also inhibits the abilityof the bacteria to combine with each other and form bioï¬lms egcg was known to attack the lipid bilayer of bacterialcell membranes leading to physical disruption of the membrane as for the cell walls results from atomic forcemicroscopy suggested that the subminimum inhibitory concentrations of egcg treatment mgl to e colio157h7 strains could lead to temporary changes in the cellwalls cui such changes were due to the damagecaused by h2o2 generated from egcg moreover egcgcaused cell membrane damage via increased intracellularros level and led to potassium leakage these are potentiallyconducive to the antibioï¬lm eï¬cacy of egcg against vibriomimicus which is a foodborne pathogen in seafood andwater in addition egcg also regulates the expression of oxidative stressrelated genes oxyr and soxrs systems are activated upon exposure to oxidative stress oxyr induces katgand soxrs induce soda strongly when cells are stressed byexogenous h2o2 egcg treatment upregulated katgand soda expression in e coli these results veriï¬ed the roleof ros in egcgmediated bacterial inhibition the cpxsystem is thought to control protein homeostasis in the cellenvelope when e coli was exposed to egcg apoptosis happened because of ros formation by the cpx system rpos is a general stress regulator in response to oxidativestress egcg could cause a reduction in the expression forrpos indicating that egcg induced oxidative stress in bacterium models the potential prooxidant properties of egcg could beattributedin part to its suppressive eï¬ects on bacteriamore broadly research is also needed to determine relativesignaling pathways and proteomic factors egcg is superexcellent natural products it could increase the eï¬cacy ofbactericidal eï¬ect when it aids other fungicides morerecent attention has been focused on the impact of greentea and green tea polyphenols on the intestinal microï¬orawhether egcg intervention would change the diversity ofmicrobiota and lead to microbiota death is also in need offurther investigation adverse effectsin recent years egcg has become one of the most aggressively promoted food supplement products in daily lifeegcg entered the market and its safety has raised queriesthe prooxidant eï¬ect of egcg is not necessarily advantageous they might have implications regarding potential toxicity hence it is necessary to systematically explore theharmful eï¬ects of egcg and the mechanisms prooxidant and hepatotoxicity eï¬ects of egcg a considerable amount of literature has been published on hepatotoxicity of green teaderived products it is noteworthythat the hepatotoxicity of green tea and its derived productswas initially found in some diet products in after beingthe cause of liver injury in subjects france and spain governments have suspended the marketing of exolise whichwas a weightloss phytotherapeutical drug in the pasttwo decades reports on liver disorders caused by green teaingestion with overdose of egcg content have graduallyemerged the liver is a major drug metabolic organ in the bodythe bioavailability of egcg in rats was determined after min of oral administration mgkg by detecting theconcentration of egcg in plasma and diï¬erent tissuesincluding the liver the results showed that the concentrationof egcg in the liver μmolkg was four times higherthan in that in the blood plasma μmolkg moreover utilizing anatomy egcg could trigger liver damagewhereas no visible abnormalities were found in other tissuesand organs [ ] hence it could be preliminarily concluded that the liver is the toxic target organ of egcgat the cellular level egcg demonstrated cytotoxic eï¬ectin cultured rat hepatocytes it was shown that μm egcgtreatment on freshly isolated rat hepatocytes caused timedependent cytotoxicity the hepatocyte was incubatedwith egcg for h resulting in liver cell function reduceddose dependently the decrease of lactate dehydrogenase ldh a marker of cell membrane damage wasobserved in rat hepatocytes egcg also caused damageto the outer mitochondrial membrane by the fact that mitochondrial membrane potential collapsed in animal experiments table the severity of egcginduced toxicity is relevant with dose route of administration and period of treatment [ ] biochemicaland histopathological analysis showed that liver samples ofmice displayed diï¬erent degrees of liver injury liver functionindexes of plasma alanine aminotransferase alt andaspartate aminotransferase ast activity increased in adosedependent mannermalondialdehyde mda and 4hydroxynonenal hne are ï¬nal products of lipid peroxidation present biochemical markers of oxidative stress metallothionein mtand γhistone 2ax γh2ax are molecular markers of oxidative stress oral high dose of egcg mgkgd to cf mice for two days significantly enhanced the formation ofmda in the liver and elevated the expression of hepaticmt and γh2ax protein and increased positive staining for4hne in liver samples intraperitoneal administrationof egcg or mgkgd for ï¬ve days raised serum hne level and western blot analysis showed that hepaticγh2ax was markedly increased all these biomarkersillustrated that egcgtriggered hepatotoxicity in vivo wasinduced by oxidative stressprevious pharmacological studies have shown that undernormal physiological conditions egcg is metabolizedthrough methylation sulfation and glucuronidation andthen excreted in urine subsequently whereas at toxicdoses these pathways might be saturated and the excessive 0canimal typefemale swissalbino micemalekunmingmiceegcgdosagemgkgd andmalekunmingmice and and male nd4micemale cf1micewistar rats ofboth sexesmale cd1micemicefemaleswisswebster miceoxidative medicine and cellular longevitytable hepatotoxicity of egcg based on animal modelsroute ofadministrationdurationresultsreferenceip and po dip treatment increased serum bilirubin markers po treatment didnot show any dosedependent changes except alt marker dtolerable dose of egcg was mgkg for ip and mgkg foripipigigpo d dserum alt ast 4hne il2 il6 and il10 and hepatic γh2axwere raised hepatic nrf2target gene expression was increasedthe fatality rate was single doseserum alt ast 4hne il6 and il10 and hepatic γh2ax wereraised hepatic nuclear and cytosolic nrf2 proteins were suppressed d dmouse growth was not aï¬ected the dosage was considered asmaximum tolerable dosehepatotoxicity occurred major hepatic antioxidant enzymes weresuppressed nrf2mediated rescue response was inducedsingle dosemice died in a dosedependent manner andthe nrf2 pathway was not activated nrf2 and its target genes were h dsuppressedalt was slightly increased histopathology of the liver showedcongestion of sinusoids and central and portal veinssingle dosealt was markedly increased histopathology of the liver showeddegenerative hepatocytes and a small number of vacuoles d dmouse survival was reduced by mouse survival was reduced by hepatic mda mt and γh2axwere increasedsingle dosealt was increased by 108fold mouse survival was reduced by egcg2²cysteine and egcg2³cysteine were detected in theurineposingle dosemice were lethargic and their respiration was labored and andipipipsingle doseplasma alt was increased mice died within h h degcg thiol conjugates egcg2²cysteinyl and egcg2³cysteinyl were detected in the urine of mice died plasma alt activity was elevated severe hepaticnecrosis occurredamount of egcg would be oxidized to form oquinonewhich could react with gsh to form egcg thiol conjugates therefore it could be inferred that high dose of egcgresults in the accumulation of oquinones and the metabolites of oquinones are biomarkers of oxidative stress twoegcg thiol conjugates egcg2²cysteinyl and egcg2²²cysteinyl were detected in the pooled h urine of micetreated with a dose of or mgkg intraperitonealip injection of egcg however egcg thiol conjugateswere not found when the dose was or mgkg bwip when cf1 mice were treated with a single doseof mgkg intragastric ig administration of egcgboth egcg2²cysteinecysteine weredetected in the pooled h urine gsh conjugate ofand egcg2²²egcg was also detected in hepatocytes incubated withegcg these ï¬ndings indicated that the formation ofdetectable amounts of egcg thiol conjugates appears toresult from the administration of toxic doses of egcgnuclear factor erythroidrelated factor nrf2 an essential antioxidant transcription factor regulates the expressionof many antioxidant and phase ii detoxifying enzyme genessuch as ho1 and nadphquinone oxidoreductase1nqo1 through antioxidant response element are undernormal metabolic and physiologic states nrf2 is repressed inthe cytoplasm by kelchlike echassociated protein1keap1 while under oxidative stress conditions nrf2 dissociates from keap1 and translocates to the nucleus to bind toare the activation of the nrf2are signaling pathway 0coxidative medicine and cellular longevityrepresenting a major cellular defense against oxidative stresscould stimulate the expression of downstream antioxidantenzymes a previous study revealed that toxic doses ofegcg and mgkg ip inhibited hepatic antioxidantenzymes sod cat and glutathione peroxidase and exacerbated oxidative damage in hepatocytes after treatmentwith egcg the expression of nrf2 decreased in the cytosoland increased in the nucleus indicative of nrf2 activationas a result mrna expression of ho1 nqo1 and otherhepatic nrf2target genes was induced in a dosedependentmanner accordingly a conclusion could be made that themolecular mechanisms underlying highdose egcg potentialtoxicity involve activation of the nrf2are signaling pathwayand suppression o | Colon_Cancer |
" triple negative breast cancer tnbc remains recalcitrant to most targeted therapy approaches however recent clinical studies suggest that inducing tumor damage can render tnbc responsive to immunotherapy we therefore tested a strategy for immune sensitization of murine tnbc 4t1 tumors through combination of focused ultrasound fus thermal ablation and a chemotherapy gemcitabine gem known to attenuate myeloid derived suppressor cells mdscsmethods we applied a sparse scan thermally ablative fus regimen at the tumor site in combination with systemically administered gem we used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity we also tested this combination in rag1 mice or t cell depleted wild type mice to determine the essentiality of adaptive immunity further we layered programmed cell death protein pd1 blockade onto this combination to evaluate its impact on tumor outgrowth and survivalresults the immune modulatory effect of fus monotherapy was insufficient to promote a robust t cell response against 4t1 consistent with the dominant mdsc driven immunosuppression evident in this model the combination of fusgem significantly constrained primary tnbc tumor outgrowth and extended overall survival of mice tumor control correlated with increased circulating antigen experienced t cells and was entirely dependent on t cell mediated immunity the ability of fusgem to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti pd1 thermally ablative fus in combination with gem restricts primary tumor outgrowth improves survival and enhances immunogenicity in a murine metastatic tnbc model this treatment strategy promises a novel option for potentiating the role of fus in immunotherapy of metastatic tnbc and is worthy of future clinical evaluationtrial registration numbers nct03237572 and nct04116320 metastatic breast cancer brca particularly the triple negative breast cancer tnbc phenotype is resistant to most chemical and molecularly targeted therapeutic approaches interestingly tnbc is often infiltrated with immune cells and the presence of these cells has been shown to have a favorable prognosis in patients treated with neoadjuvant chemotherapy1 early studies in the use of immunotherapies targeting the pd1programmed death ligand pd l1 checkpoint inhibitory axis showed some efficacy2 in tnbc compared with other brca subtypes which are generally recalcitrant to checkpoint blockade activity in the tnbc subtype may be related to the relatively high immune infiltration and correlated with the higher mutational burden observed in tnbc greater immunotherapy efficacy in tnbc has been recently observed with the use of antibodies targeting the pd1pd l1 checkpoint inhibitory axis in combination with nab paclitaxel5 this outcome suggests that inducing tumor damage augments antitumor immunity either by promoting antigen availability or disrupting the immunosuppressive tumor microenvironment tme found in tnbcamong the potential networks in tnbc that could constrain the activity of antitumor immunity is the presence of immunosuppressive myeloid cell subsets these have the capacity to impair adaptive immunity and promote tumor growth and metastasis among these cell types myeloid derived suppressor cells mdscs prevail as a heterogeneous population of immature myeloid cells which serve the eponymous role of suppressing the antitumor immune response limiting both t cell activation and effector functions6 increased levels of this cell type have been demonstrated in tumor tissues of patients with primary brca while those with metastatic disease bear the highest abundance of circulating mdscs8 studies have sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access shown that approaches that either stimulate myeloid cells with inflammatory mediators or eliminate mdsc can improve antitumor immunity9to this end the central premise put forth in this study is that focused ultrasound fusa safe noninvasive and nonionizing strategy for localized acoustic energy deposition into tissuescan synergize with immunotherapy in a murine model of metastatic tnbc fus is capable of rapidly heating tumors to thermally ablative temperatures its extracorporeal application obviates the need for catheterization injection or implantation fus can be targeted with millimeter precision under mri or ultrasound guidance thereby allowing for thermal damage and destruction of tumor tissue without compromising healthy intervening or peripheral tissues the bioeffects of fus hold distinct implications for tumor antigenicity immune cell activation and trafficking13 thermally active fus regimes have elicited antitumor immune responses in implantable models of melanoma15 pancreatic16 prostate17 colon20 kidney21 and brca23 pertaining to the challenge of myeloid cell immunosuppression in tnbc thermally ablative fus has been shown to induce the expression of heat shock proteins24 and proinflammatory cytokines including interleukin il12 interferonÎ ifnÎ and tumor necrosis factorα tnfα from a variety of cancer cell lines and after in vivo treatment of tumors26 whether the ability of fus to induce these inflammatory mediators is sufficient to overcome myeloid suppression in the context of brca is currently under debate with some studies showing activation of antigen presenting cells and t cell recruitment in patients with brca treated with thermally ablative fus28 while others show that additional innate stimuli are needed to support antitumor immunity23 notably some studies have suggested that a sparse scan thermal ablation regimen more effectively recruits and activates dendritic cells dcs and antitumor immunity than total thermal ablation perhaps by limiting thermal denaturation of tumor antigens and innate stimuli31based on the improved myeloid cell maturation that occurs with sparse scan regimens we herein tested the ability of a sparse scan partial thermal ablation fus regimen as a monotherapy to promote antitumor immunity in an aggressive syngeneic model of metastatic murine tnbc with extensive granulocytic mdsc involvement that is recalcitrant to anti pd1 while some activity is evident with the partial ablation approach significantly greater control was achieved by targeting mdsc inhibition in combination with thermally ablative fus this control was completely dependent on the adaptive immune responsemoreover we demonstrate that layering anti pd1 immune checkpoint blockade onto this combinatorial regimen moderately improves tumor growth restriction these data suggest that in disease settings where myeloid allied approaches to attenuate myeloid immunosuppression may be employed to reveal the full immunotherapeutic immunosuppression predominates potential of thermally ablative fus once immunosuppressive myeloid cells are accounted for fus treatment can promote adaptive immunity that in turn potentiates immune checkpoint blockademethodscell line maintenance4t1 and e0771 cell lines were maintained in rpmi l glut or dulbeccos modified eagles medium dmem gl d glucose l glutamine respectively supplemented with fetal bovine serum fbs at °c and co2 thawed cells were cultured for up to three passages and maintained in logarithmic growth phase for all experiments cells tested negative for mycoplasmaeight week old to week old female balbc or c57bl6 mice were obtained from nci charles river nci crl or the jackson laboratory female balbc rag1 mice were obtained from the jackson laboratory 4t1 or e0771 cells were subcutaneously implanted into the right flank of mice mice were housed on a hour12 hour lightdark cycle and supplied food ad libitum tumor outgrowth was monitored via digital caliper measurements tumor volume was calculated as follows volume lengthwidth22 approximately days 4t1 or days e0771 following tumor implantation mice were randomized into groups in a manner that ensured matching mean starting tumor volume across experimental groupsin vivo ultrasoundguided fus partial thermal ablationmice were treated with fus either days 4t1 cohorts or days e0771 postimplantation on treatment day mice were anesthetized with intraperitoneal injection of ketamine mgkg zoetis and dexdomitor mgkg pfizer in sterilized saline mouse flanks were shaved and depilated following which ultrasound guided fus thermal ablation was performed using one of the two systems system and treatment details are provided in online supplementary materials and methods mice that did not receive fus treatment consistently underwent anesthesia and depilation of the flank additionally these mice underwent a sham treatment consisting of exposure to the °c degassed water bath exposure for min following sham or fus treatment all mice were moved to a heating pad and given antisedan for anesthesia reversal and recoverygemcitabine therapygemcitabine gem mgmouse in µl volume mylan diluted in saline and filter sterilized through a µm syringe filter was administered intraperitoneally once a week on the day of fus treatment following which administration was repeated for an additional weeks administration of gem doses was based on existing literature demonstrating the use of gem for inhibition of mdscs in 4t112 the initial dose of gem was administered immediately prior to sham or fus treatment sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0cmice that did not receive gem received an intraperitoneal injection of vehicle treatment µl of sterile saline at the time points specifiedpd1 blockade therapyfor checkpoint inhibitor therapy the rat anti mouse pd1 antibody αpd1 rmp114 diluted in sterilized saline was administered intraperitoneally every days for a total of five doses µg per mouse treatment was initiated on day early αpd1 or day delayed αpd1t cell depletionst cell depletion antibodiesanti cd8 clone bio x cell and anti cd4 gk15 clone bio x cellwere diluted in sterilized saline and administered intraperitoneally every to days starting at day days post fus for a total of seven doses µg of each antibody for a total µg per mouseimmunohistochemistryon day sham or fus exposed tumors were excised and fixed in neutral buffered formalin sigma fixed tumors were paraffin embedded sectioned and stained for hematoxylin and eosin digital images of stained slides were acquired using the vectra automated quantitative pathology imaging system akoya biosciences whole slide screening and image capture were subsequently performed using phenochart akoya biosciencesflow cytometrymice were bled at days and via tail vein and samples were rbc lysed hybri max sigma and stained for flow cytometry analysis at days post tumor implantation tissues were obtained from euthanized tumor bearing animals for immune response assessment in order to gain resolution into tissue resident versus vascular immune cell populations mice were injected intravenously with rat anti mouse cd45 fitc clone f11 bd biosciences min prior to euthanasia 4t1 tumors spleens cardiac blood axillary and brachial tumor draining lymph nodes tumor dlns pooled and nondraining inguinal lymph nodes were harvested processed and stained for flow cytometry analysis additional details are provided in online supplementary materials and methodssamples were acquired on an attune nxt flow cytometer thermofisher scientific and data were analyzed with flowjo treestar or fcs express de novo software a representative gating strategy for granulocytic myeloid derived suppressor cell g mdsc and cd44 t cells is provided in online supplementary figure statistical analysisall statistical analyses were performed in graphpad prism graphpad software a detailed description of statistical methods for each experiment is provided in the corresponding figure legendopen accessanimal study approvalall animal work was performed under a protocol approved by the animal care and use committee at the university of virginia and conformed to the national institutes of health guidelines for the use of animals in researchresultspartial thermal ablation of established tnbc tumors promotes peripheral dc activation but has limited impact on the presence of t cells and other myeloid cell subsetsto achieve partial thermal ablation of 4t1 tumors we used an ultrasound guided fus system equipped with a single element therapeutic transducer driven at mhz figure 1a online supplementary figure a grid of sonications was overlaid on the ultrasound visible tumor and ablated in a raster pattern under b mode ultrasound guidance figure 1bc the exceptionally small focus of this system rendered a low ablation fraction of total tumor volume immediately following ablation tumors displayed evidence of coagulative necrosis in the ablated zone with surrounding periablative margins figure 1d one week following fus partial thermal ablation tumors and secondary lymphoid organs were excised for immunological characterization by flow cytometry figure 1b fus partial thermal ablation of 4t1 tumors conferred a significant increase fold in the absolute number of cd11c hi dcs within the axillary tumor draining lymph node adln of mice figure 1e while this was accompanied by a nearly threefold elevation in the absolute number of cd86 dcs within the adln figure 1f the percentage of dcs expressing cd86 did not change figure 1g increased numbers of dcsand cd86 dcs in particularsuggest fus is promoting the maturation or trafficking of these cells in the dlns where they could encounter and activate t cells however this did not translate to tumor growth restriction data not shown we also did not observe significant differences in the absolute number of activated t cells in 4t1 tumors figure 1h or dlns data not shown following fus exposure suggesting limitations in the ability of fus activated dc to further drive an antitumor t cell responseimmune profiling by flow cytometry revealed that irrespective of fus exposure of the intratumoral cd45 immune cell population is comprised of cd11b myeloid cells figure 1i similarly approximately of the circulating immune cell population in 4t1 tumor bearing mice is comprised of myeloid cells a striking fold elevation in circulating myeloid burden compared with naive mice online supplementary figure notably ly6g granulocytic myeloid derived suppressor cells g mdscs significantly dominated the immune cell repertoire within 4t1 tumors relative to other myeloid including f480 macrophages ly6c cell subsets monocytic myeloid derived suppressor cells m mdscs and cd11c hi dcs figure 1j fus partial thermal ablation did not significantly alter the absolute number per sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access figure partial thermal ablation of established tnbc tumors promotes peripheral dc activation but has limited impact on the presence of t cells and other myeloid cell subsets a design overview of a custom ultrasound guided fus system consisting of a mhz single element transducer orthogonally co registered to an mhz linear ultrasound imaging array the tumor bearing flank of each anesthetized mouse was acoustically coupled to ultrasound transducers via degassed water bath maintained at °c sham mice were similarly positioned but did not undergo sonications b schematic illustration of fus partial thermal ablation scheme and study layout for evaluation of immune sequelae in 4t1 tumor bearing mice a grid of sonications was applied in a raster pattern onto the b mode ultrasound visible tumor in total two planes of sonication spaced mm apart were applied to each tumor grid points were spaced mm apart within a single plane one week following thermal ablation tumors and secondary lymphoid organs were excised for sham n6 or fus treated n5 mice and processed for flow cytometry c representative b mode ultrasound images of ectopic 4t1 tumors either before top or during bottom fus exposure sonication grid depicting targets red points is superimposed on b mode image during treatment subsequent to thermal ablation hyperechoic signatures yellow arrow are occasionally observed d representative he staining of either sham 4t1 tumors or those resected immediately following fus partial thermal ablation zoomed insets depict the transition from necrotic to intact tumor tissue within the periablative zone scale bars400 µm and µm on left and right inset respectively e absolute number of cd11c hi dcs in the axillary tumor draining lymph node adln of 4t1 tumor bearing mice p00136 vs sham f absolute number of cd86 cd11c hi dcs in the adln p00063 vs sham g percentage of cd86 subset out of total cd11c hi dcs within adln h absolute number of intratumoral cd44 cd8 and cd44 cd4 t cells and regulatory t cells tregs per gram tumor i percentage of cd11b myeloid cells out of total cd45 immune cells across tumor spleen adln inguinal dln idln and nontumor draining axillary and inguinal lns ndlns p005 vs all other groups irrespective of fus exposure specifically tumor vs spleen p00226 tumor spleen vs all other organs p00001 j absolute number of intratumoral myeloid cells cd11c hi dcs f480 macrophages ly6c monocytic myeloid derived suppressor cells m mdscs ly6g granulocytic myeloid derived suppressor cells g mdscs per gram 4t1 tumor p00001 vs all other cell types irrespective of fus exposure all data represented as mean±sem significance assessed by unpaired t test fh or two way analysis of variance followed by tukey multiple comparison correction ik nsnot significant dcs dendritic cells fus focused ultrasound hifu high intensityfocused ultrasoundsheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0cgram tumor of these myeloid cell subsets these observations led us to formulate the hypothesis that widespread immunosuppressive mechanisms associated with the 4t1 tme must be addressed in order to facilitate the t cell response to fusfus partial thermal ablation in combination with gem constrains primary tnbc tumor outgrowth and extends overall survivalour observation of the overwhelming mdsc burden following 4t1 tumor implantation warranted implementation of an allied therapeutic strategy in order to counter this immunosuppressive barrier to this end we tested a combinatorial paradigm incorporating gem a myelosuppressive chemotherapy demonstrated to inhibit mdscs transiently in the 4t1 model without consequence to t cell phenotype or function12to evaluate the efficacy of fus and gem in combination we used a preclinical ultrasound guided fus system to achieve partial thermal ablation of established 4t1 tumors 14d after tumor implantation average tumor volume of mm3 in combination with the single session of fus thermal ablation we initiated gem therapy mgmouse which was then readministered weekly for a total of three gem doses figure 2a combinatorial therapy synergized to produce significant constraint of 4t1 tumor outgrowth compared with sham and monotherapy groups figure 2bcby termination of treatments at day 4t1 tumors exposed to fusgem combination saw nearly and reductions in average volume compared with sham or gem exposed tumors respectively figure 2b two dimensional tumor projections at day postimplantation saw a nearly fold reduction in area from sham to combinatorial therapy setting figure 2de in a fraction of mice treated with fusgem we observed complete regression of 4t1 tumors although transient figure 2c tumor outgrowth eventually rebounded after termination of treatments 4t1 tumor bearing mice receiving fusgem treatment additionally saw the greatest extension in overall survival with and increases in median survival time compared with sham and gem groups respectively hrs and for fusgem relative to sham and gem groups respectively figure 2f we additionally observed that fusgem significantly constrained outgrowth in a separate c57bl6 metastatic mammary carcinoma model e0771 online supplementary figure to further the clinical relevancy of these findings we applied this combinatorial strategy with the research grade analog of a clinical ultrasound guided fus system theraclion echopulse that is already ce marked for applications in breast fibroadenoma thyroidparathyroid gland and varicose vein ablation and currently in use for multiple clinical trials leveraging fus thermal ablation in combination with cancer immunotherapy we observed that partial thermal ablation using the theraclion visualization and treatment unit mhz in combination open accesswith gem controlled 4t1 tumor outgrowth to a degree comparable with that observed with the custom in house system online supplementary figure these findings lend credence to the notion that the impact of combining gem with fus may be conserved across partial thermal ablation regimens moreover they demonstrate that the efficacy of fus partial thermal ablation in combination with gem can be recapitulated on a system with a larger focus and in line image guidance that is currently in use clinicallycombination of fus partial thermal ablation with gem increases the levels of circulating t cellslymphocytesin particular cd8 and cd4 t cellsplay an important role in responding to tumor antigen and generating a durable antitumor response based on the extended protective effect observed in mice treated with fusgem flow cytometry analysis was performed to evaluate the contribution of t cells in generating systemic and local tumor control we sampled the circulating immune cell repertoire in 4t1 tumor bearing mice via serial tail bleeds days and prior to readministration of gem and a terminal cardiac bleed at the time of spleen harvest day figure 3a combinatorial therapy significantly elevated absolute number of cd8 and cd4 t cells in the circulation at days and figure 3bc and ef moreover a trend threefold to fivefold increase in circulating t cells was noted in the fus group relative to sham figure 3bc and ef from days to systemic cd44 expressing antigen experienced t cell populations both cd8 and cd4 saw a steady significant increase after combinatorial therapy figure 3d and g a similar modest trend was noted for the fus monotherapy group relative to sham and gem figure 3d and g these changes were concordant with a decrease in circulating myeloid cd11b cells in gem recipient groups demonstrating the ability of gem to partially alleviate circulating myeloid burden figure 3hsplenomegaly is a common signature that arises in parallel with the leukemoid reaction to 4t1 tumors that is the expansion of immunosuppressive myeloid cells during tumor progression we observed that combinatorial therapy most significantly reverses splenomegaly online supplementary figure 6ab consistent with this observation immunological characterization of spleens revealed a significant decrease in cd11b myeloid cellsa reduction in fusgem spleens relative to sham or monotherapy figure 3i while there appeared to be a trend toward more cd11b cells in the monotherapy groups compared with the sham this difference was not significant and there was no difference between these groups in terms of absolute cd11b cell numbers within the spleen data not shown the decrease in myeloid cells in the combination treatment group was accompanied by a significant corresponding elevation in lymphocytes in the spleen following fusgem treatment relative to these sham and gem groups combination therapy elevated splenic cd8 t lymphocytes by fold and sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access figure combination of focused ultrasound fus partial thermal ablation with gemcitabine gem constrains primary triple negative breast cancer outgrowth and extends overall survival a overview of experimental design for evaluation combination of fus with serial gem treatment in murine mammary carcinoma b average 4t1 tumor outgrowth in sham n7 fus monotherapy n5 gem monotherapy n10 and combinatorial fusgem therapy groups n10 data are represented up to select time points corresponding with mouse dropout due to humane endpoints all data represented as mean±sem significance assessed on outgrowth up to day by repeated measures mixed effects model implementing restricted maximum likelihood method followed by tukey multiple comparison correction p005 vs all other groups specifically sham vs fusgem p00001 fus vs fusgem p00001 shamgem vs fusgem p00026 c 4t1 tumor outgrowth from individual mice in sham fus shamgem or fusgem groups data represent outgrowth from initiation of treatments at day up to removal of mouse from study for meeting a humane endpoint d representative images of 4t1 tumors excised at day scale bar1 cm e quantification of 2d tumor areas from images in previous panel f kaplan meier curve depicting overall survival of sham treatment n9 fus monotherapy n6 gem monotherapy n10 and combinatorial fusgem therapy n10 recipient mice significance assessed by log rank mantel cox test p005 vs all other groups specifically sham vs fus p02154 sham vs fusgem p00001 sham vs shamgem p00050 fus vs fusgem p00021 fus vs shamgem p00312 fusgem vs shamgem p00041sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen accessfigure combination of focused ultrasound fus partial thermal ablation with gemcitabine gem increases the levels of circulating t cells a overview of experimental design to understand the impact of fus andor gem treatment on circulating immune cells bc absolute number of circulating cd8 t cells at day b and day c d percentage of circulating cd8 t cells expressing cd44 from days to ef absolute number of circulating cd4 t cells at day e and day f g percentage of circulating cd4 t cells expressing cd44 from days to h percentage of cd11b myeloid cells out of total cd45 immune cell in circulation from days to ik percentage of myeloid cells i cd8 t cells j and cd4 t cells k out of total cd452 immune cells all data represented as mean±sem all data representative of sham n6 fus monotherapy n4 gem monotherapy n9 and combinatorial fusgem therapy n6 groups significance assessed by analysis of variance followed by tukey multiple comparison correction for b c e f or fishers least significant difference lsd without multiple comparisons correction for ik significance for d g and h assessed by repeated measures mixed effects model implementing restricted maximum likelihood method followed by fishers lsd without multiple comparisons correction p005 vs all other groups unless otherwise indicated p001 p0001 vs groups indicatedsheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access fold figure 3j and cd4 t lymphocytes by fold and fold figure 3k these elevations were accompanied by a modest increase in percentage of foxp3 regulatory t cells tregs online supplementary figure 6e additionally increases in percentage of nk and b cells were noted twofold to fivefold online supplementary figure 6cd these findings indicate that combinatorial therapy with fusgem promotes a systemic lymphocyte response that is discrete from the effects of either intervention alone which may account for reduced mortality associated with pulmonary metastasescombinatorial fusgem therapy does not promote robust local antitumor t cell responsesgiven the robust systemic immune signatures within the blood and spleen following fusgem we assayed 4t1 tumors at a time point within the window of tumor growth restriction and subsequent to termination of treatments ie day to interrogate whether tumor control correlates with an increase in the effector functions of the intratumoral t cell response figure 4a approximately hours prior to euthanasia mice received intravenous brefeldin a injection to inhibit cytokine secretion for subsequent intracellular cytokine staining by flow cytometry immune characterization of tumors at days postimplantationthat is days subsequent to final gem administrationrevealed no significant changes in absolute number of antigen experienced cd44 cd8 or cd4 t lymphocytes figure 4bc moreover the polyfunctionality of these t cells as denoted by ifnÎ and granzyme b expression was not significantly altered figure 4de however intratumoral functional changes were noted in the myeloid compartment gem monotherapy modestly increased il 12p40 production by dcs fold but this was not conserved in the combinatorial therapy group figure 4f moreover while fus monotherapy generated a trend in elevated tnfα production by intratumoral g mdscs gem recipient groups saw a significant increase threefold relative to sham figure 4g these findings indicate that changes in the myeloid compartment in response to monotherapy and combination therapy may contribute to tumor control but are unlikely to drive the protective response entirely interestingly intratumoral t cell representation correlates poorly with circulating lymphocytes suggesting a transitory immune response that either cannot be fully characterized at this time point or is hampered by additional modes of immunosuppressionprotection conferred by combination of fus and gem is dependent on adaptive immunitysince our findings revealed no obvious advantage or function of adaptive immunity in the local tme we next investigated the overarching role of the adaptive immune system in protection offered by combinatorial therapy with fusgem to this end we utilized an rag1 model that is deficient in t and b cells to address the hypothesis that mature t andor b cells play a role in the observed response wild type wt or rag1 mice bearing 4t1 tumors were randomized into groups in a manner that preserved similarity in average initial tumor volumes mice were subsequently treated with either gem monotherapy or the combination of fusgem the tumor growth inhibition offered by fusgem was entirely lost in rag1 mice relative to their wt counterparts with average 4t1 tumor volume in rag1 mice being over fivefold higher than that of wt mice on termination of treatments figure 5a of note despite a trend toward loss of protection in rag1 mice tumor outgrowth in response to gem monotherapy did not significantly stratify between wt and rag1 settings figure 5a we also observed a complete loss of fusgem mediated survival benefit over gem monotherapy in the rag1 setting figure 5b while these results demonstrate that an intact adaptive immune response is required for both the overall survival benefit and restriction of primary tumor offered by fusgem therapy they do not delineate the relative roles of t andor b cellsthus to address the hypothesis that the protective effect of fusgem is specifically dependent on cd4 and cd8 t cells we depleted these populations via serial coinjections of cd8 depleting and cd4 depleting antibodies in 4t1 tumor bearing wt mice on a fusgem figure 5c depletions were maintained between day and day and flow cytometry analysis of circulating immune cells at day confirmed that the target t cell populations were effectively depleted in all mice online supplementary figure consistent with the tumor escape observ | Colon_Cancer |
"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 |
moringa oleifera l from the moringaceae family is a perennial tree widely cultivated in many tropic regions and easily grown even in adverse conditions m oleifera is also known as the miracle tree which for centuries has been indicated for traditional medicine with no reports of side effects in doses achievable by ingestion different parts of m oleifera is used to treat several conditions such as malnutrition diabetes blindness anemia hypertension stress depression skin arthritis joints and a0kidney stones disorders this plant also showed capacity of helping in maintenance of the cardiovascular system health bloodglucose levels and providing antioxidant antiinflammatory and anticancer activity as well as the regulation of urinary tract and lactation in nursing women the seed and leaves powder has water purification properties through flocculation it also supplements the food in the human diet and in the fortification of livestock feed especially in developing countries so m oleifera properties have also been applied to cosmetic and byproducts industries due to the high nutritive and protective properties of its seed oil according to the holistic or traditional medicine m oleifera has very relevant therapeutic properties and applications depending on the constitution somatic and psychological needs of patients it is usually referred as a natural product that can treat different physical and psychological health aspects offering an energetic action and structural rebuilder of the body and promoting emotions of highly positive attitudes towards life the high and specific immunological potential of m oleifera leads us to suggest an indepth study to assess the hypothesis of conferring a supportive effect against covid19 diseasekeywords moringa oleifera a0· drumstick tree a0· miracle tree a0· medicinal plant a0· cosmetics a0· food supplementdiana meireles and jo£o gomes authors contributed equally to this workdianameireleslivecompt diana meireles icbas institute of a0biomedical sciences abel salazar university of a0porto rua de je viterbo ferreira no a0porto portugal yidao acupuncture and a0tcm center porto portugal ciimar interdisciplinary centre of a0marine and a0environmental research university of a0porto porto portugal cbsin center of a0biosciences in a0integrative health porto portugalintroductionmoringa oleifera l moringa pterygosperma g wellknown as the drumstick or horseradish tree is native of northwest india its main producer but can also be found in south africa northeast africa madagascar tropical asia southwest asia and latin america the moringa genus comprises species m arborea m longituba m borziana m pygmaea m hildebrandtii m drouhardii m longituba m peregrina m stenopetala m rivae m ruspoliana m ovalifolia m concanensis and m olefera rani a0 from the moringaceae family m oleifera is the most known studied and used species anwar olson with human and animal applications the various resources obtained from this plantleaves flowers seeds pods bark and rootscan be used for cooking or in traditional medicine to treat several pathologies m oleifera has the capability to survive in humid or dry hot climates vol01234567891 0c d a0meireles and poor soils anwar et a0al mainenti m oleifera is a highly nutritious plant being ideal to treat malnutrition in developing countries zongo valdezsolana et a0al gopalakrishnan debajyoti et a0al m oleifera gained the title of miracle tree and commercial attention supported on several properties such as nutritional values amino acids and flavonols content which can be used in food supplements and cosmetic industry tables a0 and in fact when compared to other plants from a0g of dry leafs of m oleifera we can obtain times more vitamin c than from oranges times more vitamin a than from carrots times more calcium than in milk a0times more protein than in yoghurt times more potassium than from bananas and times more iron than the obtained from spinach oduro et a0al a0 a0rockwood saini et a0al table a0 shows the nutritional values for the edible parts of raw pods and leaves obtained from the united states department of agriculture usda database although it is table nutritional values per a0g of the edible portion of m oleifera pods and leavescomponentsper a0graw podsaraw leavesaenergy kcalwater gprotein gtotal lipid gcarbohydrate by difference gfibre total dietary gfatty acids total saturated gfatty acids total monounsaturated gfatty acids total polyunsaturated gfatty acids total trans gcholesterol mgvitamin a rae µgvitamin d d2 d3 µgvitamin d iuthiamin mgriboflavin mgniacin mgpantothenic acid mgvitamin b6 mgvitamin b12 µgvitamin e mgvitamin c total ascorbic acid mgfolate total µgfolic acid µgsodium mgpotassium mgcalcium mgphosphorus mgmagnesium mgiron mgzinc mgcopper mgmanganese mgselenium µga information obtained from united states department of agriculture nutrient database ndbnalusdagovndbfoods in june b average values and standard deviation published by witt only two values were found witt dried leavesb ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0ctable amino acids and flavonols per a0g of the edible raw portion of m oleifera leavesper a0graw leavescomponentsamino acids a0tryptophan g a0threonine g a0isoleucine g a0leucine g a0lysine g a0methionine g a0cystine g a0phenylalanine g a0tyrosine g a0valine g a0arginine g a0histidine g a0alanine g a0aspartic acid g a0glutamic acid g a0glycine g a0proline g a0serine gflavonols a0isorhamnetin mg a0kaempferol mg a0myricetin mg a0quercetin mginformation obtained from united states department of agriculture nutrient database ndbnalusdagovndbfoods in june known that the nutrient content varies according to the plantation site aslam and seasons witt the nutritional value of dried leaves not existent in usda database is presented as an average of values with standard deviation calculated from diverse papers that was compiled and published by witt from leaves to roots it is possible to obtain good quantities of important minerals proteins vitamins carotene amino acids and phenolic compounds anwar et a0al leone et a0al 2015a b saini et a0al fahey debajyoti et a0al divya et a0al m oleifera extracts have been studied with different medicinal purposes antiinflammatory antihypertensive diuretic antimicrobial antioxidant antidiabetic antihyperlipidemic antineoplastic antipyretic antiulcer and hepatoprotectant fahey abdull razis et a0al divya et a0al anwar et a0al published a table with various traditional medicinal uses of the different parts of m oleifera anwar et a0al the attractive properties of this plant led to studies of side effects and medical interactions in animal models and humans according the revision by stohs and hartman until this date none of the human in a0vitro studies or extrapolations of animal studies to humans reported adverse effects with doses of m oleifera leaves and leaf extracts achievable by oral ingestion although there was not any report of major adverse side effects there are some important information that should be registered in fact there are some studies suggesting that m oleifera cannot be used in combination with other modern medicines in humans a research by gholap et a0al concluded that m oleifera has been noted to be a good regulator of insulin thus according sileshi et a0al patients suffering from lack of insulin will probably have adverse reductions of sugar levels when using m oleifera for medicinal purposes suggesting that it could decrease the blood sugar to even lower levels when used in combination with other modern medications another study suggests that when treating thyroids m oleifera compounds of the leaf may improve thyroid function tahiliani et a0al this well proving evidence further suggests that it can possibly conflict with other thyroid medication triggering drug interactiona research work concerning the acceptability and safety of shortterm daily supplementation in a group of malnourished girls assessed the use acceptability and safety of m oleifera on children girls in zambia barichella et a0al with regards to safety concerns supplementation of a0g per day of m oleifera powder was deemed safe for children and adolescents both in the short and long term this research also noted that mild nausea was reported in of the children at various age groups when meals were supplemented with a0g of m oleifera daily showing to be still an inadequate and symptomatic dose in childrenother studies suggest that m oleifera could adverse and slowly breaking down the pharmaceutical drugs in the liver and thus a0may develop cirrhosis and liver failure resulting in malnutrition and weight loss as well as decreased cognitive function das et a0al kelly sileshi et a0al despite the numerous positive health benefits associated with m oleifera phytochemicals there are suspicions that it contains harmful substances fahey et a0al annongu et a0al maizuwo it contains specific chemical compounds such as alkaloids and other phytotoxins which when consumed in high doses presents potentially nerveparalysing properties and other adverse effects maizuwo et a0al some of these phytochemicals include moringine moringinine estrogen pectinesterase and phenols including tannin fahey et a0al there are unconfirmed reports that m oleifera stems roots and flowers potentially contain harmful phytochemical constituents especially during pregnancy which may promote uterus contraction leading to miscarriages in pregnant women dutta it is also suspected that it can prevent implantation in women hence it must be avoided a review of a0properties nutritional and a0pharmaceutical applications of a0moringa oleifera¦ 0c d a0meireles table compilation of food supplements containing m oleifera tree parts or extracts in june tree partsproduct informationbrandproductnaturingamoringa capsulesleavesmoringa teamoringa kids multivitamin complexmoringa powderbioheradried seeds extract moringa capsulesleavesmoringa syrupleavesleavesmiracle treemoringa anic teaanic moringa superfood supplementscapsulesmoringa superfood powdermoringa superfood sticksiswarimoringa powder anicdrasanvileaves dry extract nutrabasicsmoringaregulates the gastrointestinal transit natural antiinflammatory lowers cholesterol levels improves diabetic conditiondelays the ageing process ensures proper digestion high antioxidant power helps healing process tonifies body and mindstrengthens the immune system rich in vitamins and minerals stimulates natural defensesadds nutritional value source of fiber protein vitamins and minerals improves physical conditionstrengthens the immune system helps to reverse the aging process beautifies the skin reduces the appearance of wrinkles and fine lines maintains the normal glucose level stimulates brain function and concentration increases libidoit is a nutritionally complex whole food naturally rich in vitamins minerals and amino acids daily use of moringa can help to restore your imbalances in your dietthe moringa leaf boasts a vast array of beneficial nutrients making this tree one of the highest plant sources of vitamins and minerals aroundthe richness of its active ingredients helps maintain blood glucose levels provides flavonoids and polyphenols by those attempting to conceive as it functioning as an abortifacient nath et a0al dutta finally cytotoxicity was verified in experiments with human peripheral blood mononuclear cells only at a0mgkg of an aqueous leaf extract genotoxicity on blood rats cells was verified at a0mgkg asare et a0al however all mentioned side effects were verified with doses that far exceed the amounts used in food intake asare et a0al so research on the adverse side effects with doses achievable by oral ingestion should still go on since currently there are no scientifically confirmed clear toxic and harmful effects of m oleifera extracts and products on both human and animal models adedapo et a0al stohs et a0al many studies on nutrition phytotherapy disease treatment and prevention goals have been published thus supporting scientific basis about the efficiency of traditional uses of m oleifera fahey in fact records about symptoms signs and treatment strategies in different diseases are found in several ancient texts of traditional medicines such as ayurveda and traditional chinese medicine tcm karadi et a0al kasote et a0al debajyoti et a0al as an endemic source with highly digestible protein ca fe vitamin c and carotenoids is considered as a suitable natural product to be used by undernourishment populations dixit the resources obtained from a0m oleifera tree on a0a a0conventional approachleaves and a0podsin some countries leaves and fruits are commonly used in culinary as vegetables leaves can also be dried and used in infusions or grounded into powder allowing easier conservation and consumption moyo olson et a0al in all ways of use and conservation m oleifera does not lose nutritional value mahmood leaves and pods are low in calories and rich in minerals vitamins and natural antioxidants table a0 anwar rebufa et a0al phytochemicals like flavonoids are also present in leaves as well as a significant percentage of essential amino acids table a0 m oleifera leaves contain a high quantity of polyunsaturated fatty acids and low saturated fatty acids content moyo which combined with diuretic lipid and blood pressure lowering properties from leaves and pods contribute to the maintenance 0ctable compilation of cosmetics containing m oleifera tree parts or extracts as ingredients in june cosmetic ingredientsproduct informationproduct brandnameskinsecretantiwrinkle face creamantiaging moisturizer face creamhand creambody milklushafrican paradise body conditionerqueen bee hair honeymagical moringa facial moisturizercharity pot hand and body lotionpassion fruit lip balmgo faster feet foot lotiontwinkle toes foot powderlush gardener cold pressed soaplaboratoires s©robiologiquespurisoft®body shopmoringa range shower gel oil body butter body milk body sorbet hand cream soap body scrubmoringa eau de toilette moringa body mistpurifying and protective action against environmental stress such as smoke and pollutionmoisturizing nourishingdeodorizingremove dirt moisturizing nourishingskin cleansingpurification protects skin against pollution heavy metals cigarette smokeskin feels smooth and restoreddelicately scent your skin in a crisp floral aroma with moringaleavesm pterygosperma oilm pterygosperma leaf infusionoilm pterygosperma powderactive ingredient peptide from moringa seedsm pterygosperma oilm oleifera leaf extractm pterygosperma seed extractm oleifera seeds and oilm oleifera leaf extractoilm pterygosperma seed extractm pterygosperma extractclarinsextracomfort antipollution cleansing cream eliminates traces of pollution detoxifies the epidermis and protects the skin from the harmful effects of pollutionneutralizes the effects of pollution and purifies the skin to restore its natural radiancepurifies and refines while preserving your skins natural moisture balance neutralizes the harmful effects of pollutiononestep facial cleanserexfoliating body scrubonestep gentle exfoliating cleanserwater purifycomfort onestep cleanserdaily energizer cleansing gelnaturingamoringa soap biomoringa exfoliating face scrubmoringa o2herbal moisturizing lotionfacial tonersoapherbal shampooconditionershu uemuraantioxi pollutant and dullness clarifying cleansing oilurban moisture hydronourishing shampooconditionerdouble serumdeep treatment masquehigh antioxidant value slowing skin ageing exfoliate dead cells by regenerating the tissuemoringa seeds peel and exfoliate the skin while moringa oil moisturizes and regenerates the skinrejuvenate nourish and protects skinrepairs strengthens reduces hair fallenhanced power to remove micro impurities and stubborn makeup antipollution breakthroughhighly concentrated in nutrients vitamins and antioxidants intensely hydrates deep within strandsa review of a0properties nutritional and a0pharmaceutical applications of a0moringa oleifera¦ 0c table continuedcosmetic ingredientsm oleifera seed extractproduct brandnamebiobeaut©antipollution micellar cleansing watergentle cleansing foamcleansing oil gelgentle exfoliating geldualphase waterproof eye makeup removerd a0meireles product informationremoves makeup pollution particles and excess sebum while leaving the skin well moisturized the seed of moringa extract selected contains purifying peptides which on the surface limit the adhesion of the pollution particles and in depth activate their elimination this extract acts as a protective shield capable of preserving the good bacteria from the cutaneous flora against the aggression of pollutionof cardiovascular health anwar et a0al table a0 in dried m oleifera leaves it was also found a high content in calcium and iron which is normally residual in other plants used in our diet in the leaves is found greater a variety and quantity of proteins when comparing to other tree parts rebufa et a0al wang et a0al due to its nutritional rich values m oleifera can be a good enriching food additive to human diet and also an animal feed fortifier moyo adding fresh or dried leaves to the feed of milk cows increased milk production and respectively that fact would be of great importance in developing countries to fight deficiencies in nutrition bhargave studies of acceptance by the consumer of enriched foodssnacks with m oleifera have been obtaining good results ellis jung m oleifera can also help lactating mothers produce more milk and help to treat malnutrition in young children phytosterols from m oleifera increase estrogen production that enhance the activity of the mammary glands ducts gopalakrishnan doses of a0mgg of body weight given to mice result in increased milk production also the pup weight augment with increasing doses of m oleifera leaf powder intake titi et a0al titi and nurjanah studies of toxicity in animals show that m oleifera dried leaf extract might be safe for consumption although in high doses and prolonged intakes m oleifera may cause toxicity by accumulation of some elements a0ali et a0al the amount of a0g of m oleifera dried leaf per day is the maximum recommended doseage asiedugyekye et a0 al table a0 compiles some food supplements based on m oleifera tree parts or extracts a hydroalcoholic extract of green pods increased liver enzymes involved in the detoxification of xenobiotic substances in mice table a0 suggesting a chemo preventive potential of a drumstick extract against chemical carcinogenesis bharali et a0al m oleifera pods are also valuable to treat digestive and obesity problems and thwart colon cancer gopalakrishnan et a0al carotene the major component reported from the drumsticks of the m oleifera plant as well as the presence of vitamin a and c suggest an action in the induction of antioxidant and antiinflammatory profiles geervani and devi bharali et a0al praengam et a0al it was suggested that carotene and sterols present in the plant pods acts as potent inhibitors on the formation of reactive oxygen intermediates a prerequisite for tumorigenesis and so inducing apoptosis in the mouse colon carcinoma model gupta et a0al kraiphet et a0al studies in rats showed that m oleifera leaves extract might act as potential neuroprotectant via decreased oxidative stress and the enhanced cholinergic function kirisattayakul et a0al and function as a cognitive enhancer hence being used in dementia cases sutalangka et a0al it was also found an antidepressant activity in mouse models of depression when giving orally a a0mgkgday of a m oleifera alcoholic extract plus a0mgkgday fluoxetine for a0days kaur et a0al this effect can be increased when combined with fluoxetine as a selective serotonin reuptake inhibitorssri according to sutalangka et a0al and kaur et a0al 2015the influence of m oleifera may be due to the action of antioxidants and flavonoids through radical scavenging since its action is verified in other studies on animal models with cerebrovascular diseases exerting a multiplicity of neuroprotective actions within the brain and suppressing neuroinflammation and thus suggesting a great potential to promote memory learning and cognitive function vauzour et a0al other studies with consumption of m oleifera leaf powder revealed properties in human an animal models such as decreased blood glucose levels on diabetic type two subjects william reduction on post prandial blood glucose ghiridhari increased insulin secretion in healthy subjects anthanont et a0al decreased total plasma cholesterol and increased hdl nambiar the presence of sitosterol in m oleifera leaves may be one of the reasons for decreasing plasma cholesterol since phytosterols cause less intestinal absorption of dietary cholesterol and increase its excretion on feces jain mbikay 0cnoitcudorpmubes niks ni noitcuderledom laminadna ortiv ni la a0te amrev a0 a0 pytivitca tnadixoitnaledom laminaan yehafnoitcefni la a0te ruak la a0te akgnalatuselfiorp dipil no tcapmi evitisopledom lamina a0 a0 p raibman ledomnamuh a0 a0 pyattasiri yehaf la a0te lukak la a0te ruozuav la a0te roknod la a0te la a0te eednodnesodu hanajrundna iti t la a0te itit la a0te inoras la a0te nanhsirkalapogledom lamina a0 a0 p ledomnamuh a0 a0 p la a0te tnonahtna mailliw irahdirihgnilusni esaercni doolb laidnarp tsop no noitcuder ni slihportuen gnitalucric esaercniseiduts ortiv ni a0 a0 pesoculgledom namuh a0 a0 p la a0te eurdledom laminasserts etuca a0 a0 pledom lamina a0 a0 p lariv uehritna recnac ni romutitnadna lairetcab itna msitamrsshti serpeditna wnoitanibmoc ni tnas recnahne evitingoc tnatcetorporuen ralucsavorberecairetsyh sa sredrosid suovren airalamdna diohpit fo tnemtaertsesaesid negortse noitcudorp rosrucerpdna noitatcal esaercniselpicnirp lanoitnevnoc ot gnidrocca krab dna stoor srewofl sdees sdop sevael su hcus strap eert arefielo m tnereffid fo snoitca lacigolocamrahp fo elbat yrammus elbatarefielo m fo snoitca lacigolocamrahp ledomnamuh a0 a0 pmsidioryhtrepyh etaluger ledomnamuh a0 a0 pamehtyre niks ni ecuderseiduts ortiv ni a0 a0 pa ila la a0te ila la a0te dammahumgnigaitna niksstcartxe suoeuqa fognilaehdnuow tsaerb tnadixoitna yrotammaflniitnaicrac noloc ni sisotpopa ecudni la a0te ilarahb la a0te tehpiark la a0te inailihatledom lamina a0 a0 pnoitacfiixoted dnaledom lamina a0 a0 p ledomnamuhc ila a0 a0 pamonytivitca laiborcimitnaledom lamina a0 a0 psamonicrac latceroloc tsniaga ytivitca recnacitnadna la a0te nanhsirkalapog la a0te iramsalaseiduts ortiv niselknirwitna a0 a0 pniap tnioj dna aehrraid taert la a0te ilaledom namuh a0 a0 p la a0te nanhsirkalapog a0 a0 pan raelc dna paos ot lauqe pu lortnoc eht fognihsaw tnereffiddnah ni seitreporp lairetcabitna la a0te lednorotnoitardyh niks ni esaercni ledomnamuh a0 a0 p ni laitnetop evitneverpomehcrecnac neelps dna citapeh nanhsirkalapog la a0te ilarahbseiduts ortiv ni a0 a0 p ledomnamuhb ila a0 a0 p dna tnadixoitna citebaiditnaseitreporp citirhtraitnagnisnaelc evitcetorp gnizirutsiom gnihsiruonniks tnadixoitna la a0te yebar l ednaledom lamina a0 a0 p ledomnamuhanammaflni gnul etuca fo esaerced la a0te thginkcmledom lamina a0 a0 pnoit dna setaidemretni noitadixorep dipil fonegyxo evitcaer esaerced ilarahb eddna atpugsad la a0teledom lamina a0 a0 p yebar l edna iklamlaledom lamina a0 a0 pdik lla detaroilema dna desaercnisretemarap snoitcnuf yen ecuder sisorbfi revil detaroilemaesadixorepoleymytivitca citapeh yticixototapeh decudni tibihniledom lamina a0 a0 pseiduts ortiv ni a0 a0 p azmah la a0te areiv iklamlanajaham narmi dna meedansevaelsdopsdeesa review of a0properties nutritional and a0pharmaceutical applications of a0moringa oleifera¦ 0c detneverp dna elfiorp la a0te reugiugledom lamina la a0te reugiugledom lamina inamkcurledom lamina hzimahtabni la a0te nanhsirkalapogseiduts ortiv niandipil niagdevorpm thgiewi dna ytilitom lanitsetni etomorpstceffe evitaxalyticixototapeh decudnilomatecarap no evitcetorpotapeh etatsorp fo sledom tnednepedni negordna ni ytivitca recnacitnarecnac eruc dna cimeloretselohcopyhsmelborp yraniruarefielo m fo snoitca lacigolocamrahpdeunitnoc elbat eht dna etalaxo syendik eht ni noitisoped enotsyraniru eht ecuder la a0te idarak dna ytivitca evitcetorpotycyrotercesitna recluitna la a0te yrahduohcan a0p a0evitcetorpotapehipilitna dna cimeretselohcitnaledom lamina a0 a0 p amrukledom lamina a0 a0 pledom lamina a0 a0 p ahcenesledom lamina a0 a0 pcimed tcart yraniru fo tnemeganamsmotpmys snoitcefni hgni sdna ayruam la a0te alkuhsledom lamina a0 a0 p ledomnamuh a0 a0 p serec¡cledom laminalairetcabitna a0 a0 p la a0te reffazseiduts ortiv niantceffe noitatnalpmiitna a0p a0yrotammaflniitna a0p a0yticixototapehdecudni lomatecarapno evitcetorpotapeharopsoruen tsniaga lagnufitna inamkcurledom lamina a0 a0 pseiduts ortiv ni ahj a0 a0 pd a0meireles several m oleifera studies with leaf powder or extracts on animals revealed other properties beyond the previously referred antioxidant chemoprotectant and antihypertensive stohs and hartman the antioxidant activity derives from the high amounts of polyphenols leone et a0al 2015a b verma et a0al leaves extracts have revealed anticancer properties with antineoproliferative activity by inducing reactive oxygen species ros production only in cancer cells which leads to cell apoptosis gopalakrishnan the active compounds present in extracts from leaves and bark revealed anticancer activity against breast and colorectal cancer cell lines through diverse mechanisms as decreased cell mobility decreased colony formation low cell survival high apoptosis and g2m enrichment alasmari et a0al some extract fractions with anticancer activity have already been isolated characterized and tested in a0vitro and in a0vivo rat model krishnamurthy et a0al table a0in traditional medicine a paste made of leaves is applied externally in wounds siddhuraju and becker some scientific studies have shown that leave extracts have beneficial properties in skin aqueous leaves extract increased human dermal fibroblasts proliferation leading to faster wound healing muhammad et a0al a m oleifera leave extract fraction with ethyl acetate in low concentration a0µgml showed in a0vitro effect in skin healing by increasing proliferation of human dermal fibroblasts gothai et a0al a hydroalcoholic extract of m oleifera leaves used in a cream showed antiaging characteristics due to phenolic compounds sunscreen and photo protective characteristics were studied very recently baldisserotto et a0al when applying a cream with this extract it was also verified a reduction in sebum production ali et a0al 2013a b c and in transepidermal water loss allowing to increase hydration ali et a0al 2013a b c wrinkles and other signs of lack of skin vitality where improved during a0months of using the same topic formulation with m oleifera leaf extract ali et a0al the compounds responsible for this improvement in skin surface appear to be phenolics eg kaempferol and quercetin and antioxidants such as vitamins a c and b jadoon et a0al m oleifera leaf extract cream was also tested for potential skin irritation by a a0h semioccluded patch test and proved to be nonirritant and well accepted by the volunteers also reducing skin erythema ali et a0al 2013a b c table a0 m oleifera leaf powder can be used to clean hands when four grams of wet more efficient or dried powder are applied on hands and rubbed torondel et a0al the efficacy results were the same as for nonmedicated soap revealing potential to help in hand hygiene and prevent pathogen transmission in developing countries where hygiene products are scarcea leave extract sprayed in plant crops revealed another utility for this plant having beneficial effects on the growing anyb detneserper era seulav dnuof tonelbacilppanon a pyb detneserper era eulav tnacfiingis yllacitsitats htiw seidutssrewolfstoorkrab 0crate size and resistance on those plants and fruits bhargave m oleifera leaf tea studies demonstrated alterations in blood circulating neutrophils and conclude that moringa tea has adaptogenic capabilities in cases of stress drue et a0al table a0 previous studies using dried moringa leaves tea in mouse model with acute lung inflammation showed that mice that had decreased lung inflammation marked by alterations in cytokine production leukocyte migration and neutrophil apoptosis mcknight et a0al an ethanolic extract of moringa leaves has antianxiety effect in swiss albino mice the ethanolic extract of m oleifera leaves may have produced its anxiolytic effects via multiple mechanisms bhat seedsseeds collected from pods can be eaten raw or cooked from m oleifera seeds a rich vegetable oil can be produced m oleifera seed oil or behenben oil is produced through the cold pressing of the m oleifera seeds m oleifera oil can be used to cook as a source to prepare biodiesel as a lubricant and in the cosmetic industry rashid et a0al the oil name comes from its high content on behenic acid which confers more resistance to oxidative degradation comparing to other vegetable oils ben oil is rich in oleic acid up to palmitic but also stearic behenic and arachidic anwar it is used in various cosmetic formulations as emollient and confers nourishing moisturizing antioxidant and protective properties it is also a good skin cleansing product nadeem and imran table a0 details some cosmetic brands that use m oleifera leaves oil or active extracts as ingredients in the composition of their products this oil is also used in the enfleurage process allowing the extraction of fragrances and active compounds from difficult sources as flower petals milled m oleifera seed shells can be used as a natural exfoliating agentmoringa oleifera seeds can also help diabetic patients table a0 some studies by almalki and el rabey showed its antidiabetic activity by reducing the blood glucose level when rats where treated with or a0mg of m oleifera seeds powderkg body weight during a0weeks at the same time ingestion lead to an increase in antioxidant enzymes and consequently the compound content such as glucomoringin phenols and flavonoids moreover the same authors treating these diabetic rats significantly increased and ameliorated all kidney functions parameters in fact m oleifera seeds ameliorated liver fibrosis in rats reducing liver damage and symptoms of liver fibrosis decrease the ccl4induced elevation of serum aminotransferase activities and globulin level as well as reduce the elevated hepatic hydroxyproline content and myeloperoxidase activity hamza improving the indices of hepatoxicity in rats such as malonialdehyde level and total antioxidant capacity glutathione content catalase and superoxide dismutase activities hamza treatment with m oleifera seeds also altered oxidative stress in relation to its antiinflammatory activity histopathological observations showed mild or less infiltration of lymphocytes angiogenesis and synovial lining thickening from all above results and observations it can be concluded that the seeds possess promising antiarthritic property mahajan et a0al these seeds have others appeals to the daily life and industry seed powder showed capacity to purify water and remove heavy metals and anic compounds sharma et a0al through low molecular weight cationic proteins mediated precipitation kansal and kumari there was a reduction of in the turbidity of the water and of bacterial reduction bhargave lea the remaining paste after the oil extraction still has the same flocculation properties serving both purposes and adding value lea compounds such as pterygospermin moringine and benzyl isothiocyanate isolated from m oleifera seeds showed antimicrobial effect viera et a0al accordingly there is applicability for m oleifera seeds in the prevention of microbial diseases table a0 m oleifera oil has also been tested for its potential to produce biodiesel contributing as an alternative to the conventional diesel fuel rashid et a0al flowers and a0rootsm oleifera flowers are used directly as part of the diet but also to make infusions which ha | Colon_Cancer |
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 |
"objectives to describe the benefits and limitations of using individual and combinations of linked english electronic health data to identify incident cancersdesign and setting our descriptive study uses linked english clinical practice research datalink primary care cancer registration hospitalisation and death registration dataparticipants and measures we implemented case definitions to identify first site specific cancers at the most common sites based on the first ever cancer diagnosis recorded in each individual or commonly used combination of data sources between and we calculated positive predictive values and sensitivities of each definition compared with a gold standard algorithm that used information from all linked data sets to identify first cancers we described completeness of grade and stage information in the cancer registration data setresults gold standard cancers were identified positive predictive values of all case definitions were ¥ and ¥ for the four most common cancers breast lung colorectal and prostate sensitivity for case definitions that used cancer registration alone or in combination was ¥ for the four most common cancers and ¥ across all cancer sites except bladder cancer using cancer registration alone for case definitions using linked primary care hospitalisation and death registration data sensitivity was ¥ for the four most common cancers and ¥ for all cancer sites except kidney oral cavity and ovarian cancer when primary care or hospitalisation data were used alone sensitivities were generally lower and diagnosis dates were delayed completeness of staging data in cancer registration data was high from minimum in and in for the four most common cancerss ascertainment of incident cancers was good when using cancer registration data alone or in combination with other data sets and for the majority strengths and limitations of this study º this is the first study to present comprehensive information on the implications of using different individual and combinations of linked electronic health data sources in england to identify cases of the most common incident cancers º using a gold standard algorithm that combined all available data from multiple sources as a comparator we were able to estimate both positive predictive values and sensitivity values for a range of pragmatic case definitions º we described similarities and differences in values between age groups sexes and calendar years the impact of choice of sources on diagnosis dates and mortality rates and completeness of stage and grade in cancer registration data º a key limitation was that our gold standard algorithm is not validated and may be affected by differences in clinical diagnosis and coding of invasive cancers between data sourcesof cancers when using a combination of primary care hospitalisation and death registration dataintroductionthe clinical practice research datalink cprd provides de identified primary care data linked to additional secondary health data sources under a well governed framework1 use of linked data helps researchers to answer more epidemiological questions and increase study quality through improved exposure outcome and covariate classification2 in the field of cancer epidemiology cprd primary care data linked to hospital episode statistics admitted patient care strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access data hes apc office of national statistics ons mortality and national cancer registration and analysis service ncras cancer registration data are used to analyse factors contributing to the risk of cancer and the consequences of cancer and its treatment use of linked data reduces the sample to the common source population and data coverage period for each included data set and has cost and logistical implications which are greatest for ncras data research teams therefore commonly choose not to use all available linked data3 cancer epidemiology studies can also be conducted using ncras and hes apc data provided by national health service nhs digital and public health england phe without linkage to cprd primary care data4 this provides national coverage at the expense of the detailed health data that are available in primary care recordsvalidation studies assessing concordance between cprd gold hes apc and ncras data have estimated high positive predictive values ppvs for cprd gold data and varying proportions of registered cancers that are not captured in cprd gold and hes apc5 the most up to date analysis by arhi et al included the five most common cancers and all papers focussed on concordance between cprd gold only and ncras existing evidence therefore does not provide a complete assessment of the benefits and limitations of using different combinations of data sources within the context of practical study designs national data are available describing completeness of data fields within the cancer registry data in each collection year9 and over time for all cancers combined4 missingness for individual years has been associated with age comorbidities and clinical commissioning groups10 we aim to describe and compare the benefits and limitations of using different combinations of linked cprd primary care data hes apc ons mortality and ncras cancer registration data for conducting cancer epidemiology studies our analyses focus on incident cancer ascertainment as it is a common and important outcome in cancer epidemiology and it is more difficult to distinguish between secondary recurrent and primary cancers at a second site in these data sets we have compared definitions of the most common cancers based on the first ever cancer recorded in individual or combinations of data sets with a gold standard definition comparing information from all four data sets we also describe the availability of stage grade and treatment variables over time in the cancer registration data for the cprd linked cohort this reflects real life study design and will help researchers to decide which combination of data sources to use for future studiesmethodsstudy design and settingwe completed a concordance study using linked2 english cprd gold hes apc ons mortality and ncras data cprd gold data were extracted from the january monthly release and the 13th update to cprds linked data the study period was from january to december with december matching the end of the ncras coverage periodthe cprd gold database includes de identified records from participating general practices in the uk who use vision software1 general practice staff can record cancer diagnoses using read codes or in free text comments boxes though the latter are not collected by cprd diagnoses will typically be entered duringfollowing a consultation or from written information that is returned to the practice from secondary care cprd gold data are linked to hes apc ons mortality and ncras through a trusted third party for english practices that have agreed to participate in the linkage programme2 hes apc data are collected by nhs digital to co ordinate clinical care in england and calculate hospital payments12 admissions for and related to cancer diagnoses are recorded using international classification of diseases version icd10 codes national cancer registration data are collected by ncras which is part of phe in accordance with the cancer outcomes and services data set13 which has been the national standard for reporting of cancer in england since january data include icd10 codes to identify the cancer site and more detailed information such as stage and grade ons mortality data includes dates and causes of deaths registered in england recorded using icd10 codesparticipants exposures and outcomesour underlying study population included male and female patients registered in cprd gold practices who were eligible for linkage to hes apc ncras and ons mortality data and had at least days of follow up between january and december start of follow up was defined as the latest of the current registration date within the practice and the cprd estimated start of continuous data collection for the practice up to standard date end of follow up was determined as the date the patient left the practice ons mortality date of death or practice last collection dateidentification and classification of cancer codeswe used code lists to classify cancer records in each of cprd gold hes apc and ons mortality data as one of the most common sites other specified cancers history of cancer secondary cancers benign tumours administrative cancer codes unspecified and incompletely specified cancer codes https org data incompletely specified cancer codes could be mapped to cancer site eg icd10 code c689 malignant neoplasms of urinary organ unspecified was considered consistent with both bladder and kidney cancer for ncras we accessed coded records for the most common cancers we included cancers recorded in the clinical or referral file for cprd gold cancers recorded in any diagnosis field for hes apc and the underlying or strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure gold standard algorithm to identify incident site specific cancers using all data sources hes hospital episode statistics ncras national cancer registration and analysis service ons office of national statisticsmost immediate cancer cause of death in ons mortality datacancer case definitions based on individual sources and combinations of sourceswe developed alternative cancer case definitions mirroring those commonly used in epidemiology studies based on identifying the first malignant cancer excluding administrative codes and benign tumours recorded in various combinations of data sources ncras alone ncras and hes apc all sources cprd gold hes apc and ons mortality cprd gold alone hes apc alone multiple malignant cancers recorded on the index date in cprd gold or hes apc were reclassified as multiple site cancer and were not considered as individual site cancer records for positive predictive value and sensitivity calculations multiple codes recorded in different sources on the same date were reclassified as the site identified in the ncras data if available and as multiple site cancer if not for each case definition we only examined the first malignant cancer per individual where this occurred within the study period and at least year after the start of follow upgold standard cancer case definitionwe developed a gold standard algorithm that classifies incident records of the most common cancers by comparing the first malignant cancer identified in each individual source figure cancers recorded in ncras alone with no contradictions ie records for first cancers at different sites were considered true cases whereas cancers recorded in hes apc alone or gold alone required internal confirmation within that source in the form of another code for cancer consistent with the same site or with site unspecified within months and no contradictory codes eg for cancers at other sites in this period where cancer records were present in data source we considered a site specific cancer to be a true case a if it was recorded as the first cancer in ncras and the total number of data sources with records for cancer at that site was equal to or greater than the number of data sources with contradictory records ie records for first cancers at different sites or b where the cancer was not present in ncras if there were more data sources in total with records for cancer at that site than data sources with contradictory recordswe used ncras data to identify stage grade and treatment where available in the cancer registry only cohort binary surgery chemotherapy and radiotherapy variables were derived using individual records of treatment from the first year after diagnosisstatistical analysisfor each cancer site and each individual or combined data source we combined our applied study definitions strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access with our gold standard definition to classify each applied study definition as a true positive false positive or false negative recordwe used these categories to calculate sensitivity and positive predictive value overall and stratified by age categories to and calendar year and sex we calculated differences in diagnosis dates for true positives by subtracting the gold standard index date from the index date for each source and combination of sourceswe used kaplan meier methods to describe mortality over time for cancers identified using each definition the ons mortality death date was used for these analyseswe used the ncras only definition to calculate proportions of patients with complete stage and grade and recorded cancer treatment modalities over timepatient public involvementpatients and the public were not involved in conceiving designing or conducting this study and will not be consulted regarding the dissemination of study resultsresultsof research quality patients in the cprd gold january build were eligible for linkage to hes ons mortality and ncras data in set were excluded due to unknown sex of the remainder and had at least year of follow up between january and december and were included in the study population using the gold standard algorithm incident cases of cancer were identified the number of patients identified with each cancer is presented in online supplementary appendix table half n82 of these patients were male aged to aged to and aged or olderfigure presents ppvs for each case definition comparing the first recorded cancer in each combination of data sources with the gold standard algorithm when using ncras data alone to of cancers were confirmed by the algorithm for out of cancer sites the ncras only case definition gave the highest ppv case definitions using data sources not including ncras generally had lower ppvs ranging from to for individual cancer sites for the four most common cancers breast lung colorectal and prostate ppvs were at least for all case definitions minimal differences in ppvs were observed between age groups years and sexes online supplementary appendix figures figure presents sensitivity values for each case definition sensitivity was generally higher for the case definitions that included ncras data ranging from to for individual cancer sites except bladder cancer identified using ncras data alone and ¥ for the four most common cancers breast lung colorectal and prostate sensitivity was also generally high for definitions using a combination of cprd gold hes apc and ons mortality data ranging from to ¥ for the four most common cancers sensitivity was lower for case definitions that used cprd gold alone range to for individual cancer sites or hes apc alone range to sensitivity values for cprd gold alone and hes apc alone increased slightly in younger patients and more recent years no differences were observed between men and women online supplementary appendix figures post hoc analysis suggested that the low sensitivity of cprd gold only definitions for kidney cancer sensitivity n false negatives was driven by missing n1136 or incompletely specified urinary organ cancer codes n1108 in cprd gold rather than contradictory information about the first cancer record n625 these incompletely specified codes are less likely to be used for bladder cancers n85 than kidney cancers n1108 bladder cancers that were not recorded in ncras data n3445 were commonly recorded in both hes apc and cprd gold n2228 or in hes apc only with a subsequent unspecified or bladder cancer record in hes apc within months n995 table describes the number of days median iqr and 5th95th percentile lag between the date of incident cancers from the gold standard definition and the date of cancer arising from each case definition ie the first record within the specific combinations of data sources used case definitions using ncras alone and combinations of ¥ data sources captured cancers close to the gold standard date median lag ¤ days for all cancer sites whereas median lags were generally longer for the case definitions using cprd gold alone and hes apc alonefigure describes mortality over time following incident cancer diagnoses ascertained from each case definition minimal differences in mortality were observed between cancers identified from different case definitions where variability was observed cancers identified using cprd gold only had the lowest mortality rates eg kidney cancer and cancers identified using hes apc only or ncras only had higher mortality rates eg prostate cancer and bladder cancer respectivelyfigure describes completeness of grade and stage for cancers identified using ncras only recording of grade was highly variable between cancers with gradual increases in completeness over time completeness of staging information was low in earlier calendar years but improved substantially from around especially for the four most common cancers minimum in and in post hoc logistic regression models adjusted for year and cancer site indicated that completeness of stage and grade were associated with each other and these variables were least complete in patients aged stage data was more complete for higher grade tumours whereas grade data was more complete for lower stage tumours online supplementary appendix figure online supplementary appendix figure describes recording of treatment modalities identified using ncras strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure positive predictive value of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers defined using the first ever record in each combination of sources confirmed by a gold standard algorithm that considers confirmatory and contradictory data from each source cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure sensitivity of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers identified using the main gold standard algorithm that considers confirmatory and contradictory data from each source that are identified using the first ever record in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service onsoffice of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0celitnecrep hthtrqi inademelitnecreprqi inadem hthtelitnecrep htht inademrqielitnecrep hthtcpaseh dlogdrpc ytil atromsnodna cpaseh dlogdrpc cpasehdnasarcn secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emti l ebatopen access recnac lanoitan sarcn atad erac tneitapdetti mda scitsitats edospei latipsoh cpaseh knil atadhcraeser ecitcarp lacniilc drpc metsys suovren lartnec sncl tluafed ybnoitinfieddradnats dog eht sa emas eht si etad ssongad sa nwohs tonnoitinfied secruos ll iia setis recnac nommoc tsom ruofdcii nosrev sesaesdi fonoitacfissacliscitsitats lanoitan rof ecfifo snoi atadnoitartsgerrecnac ecvres ssyanadna liinoitartsgeri lanoitanretni eht imorf sedoc gndnopserroc ot gndrocca deredro era setis recnaci snoitinfiedde ilii ppa dna etad ssongaddradnats dog nam neewteb syadil fo rebmun ot ot ot ot ot cl amoeym epitluml ot ci ameakuel ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot elitnecrep hthtsarcn inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot c ytivac laroc laegahposeoc hcamots cc latcerooclrecnac amonaeml tnangilamc saercnapc gnulc suretuc etatsorpc seiravoc yendkic tsaerbci xvreccc sncnarbic reddablc amohpmyl s'inkgdo hnonc idoryhtc revlistrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure mortality following first ever record of cancer in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service nhl non hodgkin's lymphoma ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure completeness of grade and stage for cancers identified using ncras data only cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites grading information is not applicable to braincns sarcoma or haematological cancers and not required by in the national data standard cosd for prostate cancer core staging is not applicable to haematological and gynaecological cancers other types of staging are recommended by cosd cns central nervous system cosd cancer outcomes and services data set ncras national cancer registration and analysis service nhl non hodgkin's lymphomastrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access only missing records may indicate that the patient did not receive that treatment modality or that the treatment modality was not recordeddiscussionstatement of principal findingswe investigated the use of different sources of electronic health record data to identify incident cancers for all case definitions using individual or combined data sources a minimum of of incident site specific cancers were confirmed using the gold standard algorithm this rose to of the four most common cancers use of cancer registration data alone or in any combination of data sources captured at least of site specific cancers identified by the gold standard algorithm excepting bladder cancer and of cases for the four most common cancers combining cprd gold hes apc and ons mortality data captured at least of site specific cancers excepting kidney oral cavity and ovarian cancers and captured of cases for the four most common cancers sensitivity was much more variable when using primary care or hospital data alone and dropped to when identifying bladder cancers using cancer registration data alone use of primary care or hospital data alone resulted in a small lag in identifying cancers of interest compared with the gold standard dates but other case definitions captured cancers close to the gold standard date finally while we observed minimal changes in ppvs and sensitivities between and completeness of ncras cancer registration stage and grade data increased markedly from onwards for specific cancer types demonstrating that initiatives to improve data can have a profound impact on the quality of the data4 completeness of cancer treatment recording was difficult to assess due to the absence of a missing categorystrengths and weaknesses of the studythe main strength of this study is that we have developed a gold standard algorithm using the entirety of the evidence available from cprd to demonstrate the impact of choice of data sets in identifying incident cancers for real life studies we have also assessed the value of using ncras cancer registration data to measure stage grade and cancer treatment modalitiesa limitation of the study is that our gold standard algorithm is not validated we feel that we were justified in pre weighting ncras data as more reliable that other data sources as ncras is a highly validated data set that matches merges and quality checks data from multiple sources4 we did not consider ncras to be the outright gold standard as it is plausible that ncras does not identify all tumours diagnosed and treated in primary and secondary care for most cancer sites our gold standard algorithm identified a small proportion of cancers that are recorded in hes apc cprd gold or ons mortality data but not in ncras these tumours may have been diagnosed and coded as invasive in primary or secondary care but not by ncras been incorrectly coded in hes apc cprd gold or ons mortality data not have been notified to ncras eg tumours treated in private hospitals or be the result of linkage errors between the data sets the proportion of cancers identified in hes apc but not in ncras is particularly high for bladder cancer this is likely to be the result of difficulties inconsistencies and changes in the pathological definition and coding of cancers over time in ncras which are greatest for bladder cancer4 this explanation is supported by the higher mortality rates that we observed in bladder cancer cases identified in ncras compared with other data sources to identify incident cancers we required months of research quality follow up in cprd gold prior to inclusion in the study previous research has demonstrated that historic data is generally incorporated within the patient record with this time frame15 the identification of first ever cancers will also have been affected by different lengths of follow up data available in linked data sources as ncras data collection started in hes apc in and ons mortality data in and by the inclusion of all diagnostic codes in hes apc assuming that the first ever primary or secondary record identified incident cancer reassuringly ppvs for liver and brain cancer were high for all individual and combinations of data sets suggesting that these were not unduly misclassified as primary incident cancers despite being common sites for metastases requiring internal confirmation within months for cancers recorded in cprd gold alone in our gold standard definition is more likely to discount cancers with poorer prognoses and those recorded in the last months of follow up our data cut only included ncras data for the top cancers earlier cancers at other sites will have been missed in this studyit is also important to note that as the gold standard algorithm uses data recorded after the first record of the cancer site in any source index date it cannot be used to identify outcomes in applied studies and follow up of cohort studies with cancer as an exposure would need to start at least months after diagnosis our first ever cancer record in any source definition would be more appropriate for most studiesstrengths and weaknesses in relation to other studies discussing important differences in resultsthe most up to date study describing concordance between linked cprd gold hes apc and ncras data sets demonstrated that to of the five most common cancers recorded in cprd gold are not confirmed in either hes apc or cancer registration data and to of registered cancers are not recorded in cprd gold8 for cancers recorded in both sources the diagnosis date was a median of to days later in cprd gold than in the cancer registration data using cprd gold alone to identify these strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0ccancers marginally over represented younger healthier patients and identified to fewer deaths in the first years after diagnosis use of hes apc only identified a higher proportion of patients with the correct diagnosis date than cprd gold but over represented older patients and those diagnosed through the emergency route the majority of registered cancers were picked up using both cprd gold and hes apc ranging from for lung cancer to for breast cancer previous research demonstrated similar results with substantial differences between cancer types5 additionally a study using data from to found that using hes data in addition to ncras data identified an additional and of surgically treated colorectal lung and breast cancer cases respectively16our study is consistent with these results and provides more complete and practical evidence of the strengths and limitations of using individual and combinations of linked data sets to identify and characterise the most common incident cancerswe have also demonstrated the added value of using cancer registration data to measure stage and grade of incident cancers from about onwards levels of data completeness of staging information in the cprd extract in were similar to those reported by the united kingdom and ireland association of cancer registries ukaicr9meaning of the study possible explanations and implications for clinicians and policymakersuse of ncras cancer registration data maximised the proportion of cases confirmed as true positive based on all available linked information and captured the highest proportion of true positive cases highly complete staging and grading information is available from this source from approximately case definitions based on a combination of cprd gold hes apc and ons mortality data also had acceptable validity for the majority of cancer sites including the four most common cancersthese findings should be considered when deciding which data sources to include in research studies and which sources to use to define cancer exposures outcomes and covariatesunanswered questions and future researchfurther research is required to investigate the validity of cancer recorded in cprd gold and hes apc that are not recorded in the ncras data and to understand differences in cancer data recording with cprd gold and cprd aurum cprds recently launched primary care database based on records from practices that use emis software17 further investigation would be required to confidently identify subtypes of cancer either using codes available in each data set eg colon and rectal cancer or additional information available in hes apc or ncras data use of ncrass recently open accesslaunched systemic anti cancer therapy sact18 and national radiotherapy data sets will also improve ascertainment of therapies for futu | Colon_Cancer |
edited byfang zhoucas lamvac biotech co ltd chinareviewed bybart evertsleiden university medicalcenter netherlandsmaria rosa bonouniversity of chile chileandrew john staggqueen mary university of londonunited kingdomcorrespondencedipayan rudradipayanrudragmailcomrudradpostechackrsinhyeog imiimshpostechackr present addresshyunja kokobiolabs inc seoul south koreasungwook hongdepartment of microbiology andimmunology centre for immunologyuniversity of minnesota medicalschool minneapolis mnunited states¡these authors have contributedequally to this work§deceasedspecialty sectionthis was submitted toimmunological tolerance andregulationa section of the frontiers in immunologyreceived april accepted july published august citationko hj hong sw verma r jung jlee m kim n kim d surh cdkim ks rudra d and im sh dietary glucose consumptionpromotes raldh activity in smallintestinal cd103cd11b dendriticcells front immunol 103389ï¬mmu202001897intestinal dendritic cells dcs are critical for the initiation and regulation of innate and adaptiveimmunity by delivering self or foreign antigens to t cells the intestine is spontaneouslyexposed to innumerable antigens comprising of intestinal microbes as well as dietarycomponents to maintain immune homeostasis intestinal dcs regulate the balance betweenthe tolerogenic immune response by inducing cd4foxp3 regulatory t cells treg cells and the protective immune responses by inducing eï¬ector t cells dysregulation of thisbalance by harmful pathogens or dietary intake results in ammatory disorders such asammatory bowel disease ibd celiac disease and food allergy frontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityintestinal dcs are located in the peyers patches ppsmesenteric lymph nodes mlns and lamina propria lp andcomprise cellular subsets that have diï¬erent origins and functions among these dc subtypes intestinal cd103 dcshave the unique function that metabolizes vitamin a to retinoicacid ra through the activation of aldehyde dehydrogenase member a2 [aldh1a2 also called retinaldehyde dehydrogenaseraldh2] enzyme the ra produced by intestinaldcs play an important role in orchestrating immune responsesimprinting guthoming speciï¬city on t cells b cells andinnate lymphoid cells ilcs inducing igaproducing b cellspromoting tgfdependent diï¬erentiation of induced tregcells suppressing the diï¬erentiation of th17 cells enhancing il production by Îδ t cells and ilcs as well as inducing eï¬ectorfunctions in t cells while vitamin a derived from dietary intake can induceraldh enzymatic activity the ra produced from intestinalepithelial cells iecs by raldh1 and stroma cells in lp andmln by raldh2 in a trans activating mechanism is alsocapable of inducing raldh expression in intestinal dcs furthermore recent data suggest that ra is also involvedin the development of a gut homing precursor for intestinaldcs in the bone marrow as well as is required for theirtranscriptional programming and maturation severalendogenous factors that regulate raldh expression in lpdcs are also reported cytokines such as il4 and granulocytemacrophage colonystimulating factor gmcsf induce orenhance the expression of raldh enzymes in lpdcs whileprostaglandin e2 pge2 negatively regulates raldh activitythrough the induction of inducible cyclic amp early repressoricer despite these ï¬ndings whether additionalcomponents in diet can induce raldh activity in the intestineand promote immune tolerance remains unknown in this studywe uncover a hitherto unknown role of dietary glucose in shapingup intestinal immunological tolerance by facilitating raldhexpression speciï¬cally in intestinal lpdcsresultsmice administered antigen free diet havedefects in development and raldhactivity in cd103cd11b silpdcsto investigate the uence of commensal microbiota and foodcomponents on intestinal immunity we utilized the previouslyestablished antigen free af mice model where germfreegf mice are raised on welldeï¬ned elemental diet [termedantigen free diet afd] devoid of macromolecules such asproteins and starches when dcs in small intestine wereassessed we observed comparable frequencies of cd11cmhcii silpdcs in speciï¬c pathogen free spf gf and af micefigures 1ab however indepth analyses revealed alterationin the frequencies of tolerogenic dc subtypes the frequencyof cd103cd11b silpdcs a subset known to be a majortolerogenic dc population was slightly but significantly lowerin af when compared to spf and gf mice figure 1c acompensatory increase on the other hand was observed inthe cd103cd11b silpdc compartment more interestinglywhile the expression of the characteristic dc surface markerslargely remained comparable in all three groups figure s1athe expression of all three representative genes tested namelyaldh1a2 indoleaminepyrrole 23dioxygenase ido1 andtransforming growth factor beta tgfb1 that are functionallyimplicated in tolerogenic phenotype of cd103cd11b dcswere dramatically reduced in af mice figure 1d interestinglythe expression of aldh1a2 was found to be speciï¬cally reduced inmice raised under afd a phenomenon that was not observedin gf mice figure 1d left panel on the other hand theabsence of gut microbiota appeared to partially uence theexpression of ido1 and tgfb1 which was further enhanced byafd figure 1d middle and right panel these results indicatedthat certain dietary components otherwise absent or underrepresented in afd have most speciï¬c and the largest uenceon the expression of aldh1a2 for this study we therefore focusedon the uence of normal diet on raldh activity in silpdcsraldh is an enzyme that irreversibly metabolizes vitamin ato ra which in turn acts as a key modulator of mucosal immuneresponses to determine whether in concert to itsreduced expression the function of raldh in lpdcs fromaf mice was also negatively aï¬ected we next examined raldhenzyme activity in lpdcs from spf gf and af mice usingthe aldefluor assay in this assay which has been previouslyemployed in the context of cd103 lpdcs and mlndcs the raldh enzyme activity is measured in individual cellsby ï¬ow cytometry with a ï¬uorescent substrate based assay system in agreement with the results obtained by realtime pcranalysis cd103 silpdcs both cd11b and cd11b subsetsfrom af mice displayed significantly lower enzyme activity whencompared with spf and gf mice figures 1ef figure s1b ofnote the characteristic frequencies of the aforementioned silpdc subtypes in spf gf and af mice remained unaltered evenafter performing this assay suggesting that this enzyme assay didnot interfere with the phenotype of silpdcs figure s1cin order to further understand the role of dietary componentson raldh activity we next analyzed silpdc raldh activityin mice at diï¬erent stages of their lives after subjecting them tospeciï¬c dietary conditions we observed that raldh activityin preweaned gf mice weeks of age was significantlylower than in adult gf mice and comparable to af mice thiswas dramatically restored to the level equivalent to adult gfmice within a week after weaning figure 2a furthermorewhen mice raised in af condition were fed with normal chowdiet ncd raldh activity in cd103cd11b silpdcs waspromptly recovered within a week figure 2b mirroring thisan opposite phenomenon was observed when ncd in gf micewas replaced with afd figure 2c these results suggested thatdietary components in ncd absent in afd is required as theinitial trigger for raldh gene expression after mice are weanedthereby promoting enzyme activity as well as homeostasis ofcd103cd11b silpdcsthe above results also implied that supplementing af micewith ra the ï¬nal product of the enzymatic reaction and a knownfeedback inducer of raldh activity would be suï¬cient indriving raldh activity in these mice indeed when adult affrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure dietary intake affects differentiation and raldh activity in cd103 silpdcs cell suspensions were prepared from silp harvested from agematchedadult ¼12weekold spf gf and af mice and phenotypic and raldh activity analyses of silpdcs were carried out a representative ï¬uorescenceactivatedcontinuedfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure cell sorting facs plots of silpdc subpopulations gated on lin cd3b220 cells b statistical quantiï¬cation of percentage left and total numbersright of cd11cmhcii cells in mice from indicated experimental groups c graph displays percentage of silpdc subpopulations in cd11cmhcii cells bcdata are combined from four independent experiments d realtime analyses of mrna expression of indicated gene products normalized against hprt mrna levelsef representative facs plots left and quantiï¬cation right of relative mean ï¬uorescence intensity mfi of aldeï¬uor in cd103cd11b e and cd103cd11bf silpdc subpopulations from indicated groups deab is a raldh inhibitor 01mfi is calculated by subtracting background deab mfi from aldeï¬uor mfi relative 01mfi indicates ratio of 01mfi in experimental samples vs control data are combined from six independent experiments mean ± sem are indicated statisticalsigniï¬cance was determined by oneway anova bef and twoway anova c with turkeys multiple comparison tests p ns not statisticallysignificantmice were supplemented in their diet with ra it resulted incomplete recovery of raldh activity figure 2d interestinglyin all the cases changes in raldh activity also correlatedwith the frequencies of cd103cd11b silpdcs suggestingits role in diï¬erentiation as well as function of these cells ofnote while the above results were obtained in a gf setting thebasic ï¬ndings from these experiments could also be recapitulatedin mice raised in spf conditions thereby conï¬rming thatmice with normal repertoire of gut ï¬ora are equally aï¬ectedby dietary components with respect to raldh activity insilpdcs figures s2acraldh activities in different intestinalraproducing cells are differentiallyaffected by dietwe next sorted to understand whether the uence of dieton raldh activity is an lpdc speciï¬c phenomenon orwhether other raldh expressing cells are also aï¬ected itis wellestablished that within the gut associated lymphoidtissues besides lpdcs ra converting enzymes are alsoexpressed in lp associated stroma cells lpscs small intestineepithelial cells iecs as well as mlndcs the ra producedfrom these cellsis known as a localsource of ra for inducing the raldh expression in cd103silpdcs in particular iecsthe nonhematopoietic scs in secondary lymph nodescomprise three diï¬erent cell types based on the expressionof surface markers lymphatic stroma cells [lscs also calledï¬broblast reticular cells frc]lymphatic endothelial cellslecs and blood endothelial cells becs amonglpscs cd45epcam in smallintestine the lscs thatexpressed podoplanin pdpn and are cd31 were foundto be capable of activating raldh enzymes figure 3a aspreviously reported interestingly unlike lpdcstheraldh activity in lpscs remained comparable between gfand af mice figure 3b in contrast when iecs were analyzedthe expression of aldh1a1 raldh1 the major gene encodingfor raldh enzyme in these cells was found to be reducedin af mice figure 3c of note while the iecs are wellestablished to have raldh activity the baseline ofthis activity in these cells is known to be significantly lower thanlpdcs therefore our attempt to measure raldhactivity in iecs was unsuccessful due to lower sensitivity ofthe aldefluor assay however albeit comparatively lowerraldh activity on a per cell basis the cumulative contributionof iecs in ra production is understandably of larger signiï¬cancesince numerically there are many more iecs than the other celltypes in the intestinefinally when mlndcs were analyzed the raldh activityin particular within the cd103cd11b dc population in afmice was found to be significantly albeit to a lesser extentlower than that of gf mice figure 3d taken together theseresults suggested that dietary components diï¬erentially uenceraldh activity in diï¬erentregulatory dc populationswhereas mlndcs and iec are aï¬ected lpscs appeared toremain unaï¬ected from dietary contributions these results alsoimplied that the overall reduction of ra synthesis cumulativelyamong these cell types eventually contributed to the reducedraldh activity in the lpdcs in af miceproteins starches and minerals in diet donot uence raldh activity incd103cd11b silpdcssince for this study we focused on raldh activity insilpdcs we next wished to deï¬ne which dietary factorswere required to trigger the initial raldh activity in thesecells while in our initial ï¬ndings we observed both thesubtypes cd103cd11b and cd103cd11b silpdcs tohave reduced raldh activity in af mice figures 1ef thecell recovery of cd103cd11b silpdcs from spf and gfmice were low and the level of enzyme activity in this celltype showed variability among individual mice figures 1cftherefore henceforth in this study we focused on the raldhactivity in cd103cd11b silpdcswe ï¬rst conï¬rmed that the reduction in raldh activityin cd103cd11b silpdcs was not a consequence of lowvitamin a contentin af diet thereby compromising theprecursor for the assayed reaction based on information offood compositions from the suppliers and from our perviousreport ï¬nal consumption of vitamin a per day by gf andaf mice were comparable [table s1 and ] nonethelessthere remained a possibility that albeit equal consumptionthe absorption of vitamin a into the small intestine may becompromised in af mice however when gf mice were weanedon af diet supplemented with times more vitamin a inthe usual form of oil mix or were administrated additionaloil mix by oral gavage failed to recover the reduction inraldh activity in cd103cd11b silpdcs figure s3we thus concluded that mere unavailability of the precursorvitamin a was not a cause of reduced raldh activity inthese cellsto this end we modiï¬ed the compositions of puriï¬eddiet by removing individual food components table s2 withfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure dietary components in normal chow readily trigger and maintain raldh activity in silpdcs in mice after weaning cell suspensions prepared from silpwere subjected to aldefluor assays and raldh activity in cd103cd11b silpdcs and percentage of silpdc subpopulations in cd11cmhcii cells wereanalyzed by ï¬ow cytometry a gf mice 3weeks old before weaning preweaned gf mice weaned onto ncd for days and adult gf mice were analyzed braldh activity and frequencies of silpdc subpopulations in cd11cmhcii cells in adult af mice ¼12weekold after feeding ncd for and days c raldh activity and frequencies of silpdc subpopulations in cd11cmhcii cells in adult gf mice ¼12weekold after feeding afd for and weeks d raldh activity and frequencies of silpdc subpopulations in cd11cmhcii cells in adult gf af or af mice that were administered intraperitoneal injection of alltrans ra µg per mouse in soybean oil every other day for days data are combined from two to three independent experimentsmean ± sem are indicated bc statistical signiï¬cance was determined by oneway anova with turkeys multiple comparison test p p p ns not statistically significantthe presumption that taking out or adding back individualcomponents in otherwise welldeï¬ned diet may lead us towardidentifying the dietary component required to trigger raldhactivity to obtain relatively accurate results under in vivosettings the modiï¬ed diets were designed to contain similaramount of vitamin a as in ncd tables s1 s2 and thefrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure raldh activity in intestinal raproducing cells is differentially regulated by diet ac cell suspensions from mln and silp harvested from agematchedadult ¼12weekold gf and af mice were subjected to aldefluor assays and raldh activity of lpscs and mlndcs were analyzed by ï¬ow cytometry arepresentative facs plot of lpsc distinguished by cd31 and pdpn from cd45epcam cells red dots in facs plot indicate aldeï¬uor positive cells histogramsdepict the mfi of aldeï¬uor in lpsc subtypes b graph displays the level of raldh activity in lscs data is combined from two independent experiments c iecswere isolated from small intestine si by stripping with edta and analyzed for expression of aldh1a1 raldh1 by realtime pcr upon normalization to hprt mrnalevels fold change indicates ratio target gene in experimentcontrol data are combined from four independent experiments d representative facs plots ofmlndc subpopulations distinguished by cd103 and cd11b from cd11cmhciilin cells and the raldh activity in cd103cd11b mlndcs andcd103cd11b mlndcs data are combined from four independent experiments mean ± sem are indicated statistical signiï¬cance was determined bytwotailed unpaired ttest p p ns not statistically significantexperiments were performed primarily in gf condition in orderto eliminate the uence of microbiota in addition to avoidany uence from ncd during the preweaned period neonateaf mice were utilized and these mice were weaned onto eachmodiï¬ed diet for weeksas a starting point we took advantage of two commerciallyavailable diets with welldeï¬ned dietary compositions in the socalled amino acid deï¬ned diet aad like the afd employed sofar proteins were replaced with amino acids however there wereseveral diï¬erences between their compositions table s2 whilefrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure depletion of macromolecules from puriï¬ed diet do not alter the raldh activity in cd103cd11b silpdcs af mice ¼4weekold were weaned ontospeciï¬c diets for ¼ weeks following which the indicated analyses were carried on a a cartoon depicting experimental scheme left panel aad is a sterilized formcontinuedfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure of amino aciddeï¬ned diet aad that contains three times more vitamin a than aad where protein macromolecules are replaced with amino acidsaad_stf indicates aad from which cornstarch and maltodextrin are removed the level of raldh activity in cd103cd11b silpdcs in the indicated groups arepresented by relative aldeï¬uor 01mfi right panel b representative facs plots left panel frequencies middle panel and absolute numbers right panel of totalcd4foxp3treg cell and cd4foxp3nrp1lo peripheral treg ptreg cell populations in silp of mice subjected to the indicated diet regimes c experimentalscheme left panel and relative aldeï¬uor 01mfi of silpdcs in the indicated experimental groups right panel min_mix afd indicates afd mixing with the mineral mixpowder td94049 data are combined from four independent experiments mean ± sem are indicated statistical signiï¬cance was determined by oneway anovawith turkeys multiple comparison test p p p ns not statistically significantafd is a liquid diet where the ï¬bers are provided as cellulosebedding aad diet are edible solid pellets with cellulose mixedwith the food components unlike afd aad diet containedstarches in the form of maltodextrin and corn starch and whilethe source of sugar in afd was glucose that in aad was sucrose furthermore in terms of mineral compositionthere are substantial diï¬erences between the groups the secondcommercially available diet is aad_stf which was largelysimilar to aad but was devoid of starches note the inaad indicates a sterilizable form of the diet which is otherwisesimilar to its traditional form aad but with three times morevitamin a to account for presumed losses during sterilization byirradiation surprisingly the mice groups weaned in both aadand aad_stf showed a complete recovery of raldh activityin cd103cd11b silpdcs to an extent similar to the controlncd fed group figure 4a these results were independentof microbiota since the characteristic drop in raldh activityof cd103cd11b silpdcs in mice raised in spf conditionscould also be recovered by aad figure s2d taken togetherthese ï¬ndings led to two important conclusions first starchesare not involved second antigens in the form of peptidesderived from proteins are also dispensable as far as raldhactivity in cd103cd11b silpdcs is concerned to this endwe also considered a possibility that an artifact arising fromunaccounted protein contamination in the amino acid deï¬neddiets may be responsible for the observed recovery of raldhactivity we therefore quantiï¬ed the generation of peripheralregulatory t ptreg also referred to as itreg when inducedin vitro cell population in the silp of these mice ptreg cells area type of treg cells that are extrathymically generated primarilyat mucosal sites and are distinguished from their thymic ttregcounterparts by the lack of expression of the membrane boundcoreceptor neuropilin1 nrp1 notably in a previousreport we have demonstrated that diet derived proteins are theprimary cause for the generation of cd4foxp3nrp1 ptregcells in the small intestine and af mice display dramaticallyreduced ptreg population in silp indeed we found that whiletotal foxp3 treg populations comprising of ttreg and ptregcells remained comparable the frequencies and numbers offoxp3nrp1 ptreg cells among total treg population couldonly be recovered in mice fed with ncd and not aadand aad_stf figure 4b therefore the recovery of raldhactivity in aad and aad_stf groups were not due to anyprotein contamination in the aad dietwe next wished to exclude the possibility that diï¬erencesin minor food components such as minerals between afdand puriï¬ed diet table s2 was responsible for diï¬erences inraldh activity for that we prepared afd by supplementingwith mineral mix powder td94049 derived from aadmin_mixafd as shown in figure 4c min_mixafd failedto induce raldh activity taken togetherthese resultsconcluded that proteins starches and minerals in diet werenot responsible for the induction of raldh activity incd103cd11b silpdcsoptimum glucose level in diet is requiredto induce raldh activity incd103cd11b silpdcsif the macromolecules and micromolecules in diet were notinvolved lack of which factors in afd compromise raldhactivity in cd103cd11b silpdcs to investigate furtherwe compared the food compositions between aad_stf andafd table s2 which were the closest among the four typesof diets tested in case of aad_stf this diet does not containproteins and starches as in afd but does contain unknowndietary factors responsible for the induction of raldh activityin silpdcs there are few diï¬erences between the compositionof these two diets the diet forms pellet vs liquid thecarbohydrate sources sucrose vs glucose the amount ofcarbohydrate sucrose ¼ vs glucose ¼ based on thisobservation as well as in order to minimize the diï¬erencesin diet forms we ï¬rst generated a liquid form of afd with of sucrose afd_s500 or glucose afd_g500germ free diet gfd or aad_stf were used as positivecontrols afd with usual glucose designated as afd_g220here was used as negative control the neonate af micewere weaned onto each diet for weeks and were analyzedfor raldh activity in cd103cd11b silpdcs figure 5aleft panel indeed afd supplemented with sucroseresulted in significantly enhanced raldh activity in these cellsmore interestingly we observed a similar increase in raldhactivity in cd103cd11b silpdcs even when the source ofcarbohydrate was changed to glucose instead of sucrosefigure 5a middle panel these eï¬ects were found to be speciï¬cfor cd103cd11b silpdcs since raldh1 expression iniecs remained unaltered upon carbohydrate supplementationfigure 5a right panel these results suggested that regardlessofits optimum concentrationis important and glucose being a monosaccharide unit ofcarbohydrate is suï¬cient for the initial triggering of raldhactivity in silpdcsthe source of carbohydratein order to further understand whether the positive eï¬ectof dietary carbohydrate supplementation is speciï¬c for raldhactivity or if it can also aï¬ect diï¬erentiation of other immunecells we determined the frequencies of th1 th2 and ptregfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure supplementing glucose in af diet restores raldh activity in cd103cd11b silpdcs a cartoon depicting experimental scheme left panelneonate af mice were weaned onto speciï¬c diets for ¼ weeks afd contains of glucose afd_g220 afd_g500 indicates of glucose in afd andafd_s500 indicates of sucrose in afd aad_stf was utilized as a positive control of raldh activity in lpdcs aldefluor assays were performed on cellsuspensions from silp and raldh activity of silpdc was analyzed by ï¬ow cytometry the level of raldh activity in cd103cd11b silpdcs is represented asrelative 01mfi of aldeï¬uor middle panel aldh1a1 expression was also determined in sorted iecs relative to hprt control right panel data are combined from twoindependent experiments bd graphs display the percentage of tbet in cd4 t cells th1 b gata3 in cd4 t cells th2 c and nrp1lo populations amongin cd4foxp3 treg cells ptreg d data are combined from at least two independent experiments mean ± sem are indicated statistical signiï¬cance wasdetermined by oneway anova with turkeys multiple comparison test p p p ns not statistically significantcells in these mice supplementation of afd with additionalsucrose or glucose did not aï¬ect tbet th1 or gata3 th2 cellsfigures 5bc neither recovered the characteristically reducedptreg population in silp figure 5dwe next directly investigated the eï¬ect of glucose on raldhactivity by employing in vitro culture conditions spldcsmlndcs and cd11c silpdcs were magnetically puriï¬edand cultured either without glucose in commercially availableglucosefree media without glucose but in the presence of rawith glucose or in the presence of glucose and ra for hand then measured for raldh activity in terms of baselineraldh activity as expected spldcs displayed the least whichwas significantly increased in the presence of ra glucose onthe other hand had minimal eï¬ect figure 6a although mlndcs had the highest baseline raldh activity among the threegroups tested neither ra nor glucose had an impact figure 6bin contrast silpdcs derived from 2weeks old neonatal spfmice which had low basal raldh activity at steady state wassignificantly increased in the presence of glucose alone while raitself induced slight increase of this enzyme activity the highestactivity was observed when both ra and glucose was presentfigure 6c moreover this raldh activity promoting eï¬ect ofglucose was also observed when silp mixed lymphocytes derivedfrom 8weeks old adult mice were cultured with glucose for hunder the culture conditions tested ra by itself was found tohave minimal eï¬ect on the already high raldh activity whereasthe addition of glucose in the media was able to further boost thisactivity figure 6dfinally in order to ascertain functional relevance of theseï¬ndings we sorted to determine whether supplementationfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure glucose induces raldh activity speciï¬cally in silpdcs mlndcs and spleen dcs spldc from adult spf mice or silpdcs from 2weeks oldneonatal spf mice were either puriï¬ed ac or total single cell suspensions were isolated from adult spf mice si d cells were cultured for h in glucosefreecontinuedfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure media with or without treatments following which aldefluor assay was performed fluorescence intensities of aldeï¬uor in cd8cd11b spldcsa cd103 mlndcs b and cd103cd11b silpdcs cd were analyzed by ï¬ow cytometry raldh activity depicted in overlaid histograms and bar graphsare from the cells cultured with glucose mm or ra nm or both the line graphs indicate raldh activity from the cells cultured with different concentrations ofglucose and mm in the presence or absence of ra nm data shown is representative of at least three independent experiments mean ± sem areindicated statistical signiï¬cance was determined by oneway anova with turkeys multiple comparison test p p p ns notstatistically significantof glucose presumably through the generation of ra canfacilitate the generation of itreg cells for this we performedan in vitro itreg induction assay with magnetically puriï¬edcd11chi dcs with high cd11c expression that were pretreated with glucose and incubated with naïve t cells undersuboptimal itreg inducing condition we observed significantincrease in itreg induction when silpdcs were pretreatedwith glucose compared to mock this eï¬ect of glucose pretreatment was speciï¬c for silpdcs and was not observedwhen mlndcs were used in the assay figures 7ab andfigure s4 these results in accordance to the results presentedin figure 6b suggested that the silp dcs are particularlymore susceptible to glucose treatment and thereby presumablythrough enhanced raldh activity acquire superior itreg cellinduction capacity compared to mln dcs it is to be notedhowever that while the puriï¬ed cd11chimhcii dcs usedin this assay are presentthere aresome site speciï¬c diï¬erences with regard to the expressionof mhcii and cd11c in mln and silp compared to silpmln has significantly higher proportion of cd11cmhciiand cd11cintmchii with intermediate cd11c expressionpopulations figure s4 left panels furthermore the expressionof mhcii in puriï¬ed mln dcs is lower than that of silpdcs figure s4 right panels these observations raised theformal possibility that enhanced mhcii expression in silprather than glucose mediated enhanced raldh activity maybe responsible for increased itreg conversion however pretreatment with ra either alone or in the presence of glucoseresulted in equally eï¬cient itreg induction irrespective of thesource of the dcs suggesting that the beneï¬t of glucose pretreatment is indeed primarily due to enhanced ra productionfigures 7ab lastly when itreg induction was carried outin vitro in a dc independent manner supplementation ofthe media with excess glucose had little eï¬ect thereby furthersubstantiating the role of dc derived raldh in this processfigure s5in similar frequenciesdiscussionthere is accumulating evidence suggesting that vitamin a and itsmetabolites play a pivotal role in maintaining various biologicalprocesses dietary supplementation of ra in thecontext of cutaneous t cell lymphoma and acute promyelocyticleukemia have been shown to have beneï¬cial outcome furthermore many studies highlight antiammatoryactivities of ra at mucosal sites and tissues such as intestinalmucosa airways lung central nervous system and skin in addition to the eï¬ect of ra on cancer and ammatorydiseases vitamin a or its metabolites play an important roleto suppress dietinduced obesity and insulin resistance therefore a better understanding of the cellular and molecularparameters responsible for vitamin a metabolism is of greatbiological relevance in this study by identifying the role of dietand the importance of glucose consumption for establishingraldh activity early in life in small intestine dcs we makesubstantial contribution to our knowledge related to the role ofnutritional components in establishing immunological toleranceat mucosal sitesby feeding af diet to mice raised under germ free conditionwe found that small intestine cd103cd11b lpdcs require adietary component as an initial trigger for raldh activity sincevitamin a in diet is known to be essential to activate raldhand generate ra in lpdcs lpscs mlndcs mlnscs andiecs one explanation of this observation could bethe possibility that at steady state af mice consume less vitamina compared to gf mice raised on ncd however our furtherexperiments in conjunction with a previous report stronglyindicate that the availability of vitamin a and the way it is feddoes not account for the low raldh activity in af m | Colon_Cancer |
"as metastasis is a major cause of death in cancer patients new antimetastatic strategies are needed to improvecancer therapy outcomes numerous pathways have been shown to contribute to migration and invasion ofmalignant tumors aspartate hydroxylase asph is a key player in the malignant transformation of solid tumorsby enhancing cell proliferation migration and invasion asph also promotes tumor growth by stimulation ofangiogenesis and immunosuppression these effects are mainly achieved via the activation of notch and srcsignaling pathways asph expression is upregulated by growth factors and hypoxia in different human tumors andits inactivation may have broad clinical impact therefore small molecule inhibitors of asph enzymatic activity havebeen developed and their antimetastatic effect confirmed in preclinical mouse models asph can also be targetedby monoclonal antibodies and has also been used as a tumorassociated antigen to induce both cluster ofdifferentiation cd and cd4 t cells in mice the pan3011 vaccine against asph has already been tested in aphase clinical trial in patients with prostate cancer in summary asph is a promising target for antitumor andantimetastatic therapy based on inactivation of catalytic activity andor immunotherapykeywords asph small molecule inhibitor metastasis immunotherapy cancer is a multifactorial disease with an approximate million fatalities in worldwide it is the secondleading cause of death the complex modifications inthe genome affected by the interactions between hostand environment lead to cancer development and progression despite advancements in characterizing themolecular mechanisms of oncogenesis tumor progression and metastasis delayed cancer detection limitedsurgical options therapeutic resistance and tumor recurrence are serious obstacles in decreasing the prevalence and fatality rate of cancer since metastasis is theprimary cause of deaths from cancer the design oftherapeutictarget mechanisms oftumorcell migration and invasiveness is essential in thisapproachesthat correspondence smahelmnaturcunicz1department of genetics and microbiology faculty of science charlesuniversity biocev vestec czech republicfull list of author information is available at the end of the regard a growing number of investigations of signalingpathways involving products of oncogenes and tumorsuppressor genes in human carcinomas has helped toelucidate the mechanisms underlying malignant transformation of cells and facilitated the development ofnew and more efficient therapeutic methodsaspartate hydroxylase asph has been identified asone of the cell surface proteins associated with malignant transformation of tumor cells [ ] asph belongsamong the most important biological targets to controlmigration and invasion of tumor cells as its overexpression has been observed in of human solid tumors [] the overexpressed asph is transportedfrom the endoplasmic reticulum to the plasma membrane which results in exposure of the cterminal regionto the extracellular environment where it is accessible toantibody binding recently molecular targeted therapyhas been developed against this target using small molecule inhibitors smi that can inhibit the catalytic site the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ckanwal of experimental clinical cancer research page of in the cterminal region moreover as antigenic epitopes that reside on the asph protein can efficientlystimulate cluster of differentiation cd and cd8 tcell responses unique to tumor cells harboring asphthis enzyme can be used as a tumor associated antigentaa in immunotherapy [ ]thedioxygenasesstructure of the asph gene and isoformsasph is a type ii transmembrane protein of approxito the family of αmately kda that belongsketoglutaratedependenthydroxylated products of asph hydroxylation were firstdetected in blood coagulation proteins [] asphwas initially identified in the bovine liver as an enzymeresponsible for catalyzing the hydroxylation of aspartyland asparaginyl residues in calcium binding epidermalgrowth factor cbegflike domains of various proteins fig thereafter the human asph gene wascloned and characterized this gene spanning base pairs long region of genomic dna and containing exons is located at the position q123 of thehuman chromosome the asph sequence is highlyconserved in mammalian evolution the sequence of thehuman protein is from about identical to the sequences of rat and mouse analogs and the catalytic siteis quite conserved among proteins of these three species the whole asph protein consists of five domainsan nterminal cytoplasmic a universal transmembranea positively charged luminal a calcium binding and a cterminal catalytic domain tissue specific transcription is directed from two putative promoters p1 and p2which differ in their regulation sequences [ ] whilethe transcription from the p1 promoter was observed inmost human tissues the p2 promoter is activated by thecalciumdependent transcription factor myocyte enhancer factor mef2 particularly in muscle tissues the asph gene undergoes extensive alternative splicingresulting in four protein isoforms ie asph humbugjunctate and junctin [ ] these proteins vary in thecterminal region which affects their function [ ]the two longest asph transcript variantsthat aretranscribed from the p1 and p2 promoters and differ inthe length of the ²untranslated region encode the fulllength asph protein this protein contains the catalyticcterminal domain that catalyzes the posttranslationalhydroxylation in the cbegflike domains of numerousproteins supplementary fig including receptors receptor ligands and extracellular adhesion moleculesthat influence cell motility and invasiveness [ ] thetruncated isoforms humbug junctate and junctin sharethe nterminal part with the asph protein but lackcatalytic function they are involved in calcium homeostasis humbug has a potential role in cell adhesionand calcium flux and similar to asph its overexpressionhas been correlated with aggressive tumorcell behavior reticulummembranebound protein that is known for its functionin the regulation of the intracellular ca2 concentrationjunctin is a structural membrane protein and as an integral part of the complex consisting of the ryanodine receptor calsequestrin and triadin influences calciumrelease from the sarcoplasmic reticulum [ ]sarcoendoplasmicjunctateisalocalization in cells tissue distribution andexpression regulationasph is predominantly a cellsurface protein that isalso localized in the endoplasmic and sarcoplasmicreticulum furthermore a recent study identifiedmitochondriallocalization of asph in hepatocellularcarcinoma hcc in that study asph overexpressioncorrelated with an instability of mitochondrial dna andmitochondrial dysfunction that may lead to more aggressive pathological outcomes in hcc asph is abundantly expressed in proliferating placental trophoblastic cells [ ] and in decidua and endometrial glands and has a potential role in placentalimplantation and fetal growth on the contrary theasph expression in normal adult tissues is relativelylow or negligible however asph expression is inappropriately activated during oncogenesis when asph is required for generation of malignant and metastaticphenotypes the elevated expression of asph at bothfig asph catalytic reaction aspartyl and asparaginyl residues in cbegflike domains are hydroxylated 0ckanwal of experimental clinical cancer research page of transcription and translation levels has been shown in awide range of transformed cell lines as well as humancarcinoma tissues including hepatocellular pancreaticcolon prostate lung breast ovarian and cervical carcinoma cholangiocarcinoma neuroblastoma and gastriccancer table the first study that demonstrated thesignificantly higher expression of both asph mrnaand protein in hcc and cholangiocarcinoma relative totheir normal adjacent tissue counterparts was by lavaissiere subsequently they verified the role of upregulated asph protein production and its enzymaticfunction in the malignant transformation on biliary epithelium the nih3 t3 cell line and animal models the level of asph also correlated with cell motility andinvasiveness in in vitro experiments [ ] in thestudy by maeda the overexpression of theasph protein was in accordance with worse clinical andhistopathological characteristics of the intrahepatic cholangiocarcinomas and prognosis of patients similar findings were obtained in other studies for hepatocellular[ ] nonsmall cell lung and colon carcinomas and glioblastoma multiforme recentlytheprognostic significance of 2oxoglutaratedependent oxygenase expression was demonstrated by analysis of expression profile datasets of tumor samples and nontumor samples asph has been identified asone of the genes which upregulated expression couldserve for risk stratification of patients with cancertypes in glioblastoma the prognostic significance ofasph was suggested by profiling of alternative mrnasplicing asph gene expression is upregulated via wntcatenin and insulininsulinlike growth factor igf1insulin receptor substrate irs1 signaling [ ]through extracellular signalregulated kinaseerkmitogenactivated protein kinase mapk andphosphatidylinositol3kinaseprotein kinase b pi3kakt pathways fig for review see ref insulinigf1irs1 signaling affects cell growth and survival andcan be involved in oncogenesis in various human tumorstable summary of the studies which have identified the elevated asph expression in human tumor tissuesstudypositive cases of studied samples nntumor tissuesdetectionmethodihcantibody recognized region ofasph proteinfb50 ab nterminusfb50 ab nterminusfb50 ab nterminusfb50 ab nterminusfb50 ab nterminus or 15c7 abcatalytic domainfb50 ab nterminusmab g3 hybridomapolyclonalfb50 mab nterminusihcihcihcihcrtqpcrihcrtqpcrihcihcihcrtqpcrihcfb50 ab nterminuslavaissiere hepatocellularcholangiocarcinomabreastcolonpalumbo pancreatic adenocarcinomasepe primitive neuroectodermalmedulloblastoma neuroblastomamaeda cantarini cholangiocarcinomahepatocellularmonte hepatocellularyang wang dong tang lin types of tumor tissuesahepatocellularpancreatic cancerhepatocellularbreast 75fold higher level of mrnacompared to normal tissue 7fold higher level of mrnacompared to normal tissuepancreatic ductal adenocarcinomaogawa aliver kidney breast cervical ovarian fallopian tube laryngeal lung thyroid pancreatic thymic prostate bladder esophagus gastric gall bladder colon andrectum cancer and cholangiocarcinomafb50 ab nterminusihc 0ckanwal of experimental clinical cancer research page of fig regulation of asph expression and asph involvement in signaling pathways the expression of the asph protein can be regulated atseveral levels the asph gene can be amplified in tumor cells and its transcription activated by inigf1 and wnt catenin pathways or inducedby hypoxia at the posttranscriptional level mir200a and mir135a can downregulate asph expression stability of the asph protein can bereduced by phosphorylation with gsk3 conversely asph can enhance gsk3 activity by inhibition of its phosphorylation with akt and p38kinases asph also supports cell proliferation epithelialmesenchymal transition migration invasion and angiogenesis and consequently tumrowth and metastasis by hydroxylation of the notch receptor and ligands ex jag and interaction with prb vimentin and adams finallyinactivation of nk cells by asph has been demonstrated green arrow activation signal red bar inhibitory signal the catenindependent wnt pathway regulatescell proliferation motility and differentiation and is oneof the most frequently modified pathways in humanmalignancies upon aberrant activation of wnt signalingcatenin is accumulated in the cytoplasm and subsequently translocated to the nucleus where an interaction between catenin and tcellfactorlymphoidenhancerbinding factor tcflef proteins forms atranscriptional regulatory complex which enhances theexpression of wnt target genes including irs1 asph was proposed as a common link between wntcatenin and insulinigf1irs1 pathways and downstream signaling the regulation of asph gene expression in tumorsmight also be affected by a copy number variation inthe study by kadota the asph gene locus hasbeen identified as one of the dna regions with focalamplification in primary breast cancer in colorectal cancer asph gain or amplification was found in ofsamples next a suppressant role of the micrornamir200a in posttranscription regulation of the asphexpression in hepatoma cells has been found mir200a belongs to mir200 family which plays significantrole in preventing cancer initiation and metastasis forreview see ref similarly mir135a has beenshown to suppress asph in endometrial cancer moreover consistent with the protein sequence analysis that recognized numerous prospective phosphorylation sites of glycogen synthase kinase3 gsk3casein kinase ck2 protein kinase a pka and protein kinase c pkc on asph several studies demonstrated that phosphorylation can regulate the asphprotein expression [ ] inhibition of the gsk3activity did not modify mrna expression but increased 0ckanwal of experimental clinical cancer research page of the asph protein level direct phosphorylation ofasph by gsk3 probably decreases asph stability andthus reduces cell mobility asph protein expressionwas also increased by inhibitors of pka pkc and ck2 mutational analysis of potential sites of phosphorylation demonstrated complex and nonuniform effects ofasph phosphorylation on protein expression enzymaticactivity and subcellular localization [ ] thereforeasph phosphorylation probably regulates the functionof this protein by various mechanismsasph expression can also be regulated by hypoxia andoxidative stress in human neuronal cells this effect wasmediated by hypoxia inducible factor alpha hif1αthat is stabilized under hypoxiaoxidative stress whenthe prolyl hydroxylase domain phd proteins and factorinhibiting hif fih are inactivated consequently thehif1 heterodimer made up of subunits hif1α andhif1 functions as a transcription factor likely enhancing asph expression by binding to hypoxiaresponsiveelements in hypoxic regions of glioblastoma bothhif1α and asph were highly expressed particularly inmore aggressive mesenchymal subtype of glioblastomasuggesting a possible involvement of asph in mesenchymal transition brewitz showed reducedasph hydroxylation activity at low oxygen concentrations and suggested an asph role in oxygen hypoxiasensing asph upregulation induced by hypoxia couldcompensate for reduced enzymatic activity moreover a recent study reported an oxidative stress state ofthe castrationresistant prostate cancer cells upon asphoverexpression which was reversed by silencing asphexpression or generating hypoxic conditions resulting inimpaired cell proliferation and invasion asph protein interactions and signaling pathwaysthe asph hydroxylation consensus sequence is confined within cbegflike domains that are found in proteins of diverse function including notch receptors andligands clotting factors structural proteins of the extracellular matrix and ligands of the tyro3axl family ofreceptor tyrosine kinases the notch signaling cascade is a remarkably conservedpathway notch proteins notch1 notch4 are singlepasscell surface receptors that mediate communication betweencells and their expression is crucial for proper embryonicdevelopment notch signaling mainly results in cell differentiation but also plays a significant role in proliferationapoptosis and the maintenance and selfrenewal of stemcells dysregulation of the notch pathway is directly linkedto cancer vascular disorders and congenital defects in mammals notch signaling activated by binding ofone of two families of canonical notch ligandsjaggedjag1 and jag2 and delta like dll1 dll3 and dll4leads to the generation of the cleaved notch intracellulardomain nicd fragment and its nuclear translocation inthe nucleus the nicd fragment interacts with the dnabinding complex csl cbf1rbpjκ suh lag1 thiscomplex is then converted from a repressor into an activator leading to increased transcription of target genes suchas hes family bhlh transcription factor hes1 heswith yrpw motif hey1 cd44 epithelial cell adhesionmolecule epcam cmyc protooncogene matrix metallopeptidase mmp29 cyclin d1 cyclooxygenase vascular endothelial growth factor vegf and proliferatingcell nuclear antigen pcna [ ]upregulation of asph results in enzymatic modification of the cbegflike repeats in the notch receptorextracellular domain and its ligands which promotes thereceptor interaction with the ligands and the activationof notch signaling [ ] furthermore the interactionof asph with a disintegrin and metallopeptidase domainadam stabilizes this complex and enhances thes2 cleavage ofthe notch receptors and subsequentnicd fragment release the activation of the targetgenes in malignant cells increases cell proliferation migration and invasion through the epithelialtomesenchymal transition emt that is probably upregulated by the interaction of asph with vimentin consequentlythis activation supports tumor growthand metastasis the asphnotch axis also stimulatesthe release of exosomes that transfer proteins involvedin invasion metastasis metabolism and immunosuppression [ ]the src kinase pathway is another important pathwayin malignant cell transformation that regulates a complex signaling network promoting angiogenesis invadopodia formation and maturation and metastasis asph has been identified as an src pathway activatoroverexpressed asph directly interacts with adam12 and strengthens the src activation by these proteinswhich promotes mmpmediated extracellular matrixdegradation and tumor invasiveness asph can also contribute to malignant phenotype ofcells by interaction with other proteins iwagami revealed the interaction of asph with gsk3 that prevents gsk3 inactivation by phosphorylation with upstream kinases this mechanism was confirmed ina castrationresistant prostate cancer model gsk3 is a multifunctional kinase that is involved in variousprocesses including glycogen metabolism cell divisionand cell fate determination some types of tumors aresensitive to gsk3 inhibitors recently huang elucidated a direct binding of asph with retinoblastoma protein prb leading to prb phosphorylation they also showed that this effect was mediated byincreased binding of cyclindependent kinase cdk cdk4 and cyclins d1 and e with prb and wasdependentasasph enzymaticonactivity 0ckanwal of experimental clinical cancer research page of phosphorylation of prb inactivates its tumorsuppressorfunction asph can contribute to the progression of cellcycle via interaction with prbeffect of asph on an immune systemtumor generation and progression are influenced bycancer immunoediting that involves immunosurveillanceand escape from a host immune system in theseprocesses various mechanisms of both innate and adaptive immunity are included immune cells that infiltrate developing tumors are initially antitumorigenicbut in tumor microenvironment they can be modifiedinto cells with protumorigenic properties as potential targets of asph hydroxylation are alsoexpressed on immune cells this enzyme could affect thefunction of immune system particularly in tumor microenvironment when asph is overexpressed on cancercells indeed such effect was demonstrated for humannatural killer nk cells by using recombinant asphwhich reduced viability and cytotoxicity of these cells viaenhancing caspase signaling and decreasing the surfaceexpression of activating receptorsrespectively antibodies against asph inhibited these effectsinteraction of asph with other immune cells has notbeen studied however we suppose possible influence ofasph on different tumorinfiltrating cells this assumption comes from the involvement of notch signaling indifferentiation and function of various immune cells fibroblasts mesenchymal cells and endothelial cells forinstance notch activation contributed to stimulation ofproinflammatoryantitumorigenic m1 polarization inboth bone marrowderived primary macrophages and tumorassociated macrophages whennotch signaling was abrogated protumorigenic m2polarization was induced even by stimulators of m1polarization mir125a has been identified as adownstream mediator of notch signaling in macrophages similarly the notch pathway plays an important role in differentiation of other types of myeloidcells and probably all subsets of cd4 and cd8 t cells different notch receptors and their interactionwith different ligands contribute to these processes moreover noncanonical notch signaling is implicatedin regulation of immune cells while activation ofnotch signaling in some cells eg t helper cells cytotoxic cd8 t cells and m1 macrophages supports induction ofincluding antitumorimmunity in other cells particularly regulatory t cellsit leads to immunosuppression thus immunostimulatory effect of notch signaling is often inhibited intumor microenvironment to enable the tumor cells toescape from the host immunity therapeutics affecting notch signaling in malignant diseases are being developed and tested in clinical trials but their effects onimmune reactionsimmune reactions and possible combination with immunotherapy have not been properly studiedasph as a therapeutic targetoncogenic abilities of asph have been experimentallydemonstrated using tumor cell lines and mouse and ratmodels of different types of human tumors with asphoverexpression including cholangiocarcinoma [ ] hepatocellular carcinoma [ ]neuroblastoma pancreatic cancer [ ] glioma breast carcinoma castrationresistant prostatecancer and colorectal cancer in studies analyzingasph function various approaches were utilized to revealsignaling pathways affected by asph particularly asphexpression was diminished by using small interfering rnas[ ] short hairpin rnas [ ] or thecrisprcas9 system [ ] the importance of asphenzymatic activity in these processes was shown by the sitedirected mutagenesis [ ] or treatment by smis [ ] in vitro assays showed asph involvement in cell proliferation migration and invasion cellularalterations included emtinhibition of apoptosis andstemness acquisition tumor growth and invasivenesscould further be supported by asphinduced extracellularmatrix degradation angiogenesis and transendothelial migration notch and src signaling are probably major pathways influenced by asph fig and contributing toincreased aggressiveness of tumor cells that was verified inin vivo models thus these studies also demonstrated thatasph is a suitable target for cancer treatment especially bysmis or immunotherapysmall molecule inhibitorssmis of asph fig have been developed and used totest the role of asph in a wide range of cancer modelsincluding subcutaneous orthotopic and patient derivedxenograft in vivo models [ ] a small orallybioavailable inhibitor has severalintrinsic advantagesover immunotherapy approaches not only can these inhibitors inhibit the catalytic activity of asph unlike conventional antibodies that simply bind to the protein butthey can also penetrate into the cell and inhibit asphcatalytic activity in the endoplasmic reticulum differentcancers have different asph expression patterns andwhile surface expression is quite common in pancreaticcancer and hepatocellular carcinoma intracellular overexpression patterns have also been observed the first asph smis published were the tetronimidesmoi500 and moi1100 tetronimides were originallysynthesized in by dahn and are redoxactivemimics of ascorbic acid and 2oxoglutarate moi500 isa mixed inhibitor that inhibits both asph and the fatmass and obesity protein fto and is not onlyorally bioavailable but also can penetrate the blood 0ckanwal of experimental clinical cancer research page of fig small molecule inhibitors of asphthereand kinasesbrain barrier moi1100 is a more potent inhibitor ofasph and is also more selective despite investigation against a wide range ofirondependent dioxygenasesare no other knownenzymatic targets for moi1100 enhanced activity wasobserved by replacing the chlorine with a trifluoromethylgroup as in moi1151 and even a greater improvement in in vivo activity was found by replacing the trifluoromethyl group with a carboxymethyl group as inmoi1182 although it is not yet clear if the nature ofthis enhancement is due to increased inhibitory activityorenhanced solubility parameters moi1182 isreported to have the ability to suppress invasive activityat a concentration of nm smis of asph have acharacteristic in vitro concentration dependent profilewhere the activity of the smi plateaus at value around viability emphasizing the noncytotoxic properties of this class of inhibitorsnatural products and inhibitors of other enzymes thathave been repurposed as asph inhibitors have alsorecently been reported in the patent literature includingbosutinibcepharanthinecn2019101414327 and guaianolides related to nortrilobolidecn2019104575886cn2019101414219cn2019101414187 0ckanwal of experimental clinical cancer research page of sesquiterpene with complex anticancerbosutinib is a wellknown inhibitor of bcrabl and srctyrosine kinases approved for the treatment of chronic myelogenous leukemia cepharanthine is a natural productactivityincluding ampactivated protein kinase ampk activationand nuclear factor kappa b nfκb inhibition nortrilobolide and related compounds are reported to be potentcytotoxic agents with subnanomolar sarcoendoplasmicreticulum calcium atpase serca inhibition recently a family of potent pyridine dicarboxylates have alsobeen published utilizing a mass spectrometrybasedinhibition assay these compounds are related toknown irondependent dioxygenase inhibitors 23pyridinedicarboxylate 24pyridine dicarboxylate and 26pyridinedicarboxylate the synthesized pyridine dicarboxylateswere assayed for activity against a range of other enzymesto include phd2 fih and lysinespecific demethylase 4ekdm4e in addition to asph with varying degrees of selectivity however while cellbased activities have not beenevaluated the dicarboxylate nature of the compounds maybe useful for cell surface asph inhibitors that may nothave cell penetrating activity immunity as a target of humoralimmunotherapyasph can be used not only as a target of the inhibitors inactivating its enzymatic activity but also as a target of immune reactions leading to destruction of tumor cells andtumor growth suppression since asph is cell surface displayed on tumor cells it represents a tumorassociatedantigen that can be targeted by both cellmediated andhumoralimmunityasph on the surface of cancer cells can be bound by antibodies that mediate antibodydependent cellular cytotoxicity adcc complement dependent cytotoxicity cdcor antibodydependent cellular phagocytosis adcp when the asph antigen is processed in tumor cells orantigen presenting cells antigenic peptides are presentedon these cells by human leukocyte antigen hla class ior class ii molecules and recognized by cd8 or cd4 tlymphocytes respectively that can be stimulated byimmunization breaking tolerance to selfantigens induction of asphspecific cd4 and cd8 t cells wasexamined in blood samples of hcc patients using synthetic peptides derived from asph after prediction of hlaclass i and hla class iirestricted epitopes it has beenfound that asph is a highly immunogenic protein that activates both types of analyzed t cells thus efficientantitumor reactions could be stimulated by immunizationthe first vaccine against asph was based on matureddendritic cells dc loaded with the asph protein andtested in an orthotopic rat model of intrahepatic cholangiocarcinoma this study showed that vaccinationstimulated cytotoxicity against cancer cells in an in vitroassay and decreased tumor growth and metastasis bothcd8 and cd4 cells contributed to an antitumor effectinduced in a mouse model of hcc by immunizationwith asphloaded dcs the next antiasph vaccine was based on a bacteriophage lambda display system the viral capsid proteingpd was fused with the n or cterminus of asph andimmunogenicity ofthese nanopforming constructs was verified in two mouse tumor models the vaccine pan3011 containing these constructs hasalready been examined in a phase clinical trial in patients with biochemically relapsed prostate cancer this study demonstrated safety and immunogenicity of pan3011 and indicated an antitumor effect interms of the reduction of prostate specific antigen psaor psa doubling time asphspecific immune responseswere mediated by both antibodies and t lymphocytesas asph is a type ii transmembrane protein its cterminus carrying the enzymatic domain is exposed outside cells and can be bound by antibodies that can be usedfor diagnostic and therapeutic purposes development ofasphspecific antibodies has been described in several s [] the human igg1 pan622 recognizesthe catalytic domain of asph this antibody is not directly cytotoxic for tumor cells but is internalized and candeliver cytotoxic moieties into cells in the subsequent study with a mouse model of metastatic breast cancerandradioimmunotherapy with promising results mouseigg1 monoclonal antibody binding to the cterminalasph domain mediated adcc by human nk cells recently a secondgeneration antibody approach hasbeen disclosed the prepared antibody binds to the extreme cterminus of asph us that is involved in specific substrate recognition thereforethis antibody has direct asph inhibitory activity anddoes not require any radioisotope or cytotoxic payloadfor potential therapeutic activitypan622 wasbioimagingusedforconclusionsasph is an important enzyme in malignant transformationof cells it stimulates tumor cell proliferation migration andinvasion but it can also affect other cells in tumor microenvironment two main pathways notch and srcthrough which asph promotes the tumor growth havebeen identified it has also been shown that asph expression is induced by some growth factors and hypoxia and isregulated at various levels the overexpression of asphand its downstream targets has been detected in numeroushuman malignancies since asph is not expressed in appreciable level in normal adult tissues and the catalytic domain is localized on the cell surface it has been proposedas one of the most exciting potential therapeutic targetsfig small inhibitory molecules orally bioavailable havebeen developed and successfully tested in several cancer 0ckanwal of experimental clinical cancer research page of fig asph as a therapeutic target asph expression is upregulated by growth factors and hypoxia its enzymatic activity can be inhibited bysmis or monoclonal antibodies which results in reduction of cell proliferation angiogenesis immunosuppression and cell migration and invasionconsequently tumor growth and metastasis are also reducedmodels but they have not yet advanced into clinical trialsadditionally as asph was identified as a tumorassociatedantigen immunotherapy approaches vaccines and monoclonal antibodies were tested with promising results in preclinical experiments and results of pha | Colon_Cancer |
purpose pancreatic ductal adenocarcinomas exhibit a high degree of desmoplasia due to extensive extracellular matrix deposition encasement of mesenteric vessels by stroma in locally advanced pancreatic cancer lapc prevents surgical resection this study sought to determine if the addition of a monoclonal antibody to connective tissue growth factor pamrevlumab to neoadjuvant chemotherapy would be safe and lead to improved resectability in this surgically adverse patient populationmethods in this phase iii trial patients with lapc were randomised to gemcitabinenab paclitaxel plus arm a n24 or minus arm b n13 pamrevlumab those who completed six cycles of treatment were assessed for surgical eligibility by protocol defined criteria resection rates progression free and overall survival were evaluatedresults eighteen patients in arm a and seven in arm b completed six cycles of therapy with similar toxicity patterns in arms a and b carbohydrate antigen response as defined by ¥ decline from baseline occurred in and respectively sixteen per cent of patients were radiographically downstaged by national comprehensive cancer network criteria in arm a and in arm b positron emission tomography normalised in vs of patients in arm a vs arm b respectively and correlated with surgical exploration eligibility for surgical exploration was vs p00019 and resection was achieved in vs of patients in arm a vs arm b p01193 respectively postoperative complication rates were not different between armss neoadjuvant chemotherapy with pamrevlumab holds promise for enhancing resection rates in patients with lapc without added toxicity this combination merits evaluation in a larger patient cohortintroductionpancreatic cancer is currently the third leading cause of cancer death in the usa1 and by it will likely become the second leading cause of cancer related death after key questionswhat is already known about this subject º pamrevlumab is anti ctgf1 inhibitor highly safe when given to patients with pancreatic cancer and potentially adds to the activity of gemcitabine and erlotinib when given in metastatic diseasewhat does this study add º this study examines a the safety and activity of pamrevlumab when added to gemcitabine and nab paclitaxel in locally advanced pancreatic cancer b the impact of this regimen and surgical resection and postoperative safety c explores the utility of a novel study design for locally advanced pancreatic cancerhow might this impact clinical practice º this study has the potential to lead to practice changing activity in locally advanced pancreatic cancer via the eventual approval of pamrevlumab for use in this situation the promulgation of a new study design for locally advanced pancreatic cancer and increased potential for surgical resection and thus prolonged os curability in locally advanced pancreatic cancerlung cancer surpassing breast and colon cancer2 surgical resection is generally necessary for treatment with curative intent or to extend life expectancy3 however only of patients have disease amenable to upfront curative resection at the time of diagnosis4 approximately of patients are diagnosed with locally advanced disease5 determined surgically unresectable per national comprehensive cancer network nccn guidelines6 patients with locally advanced pancreatic cancer lapc have a prognosis similar to those with metastatic disease with a historical median overall survival os of picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen access months with recent trials demonstrating median os of months7 recent single institution retrospective studies have reported the potential for resection of lapc with neoadjuvant therapy irrespective of imaging findings with promising results8 however these are limited by significant selection bias lack of strict radiographic classification and chemotherapy standardisation current prospective trials have documented resection rates of lapc in the range of to therefore novel approaches are needed to improve patient outcomesthe tumour biology inherent to pancreatic ductal adenocarcinoma pdac significantly contributes to the poor outcomes seen in this disease notably pdac exhibits a high degree of desmoplasia often in association with elevated connective tissue growth factor ctgf expression12 ctgf appears to play a central role in the biology of pancreatic cancer affecting both its cellular biology and its extracellular matrix composition this leads to many simultaneous biological effects that promote pancreatic cancer growth including increased cellular proliferation and differentiation increased cellular adherence and migration antiapoptosis vascular permeability angiogenesis and suppression of tumour immunological responses13 this stroma may also contribute to the radiographic imaging findings of mesenteric vessel involvement or encasement that is used to determine resectability of pancreatic tumours executing a pharmacological intervention on the pancreatic cancer stromal environment is therefore a major goal of the development of novel pancreatic cancer therapeuticspamrevlumab is a human monoclonal antibody that targets ctgf preclinical studies showed that ctgf overexpression is associated with both desmoplasia and gemcitabine resistance in the kpc pancreatic cancer mouse model14 when pamrevlumab was used in combination with gemcitabine sensitivity to gemcitabine was enhanced which correlated with inhibition of xiap an antiapoptotic protein15 when tested in patients with advanced pancreatic cancer stage iv and locally advanced stage iii treated with gemcitabine and erlotinib in a phase iii study n75 pamrevlumab displayed multiple favourable outcomes16we hypothesised that through inhibition of the downstream effects of ctgf overexpression on tissue adhesion and other mechanisms pamrevlumab may influence resectability of pdac tumours with this in mind this novel phase iii randomised multicentre trial was designed to explore the safety and efficacy of pamrevlumab in combination with gemcitabinenab paclitaxel in lapc with special emphasis on surgical eligibility and safetymethodsstudy designthis was a phase iii randomised trial of safety and efficacy in patients with lapc who received gemcitabine and nab paclitaxel with or without pamrevlumab as neoadjuvant therapy the randomisation was preplanned and blinded to the investigator the study was approved by individual institutional review boards at nine us institutions and conducted according to the declaration of helsinki the trial was registered at clinicaltrials gov as nct eligibilitykey protocol eligibility requirements included biopsy proven diagnosis of pdac radiographic staging consistent with locally advanced unresectable disease as defined nccn guidelines v2 clinical stage confirmed by diagnostic laparoscopy radiographically measurable disease per response evaluation criteria in solid tumors recist v11 eastern cooperative oncology group ecog performance status of or adequate haematological renal and hepatic function no prior therapy for pdac and no concomitant cancer diagnosis within the past yearsstudy schemaeligible patients were randomised to arm a or arm b to receive a total of six treatment cycles weeks of therapy figure patients in arm a received pamrevlumab mgkg by intravenous infusion on days and of each day cycle with an additional dose given on day in the first cycle patients in both arms a and b received gemcitabine mgm2 by intravenous infusion on days and of each day treatment cycle nab paclitaxel mgm2 by intravenous infusion on days and of each day cycle doses for gemcitabine and nab paclitaxel were modified for haematological and non haematological toxicity as per standard of care soc15 patients remained on therapy for six treatment cycles weeks unless they had disease progression an intolerable adverse event ae or toxicity withdrew consent or were withdrawn at the investigators discretion all patients were followed for drug toxicity until days after the last drug dose patients undergoing surgery were followed for days following hospital discharge for surgical complications ctgf levels were obtained prior to treatment from all patients plasma samples for pamrevlumab level determination were obtained from all patients receiving this drug after all protocol specified therapy was completed patients were followed for disease progression survival and additional oncological therapy postoperative complications including day readmissions and day mortality were notedresponse assessmentpatients were evaluated for response by the following measures carbohydrate antigen ca measured at baseline first day of each cycle and end of treatment eot recist v11 read based on full body ct imaging high resolution dual phase fine cut ct imaging at baseline and every weeks thereafter fluorodeoxyglucose fdg positron emission tomography pet imaging and nccn v2 resectability criteria at baseline and eotpicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accessfigure patient flow and surgery outcomes in arm a four of the eligible subjects had their surgeries cancelled 1portal vein thrombosis 3medical issues precluding surgery in arm a four eligible subjects underwent surgery but resection was not achieved 3metastatic disease discovered 1extensive sma encasement in arm b one eligible subject underwent surgery but resection was not achieved 1extensive vascular encasement sma superior mesenteric arterysurgical assessmentsubjects who finished six cycles of combination chemotherapy were evaluated for eligibility for surgical exploration per protocol pp defined criteria given that patients included in the trial were determined to be initially unresectable by radiographic imaging and nccn criteria objective criteria were developed to standardise attempts at surgical resectionpatients were deemed eligible for surgery if one or more of the following criteria were met reduction in plasma ca level by ¥ at eot compared with baseline reduction in fdg pet maximum standardised uptake value suvmax by ¥ at eot compared with baseline radiological tumour response per recist of partial response pr or complete response cr at eot or met the definition of resectable or borderline resectable per nccn guidelines subjects were classified as ineligible for surgical exploration if any of the following occurred development of distant metastases or local progression on ct scan tumour anatomy precluding vascular reconstruction unreconstructible local complications preventing surgery eg portal vein pvsplenic vein thrombosis pancreatitis or decline in performance status to a karnofsky score ¤ or picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668absolute contraindication to surgery from comorbidity eg recovery from myocardial infarction or uncontrolled diabetes the final decision regarding whether resection was to be performed was made by the treating surgeonendpointssafety endpoints included serious adverse events sae during neoadjuvant therapy and surgical complications postresection the efficacy endpoints included surgical eligibility r0 resection r0r1 resection median os progression free survival pfs and year survival rate all patients were followed and data analysis was stratified by pp population and intention to treat itt cohortstatistical considerationsthe comparison between selected clinical characteristics toxicity profiles and eligibility for surgical exploration or completed surgical resection was performed using the ϲ test exact cis for the point estimates as well as the treatment difference were obtained from the sas proc freq procedure with the exact option the two treatment arms were compared using the cochran mantel haentzel test controlling for baseline factors tnm stage ecog ca pet suvmax 0copen accesssuperior mesenteric artery sma involvement coeliac abutment and so on as prespecified in the protocol all cause mortality was used in determining os which was analysed by the kaplan meier method survival status was updated within month before the data cut off date data from patients who were alive at the cut off date were censored for survival analysis all statistical tests were performed at the significance level of α005 using two sided testsresultspatient characteristics and dispositionthirty seven patients were randomised to study treatment to arm a pamrevlumabgemcitabinenab paclitaxel and to arm b gemcitabinenab paclitaxel alone patient characteristics at baseline are summarised in table all patients enrolled were unresectable by nccn criteria patients had tumour arterial involvement sma encasement ° coeliac abutment table patient characteristicsbaseline demographics years years ¥ years median male femaleage group sex bmi kgm2 mean sd median min maxecog grade grade tnm stage t3 n0 m0 t3 n1 m0 t4 n0 m0 t4 n1 m0 t4 nx m0location of the tumour in the pancreas non resectability per nccn criterion head body tail median tumour size mm ° sma encasement any coeliac abutment inferior vena cava invasion or encasement unreconstructible smvportal occlusion aortic invasion and encasementarm agnppn24 arm bgnpn13 totaln37 to to to · ok as isnot mutually exclusivebmi body mass index ecog eastern cooperative oncology group g gemcitabine n number of subjects nccn national comprehensive cancer network np nab paclitaxel p pamrevlumab pv portal vein sma superior mesenteric artery smv superior mesenteric veinpicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accesswithout gastroduodenal artery involvement or aortic involvement inferior vena cava invasion and unreconstructible pvsuperior mesenteric vein smv occlusion a higher percentage of patients with sma encasement ° were randomised to arm a vs arm b patient disposition is summarised in figure twenty four patients in arm a received gemcitabinenab paclitaxel and pamrevlumab patients completed six treatment cycles six patients discontinued treatment early due to progressive disease three patients aes two patients or physician decision one patient thirteen patients in arm b received gemcitabinenab paclitaxel patients completed six treatment cycles six patients discontinued treatment early due to progressive disease two patients aes two patients or patientphysician decision two patientssafetysaes are summarised in table forty one per cent of patients had a treatment emergent sae arm a arm b no individual toxicity category occurred with frequency except systemic infection patients there was no demonstrable increase in any toxicity with the addition of pamrevlumab to gemcitabinenab paclitaxel chemotherapytable summary of treatment emergent serious adverse eventssystem organ classpreferred term ascites nausea pancreatitis vomiting device occlusion drug withdrawal syndrome feverno of patients with any treatment emergent saeblood and lymphatic disorders haemolytic uremic syndrome lymphadenopathycardiac disorders cardiac failure supraventricular tachycardiagastrointestinal disorders general disorders and administrative site conditions hepatobiliary disorders infections sepsis cellulitis urinary tract infectioninjury poisoning and procedural complications respiratory thoracic and mediastinal disorders skin and subcutaneous disorders cholangitis hyperbilirubinaemia craniocerebral injury pneumonitis pulmonary embolism rasharm an24n arm bn13n overalln37n · ok as ispicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accessresponse to therapyin arm a had ¥ ca decline at eot response by recist pr ¥ decline in pet suvmax and were radiographically downstaged by nccn criteria during the treatment period the median ca decline was patients were non secretors seven out of patients had best objective recist response crpr some patients had exceptional responses defined as normalisation or ¥ decline of ca patients or normalisation pet suvmax in in arm b had ¥ ca decline at eot response by recist pr ¥ decline in pet suvmax and were radiographically downstaged by nccn criteria four out of patients had best objective recist response cr pr in arm b of patients had an exceptional ca response and had an exceptional pet response as defined by either ¥ normalized ca response normalized suv max andorradiographic downstaging post therapy completion surgical evaluationoverall of the total study patients were eligible for surgical exploration using protocol defined criteria arm a arm b p00019 resection was completed in of the patients arm a arm b p01193 details of the nine resected patients are shown in table in arm a of the patients were eligible for surgical exploration in the itt population and of the patients were eligible in the pp population patients who completed six cycles of treatment in arm a out of eligible patients ultimately underwent surgical exploration for resection five operations were cancelled four due to drug toxicitymedical comorbidity sepsis gallbladder perforation declined performance status and fever and one patient declined eight out of patients in arm a were resected r0 r1 the remaining four patients who were explored were not resected due to progression or unresectable disease intraoperatively in arm b of the patients were eligible for surgical exploration in the itt population and were eligible in the pp population of the two subjects found to be surgically eligible only one was resected as the other patient had local progressionpredictors of resectionhigh ca response ¥ decline andor normalisation was contributive to surgical eligibility vs p03 normalisation versus non normalisation of pet suvmax was predictive of surgical eligibility vs p0013 and successful resection vs p0002 combining these two criteria was highly predictive for surgical eligibility vs p0003 and completed vs p0002 resection all nine successful resections were identified by one or both of these criteria table summary of resected patientssitesubject idtreatmentarmresponse to treatmentnccnbaselinenccnend of treatmentresection statusaaaaaaaab unresectablecoeliacunresectablesma smvunresectablecoeliacunresectablecoeliacunresectablesmvunresectablesmaunresectablesma smv coeliacunresectablesmaunresectablecoeliacunresectablecoeliacunresectablesma smvunresectablecoeliacborderline resectableunresectablesmvunresectablesmaunresectablecoeliacunresectablesmaunresectablecoeliacr0r1r0r0r1r1r1r0r0protocol defined criteria ca decrease fdg pet suvmax decrease ¥ recist v11 response pr or cr nccn resectable or borderline resectable criteriaca carbohydrate antigen cr complete response fdg fluorodeoxyglucose nccn national comprehensive cancer network pet positron emission tomography pr partial response recist response evaluation criteria in solid tumors sma superior mesenteric artery smv superior mesenteric vein suvmax maximum standardised uptake valuepicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0cconversely radiographic features of response did not correlate with operative potential neither recist response nor radiographic downstaging per nccn criteria statistically correlated with completed resectionsurgical complicationspostoperative complications were summarised according to the clavien dindo classification posthoc analysis ischaemic gastritis and ulceration and right lower lung lobe collapse were reported for one patient in arm a grade ii there was one episode of clinically significant pancreatic leak in each arm grade iiia no reoperations and no day or day surgical mortality were noted one patient in arm b had a delayed gastric perforation following a distal pancreatectomy with coeliac axis resection likely due to thermal injury and was treated non operatively grade iiib no wound complications or superficial site infections were noted in either group four out of patients and out of patients in arm a and b respectively were readmitted within days and the difference between treatment arms was not clinically or statistically significantsurvivalas of the data cut off date of evaluable patients were known to be alive with a median length of follow up at approximately months pfs was months ci to and months ci to in arm a and arm b respectively one year survival and median os were and months ci open accessto in arm a and and months ci nr in arm b the median os for all patients who were eligible for surgical exploration arm a arm b vs ineligible arm a arm b was months ci nr vs months ci to p00766 the median os for resected arm a arm b vs non resected patients arm a arm b was not reached ci nr vs months ci to p00141 figure discussionthe treatment of lapc with neoadjuvant therapy remains challenging and there is no established soc several high volume centres have reported their single centre experiences with varying neoadjuvant chemotherapy and chemoradiation strategies8 the combination of more active regimens delivered over an extended period and surgeons comfort with resecting and reconstructing major mesenteric vessels has led to an increase in resection rates a meta analysis of studies using folfirinox has demonstrated resection rates ranging from to in lapc17 one of the larger studies including patients with lapc reported a resection rate of that was more dependent on duration of therapy months than chemotherapy regimen folfirinox or gemcitabine based18 recently a single institution and single arm prospective study of neoadjuvant folfirinox and losartan with selective use of radiation in patients with lapc reported an r0 resection rate of figure overall survival resected vs non resected patientspicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen access however the lack of randomisation makes it difficult to determine what aspect of therapy was responsible for this effect and only of resected patients needed any type of vascular reconstruction perhaps suggesting more favourable outcome than usually seen in locally advanced disease these retrospective studies contain significant interpretative challenges including selection bias treatment variables including radiation and the use of different definitions of lapcthe anti ctgf mechanism of action with respect to gemcitabine based therapy a recent large scale prospective trial of patients with lapc treated with induction gemcitabine with or without erlotinib followed by radiation only patients were able to undergo resection10 furthermore the addition of erlotinib to gemcitabine did not improve any downstaging capacity and there was no difference in survival with the addition of radiation more recently the la pact trial examining the role of induction gemcitabine nab paclitaxel found that only out of patients with lapc were able to undergo surgery following neoadjuvant therapy with combination gemcitabine and nab paclitaxel for six cycles by investigators choice11 last although folfirinox has been the most studied induction combination chemotherapy regimen in this population recent randomised data from european patients who received neoadjuvant folfirinox versus gemcitabinenab paclitaxel prior to resection20 showed no clear difference with respect to r0r1 to resection rate vs p0135 or os vs months p0268given for pamrevlumab its use in the neoadjuvant setting has the potential to impact tumour regression and modulate the desmoplastic niche and possibly affect tumour margins allowing for improved resection rates previous studies have looked at the ability of gemcitabinenab paclitaxel to reduce cancer associated fibroblasts resulting in a softening of tumours by endoscopic ultrasound elastography21 this stromal depletion also translated into a decrease of suv uptake on pet22 in the study reported herein we combined gemcitabine and nab paclitaxel with pamrevlumab to explore its effect in terms of therapeutic response the impact on eligibility for surgical exploration and improved resection rates in locally advanced patientsthe protocol specified therapeutic response criteria ca pet suvmax recist and nccn criteria were used as criteria to determine eligibility for surgical exploration in lapc this is a novel approach specific to the protocol and allows participating patients to be explored for resection when otherwise they may not qualify by current treatment standards nccn criteria for example by nccn conversion alone ie converted from unresectable to borderline resectable only of patients in arm a would have been eligible for surgical exploration however by protocol criteria of patients in arm a were eligible for surgical exploration a higher percentage of patients were eligible for surgical exploration by the above criteria in arm a vs arm b vs respectivelyoverall the rate of successful resections in the pamrevlumab treated group was higher than in the control group but this did not reach statistical significance most probably due to small sample size of the nine subjects that were successfully resected in this trial only one was converted by nccn criteria to borderline resectable prior to surgical exploration despite this phenomenon the data support the hypothesis that pamrevlumab a human monoclonal antibody with anti ctgf mechanism of action could alter tumour characteristics allowing resection in otherwise unresectable patients this hypothesis needs to be confirmed and patients should be stratified by coeliac andor sma involvementthe most common predictive factors for eligibility for surgical exploration and resection were ca decline and pet suv max response which are indicators of tumour response to treatment the combination of these two factors proved to be a highly sensitive objective readout for prediction of potential surgical success both the ability of ca response and the inability of radiographic response recist and nccn criteria of resect ability to predict surgical outcome has been observed by others3 and these observations deserve further examination in subsequent clinical trials in the mpact study both ca and pet response correlated to improved survival in metastatic patients treated with gemcitabine and nab paclitaxel23 recent surgical series of patients with borderline resectable and lapc have also corroborated their impact in the localised setting25 correlation of clinical response with plasma levels of endogenous ctgf and pamrevlumab exposure as shown in the prior study by picozzi et al16 may provide added prognostic and predictive insightwith regard to safety no major incremental toxicity in any category was noted with the addition of pamrevlumab to gemcitabinenab paclitaxel in addition a higher number of patients were able to complete six cycles of the three drug combination including pamrevlumab when compared with gemcitabinenab paclitaxel alone pamrevlumab is well tolerated and considered safe compared with the soc drugs for patients with pdac these observations represent a very favourable attribute when considering potential neoadjuvant chemotherapy in a patient population with the frequency of medical problems typically seen in lapc in addition there were no signals of increased surgical morbidity or wound healing problems with ctgf blockade by pamrevlumab in fact there were only two clinically significant pancreatic leaks one in each arm which is comparable to national outcome data from high volume pancreatic surgery centres similarly readmissions following resection were comparable between arms and reflected the complexity of this challenging patient populationfinally while survival data are not yet mature both patients who were eligible for surgery and those that picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0cwere ultimately resected had longer pfs and os highlighting the importance of surgical resection of the tumour therefore more investigation into newer agents targeting lapc and increased consideration of candidacy for surgery in those patients who do not progress on therapy or suffer toxicity should be of utmost importance to improve outcomes in this diseasein this is the first prospective randomised multicentre trial examining the role of neoadjuvant therapy in lapc with prespecified criteria for surgical exploration the use of pamrevlumab in combination with gemcitabine and nab paclitaxel showed a potential to enhance tumour response and increase resection rates further evaluation of this drug combination in the neoadjuvant treatment setting for lapc is warranted and a larger phase iii trial with resection and survival endpoints is ongoingcontributors fibrogen inc was the study sponsor that designed the study in consultation with the principal investigator vp and surgical co investigator fgr all authors except those of the sponsor contributed patients to the study fibrogen was responsible for data collection and analysis all authors reviewed the manuscript and signed off on its accuracyfunding the study was funded by fibrogen inc san francisco cadisclaimer the corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors an exclusive licence or non exclusive for government employees on a worldwide basis to the bmj publishing group ltd and its licensees to permit this article if accepted to be published in esmo open editions and any other bmjpgl products to exploit all subsidiary rights as set out in our licencecompeting interests mc mz sp ek and ec are employees of fibrogen and hold stock andor stock options in fibrogenpatient consent for publication not requiredprovenance and peer review not commissioned externally peer revieweddata availability statement data are available on reasonable request all data relevant to the study are included in the article or uploaded as supplementary information data will be available as presented in this manuscriptopen 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 org licenses by nc orcid idewa a0carrier http orcid org references american cancer society cancer facts figures available httpswww cancer org content dam cancer org research cancer facts and statistics annual cancer facts and figures cancer facts and figures pdf rahib l smith bd aizenberg r et a0al projecting cancer incidence and deaths to the unexpected burden of thyroid liver and pancreas cancers in the united states cancer r | Colon_Cancer |
" inflammatory bowel disease ibd is the collective term for chronic immunemediated diseases ofunknown multifactorial etiology arising from the interplay between genetic and environmental factors andincluding two main disease manifestations ulcerative colitis uc and crohns disease in the last few decadesnaturally occurring alkaloids have gained interest because of their substantial antiinflammatory effects in severalanimal models of disease studies on mouse models of ibd have demonstrated the antiinflammatory action of themain tobacco alkaloid nicotine in addition anatabine a minor tobacco alkaloid also present in peppers tomatoand eggplant presents antiinflammatory properties in vivo and in vitro in this study we aimed to evaluate theantiinflammatory properties of nicotine and anatabine in a dextran sulfate sodium dss mouse model of ucresults oral administration of anatabine but not nicotine reduced the clinical symptoms of dssinduced colitisthe result of gene expression analysis suggested that anatabine had a restorative effect on global dssinducedgene expression profiles while nicotine only had limited effects accordingly map findings revealed that anatabinereduced the colonic abundance of dssassociated cytokines and increased il10 abundances our results support the amelioration of inflammatory effects by anatabine in the dss mouse modelof uc and suggest that anatabine constitutes a promising therapeutic agent for ibd treatmentkeywords plantderived alkaloid mouse model nicotine colitis correspondence pedroantonioruizcastropmicomblainephillipspmicom juliahoengpmicom pedro a ruiz castro ulrike kogel giuseppe lo sasso blaine w phillips andalain sewer contributed equally to this work1philip morris international rd philip morris products sa quai jeanrenaud neuch¢tel switzerland2philip morris international research laboratories pte ltd science parkroad the kendall science park ii singapore singapore 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 0cruiz castro of inflammation page of crohns disease cd and ulcerative colitis uc the mainclinical phenotypes of inflammatory bowel diseases ibdare chronic relapsing inflammatory disorders that affectthe gastrointestinal tract ibd are thought to result froman inappropriate and continuing inflammatory responseto commensal microbes in a genetically susceptible hostenvironmental triggers such as increased hygiene druguse stress smoking and diet influence the onset of thedisease over the past decades naturally occurring alkaloids from plant or medicinal herb sources have sparkedconsiderable interest because of their significant antiinflammatory and antioxidant properties []alkaloidsa class of natural bioactive compoundsderived from amino acids that contain one or moreheterocyclic nitrogen atomsare produced by a widerange of living anisms such as bacteria fungi plantsand animals in plants alkaloids are produced assecondary metabolites in response to environmentalbiotic or abiotic interactions and they confer protectionthrough a range of insecticidal antimicrobial and pharmacological attributes the antiinflammatory activities ofplantderived alkaloids have been documented in severalanimal models of disease including respiratory distress spinal cord injury hepatic fibrosis cancer and ibd [ ] the protective effects of alkaloids havebeen attributed to amelioration of inflammatory responsesand colonic oxidative stress [ ] promotion of epithelial barrier function and positive regulation of gutmicrobiota pyridine alkaloids present in tobacco nicotiana tabacum have been the subject of intensive research nicotinethe major alkaloid in tobacco accounts for approximately of the total alkaloid content of tobacco while thestructurally related nornicotine and anatabine are themost abundant minor pyridine alkaloids accounting for to of total alkaloids other pyridine alkaloids intobacco such as anabasine anabaseine and cotinine arepresent in smaller amounts nicotine and all minortobacco alkaloids have been shown to be pharmacologically active upon binding to several nicotinic acetylcholinereceptors nachrs tobacco nachr agonists suchas nicotine anatabine anabasine anabaseine and cotininedisplay protective effects in animal models of several inflammatory conditions including sepsis parkinsonsdisease alzheimers disease and ibd several in vitro and in vivo studies have shown a clearnicotinedependent positive effect on inflammatory processes [ ] in a previous study oral nicotine administration reduced the severity of dssinduced colitis andreduced colonic tnfα synthesis while subcutaneous injection or minipump infusion had no effect highlightingthe crucial role of administration route for the protectiveeffects of nicotine in dss colitis nicotine has alsobeen shown to attenuate the severity of dss colitis andexpression of il6 in cd4t cells a recent studysuggested that nicotine ameliorates dssinduced inflammation through inhibition of signaltransducer andactivator oftranscription stat3 in gutinfiltratedlymphocytes and intestinal epithelial cells recentlynicotine was shown to cause a decrease in leukocyte recruitment disease activity index dai and histologicalscore in dss colitis and block tnfmediated expressionof mucosal vascular addresin cell adhesion molecule1 inendothelial cells these authors concluded that nicotinesuppressesinflammation through downregulation ofadhesion molecules in the gut nonetheless clinicalstudies on the efficacy and tolerability of nicotine haveshown thattherapeutic application of nicotine fortreatment of uc is limited because of frequent adverseeffects and nicotine inconsistent efficacy in maintainingremission in uc patients [ ]anatabine is found in plants of the solanacea familywhich includes tobacco peppers tomato and eggplant little is known about the biological properties of anatabinealthough several studies have suggested that this alkaloid is apotential candidate compound for antiinflammatory drugdevelopment [ ] anatabine was marketed in the us asa dietary supplement under the name anatabloc in aninternetbased survey approximately of all users ratedthe effect of anatabine supplementation as good or excellentfor joint pain stiffness and functionality anatabine hasbeen shown to inhibit lipopolysaccharide lpsinducedproinflammatory gene expression as well as nfκb andstat3 phosphorylation in human neuroblastoma shsy5y hek293 human microglia and human bloodmononuclear cells as well as in the brain and spleen ofmouse models of autoimmune encephalomyelitis andalzheimers disease in shsy5y cells anatabine alsoreduced the expression of betasecretase the rate limitingenzyme for amyloid peptide production which is a majorhallmark of alzheimers diseasethrough inhibition of nfκb activation in this study we aimed to assess the antiinflammatoryeffects of the tobacco alkaloids nicotine and anatabine inthe established dss mouse model of uc our resultsshow that oral administration of anatabine but notnicotine ameliorates the clinical manifestations of dsstreatment in mice the results of gene expression analysisindicated that anatabine had a partial restorative effect onglobal dssinduced gene expression profiles while nicotineonly had minimal effects moreover multianalyte profilingmap showed that anatabine but not nicotine suppressesthe production of il6 il1α tnfα granulocytecolonystimulating factor gcsf and keratinocyte chemoattractant kc while increasing il10 expression in the colon ofdsstreated mice for an overview of the study conceptand analytical procedures see online resource 0cruiz castro of inflammation page of resultsanatabine has a protective effect in the dss mousemodel of colitisto study the antiinflammatory properties of nicotineand anatabine c57bl6 mice were provided with nicotine or anatabine in drinking water for a total of dayscolitis was induced by oral administration of dssin drinking water ad libitum during days fig 1adsstreated mice developed colitis as evident from thebody weight loss fig 1b increased colon weightlengthratio fig 1c increased dai fig 1d increased stooloccult blood score fig 1e increased intestinal bleedingscore fig 1fincreased diarrhea score fig 1g andincreased colon inflammation score fig 1h in micefig clinical parameters of dsstreated mice administered nicotine or anatabine a mice were orally administered nicotine or anatabine or mgkg for a total of days colitis was induced by oral administration of dss in drinking water ad libitum during days b bodyweight changes in mice during the colitis induction and recovery phases body weight changes were calculated as percentage relative to thestarting day of dss treatment day c colon weightlength ratio are represented as mgcm of colon d dai was calculated according to weightloss colon weightlength ratio and intestinal bleeding e stool occult blood score f intestinal bleeding score g diarrhea score h coloninflammatory status data are shown as mean ± sem p p nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkg 0cruiz castro of inflammation page of not subjected to dss treatment nicotine and anatabine administration had no significant effect on these parametershowever mice treated concomitantly with anatabine anddss showed a decrease in colitis severity in particular inmice that received concomitant anatabine and dss treatment anatabine improved body weight recovery at mgkg p fig 1b caused a decrease in global dai relative to that in dsstreated mice at daily dose of p and mgkg p fig 1c and reduced the stooloccult blood score at mgkg p fig 1e interestingly nicotine but not anatabine improved the intestinalbleeding score relative to that in dssadministered mice at mgkg p fig 1f the diarrhea score fig 1gand the colon inflammation score fig 1h were notaffected by nicotine or anatabine no variation in waterconsumption was registered across the different experimental groups online resource anatabine reduces dssinduced gene expression changesin the distal colon on a global levelto complement these pathological findings we analyzed the colon transcriptome dsselicited lesions inmost mouse inbred strains including c57bl6 micehave been shown to be more pronounced in the distalcolon [ ] therefore we focused our study on thatportion of the large intestine principal componentanalysis clearly captured the effect of dss treatmenton the first principal component explainedvariance fig 2a while nicotine treatment did notproduce a pronounced effect on the first principalcomponent in the dsstreatment context anatabinetreatmentin combination with dss produced aresponse more similar to that observed in the watercontrols no dssindicating that anatabine had anameliorating effect on dssinduced gene expressionchanges the results of our differential gene expressionanalysis suggested substantial perturbation of the colontranscriptomefdr fig 2bc application of dss in drinking watercaused significant changes in nearly genes incolon tissues addition of nicotine to dsscontainingdrinking water slightly increased the number of differentially expressed genes in contrast addition of anatabine decreased the number of differentially expressedgenes upon dss treatment by approximately fig 2b and c this alleviating effect of anatabinetreatment on dssinduced gene expression profileswas also apparent in the global gene expression heatmap which showed a global reduction of expressionfold changes upon anatabine treatment fig 2d and eof note in the absence of dss anatabine treatmentdid not result in differential expression of genes andnicotine treatment alone had minor effects fdr pvalue online resource upon dsstreatmentanatabine reduces dssinduced inflammatory geneexpression in the distal colonto gain a more mechanistic understanding of the effectsof nicotine and anatabine treatment we further investigated gene expression changes at the level of functionalgene sets and a ucrelevant causal network model gsaofthe reactome database showed changes acrossmultiple functional categories fig 3b the effects onfunctional categories in dsstreated mice administerednicotine appeared rather similar to those in mice treatedwith dss alone whereas administration of anatabineresulted in a general repression of dssmediated effectswe also evaluated the interaction terms betweennicotineanatabine and dss treatment to more directlyfocus on the modulating effect of anatabine and nicotinetreatment on dss effects fig 3a online resource the following six biological categories showed significant interaction terms upon anatabine and dss treatmentsupporting asignificant suppression of dssinduced effects in thepresence of anatabine immune system extracellularmatrix anization protein localization metabolism hemostasis and signal transduction fig 3bof these we further explored immune system extracellular matrix anization and signal transductionas the most relevant categories in ibdana 20dss fdr allfiginteractiontermsp the hierarchical anization of the reactome database allowed us to investigate the underlying functionalchanges in more detail the immune system categoryincludes adaptive immune system cytokine signaling in immune system and innate immune systemin particular within these immune categories cytokinesignaling egincluding il6 family signaling andtolllike receptor cascades were modulated withsignificant3conline resource online resource within thereactome signal transduction category signaling byreceptor tyrosine kinasesby rhogtpases signaling by transforming growth factortgfbeta family members mapk family signalingcascades integrin signaling signaling by erythropoietin and signaling by gpcr were found to be significantly impacted online resource online resource within the extracellular matrix anization category almost all subcategories were perturbed includingdegradation of the extracellular matrix elastic fiberformation and extracellular matrix proteoglycansonline resource online resource signalingdegradation ofthe extracellular matrix includesbiological processes such as activation of matrix metalloproteinases mmps and collagen degradationto further follow up on the effects of nicotine andanatabine on inflammatory signaling we evaluatedthe perturbation of the ucrelevant tlril1rtnfr 0cruiz castro of inflammation page of fig transcriptomics results of colon biopsy samples a principal component pc analysis the plot displays all samples in the reduced pc1pc2plane covering of the total data variance it allows us to examine the relationships between the various groups and treatmentsremarkably pc1 captured the pure dss effect black arrow while pc2 captured the pure exposure effects of anatabine and nicotine blue andred arrows respectively b volcano plots for individual gene differential expressions the horizontal axis represents the log2 differential expressionfold changes and the vertical axis represents its statistical significance as log10 fdr c number of differentially expressed genes for theselected pairwise comparisons the bar plot displays the number of genes with positive or negative fold changes with corresponding statisticallysignificant fdr ¤ note that the lower fdr values observed for the ana dss comparisons do not necessarily prefigure a reduction ofbiological effects because the statistics underlying the fdrp values also depend on the gene expression variance within the experimentalgroups d highlevel heatmap of the statistically significant differentially expressed genes for the selected pairwise comparisons in order toprovide a comprehensible display of the large foldchange matrix we first normalized its rows to their maximum absolute values andthen reordered them by hierarchical clustering complete linkage method on the basis of their euclidean distances e scatter plot from thecomparisons between the pure and exposuremodified dss effects by using the differential gene expression results the horizontal axis of thescatter plots represents the fold changes of the pure dss effect water dss vs water pairwise comparison whereas the vertical axis containsthe corresponding values in case of anatabine or nicotine exposure anatabinenicotine dss vs water pairwise comparisons in an idealcase included as a reference [first plot] the bestfitted straight line coincides with the diagonal indicated in green and its slope is equal to the transcriptomics effects of anatabine or nicotine exposure were quantified by the slope of the bestfitted straight lines indicated on each plotnic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkgnetwork model by using an established networkenrichment approach we inferred the activationstates of the network nodes on the basis of the observedgene expression changes and calculated the overall network perturbation amplitude for each group comparisonfig 3c dss treatment had a strong activating effect onthis signaling network including activation of several coresignaling nodes such as il1rassociated kinase irak1irak4 and myeloid differentiation primary response myd88 online resource heatmap leadingnodes of note the network perturbation induced bydss treatment was reduced only in the presence ofanatabine with this the results of network analysisfurther supported the ameliorating effect of anatabineon dssmediated inflammation whereas no similareffect was apparent upon nicotine treatment 0cruiz castro of inflammation page of fig biological interpretation of the transcriptomics results a schematic representation of the effects of anatabinenicotine exposure as amodification of the pure dss effect the measured combined anatabinenicotine dss effect captured by the pairwise comparisons anatabinenicotine dss vs water can be viewed as the sum of the pure effects of the two factors dss treatment and anatabinenicotine exposuretogether with the adjusting twofactor interaction term anatabinenicotinedss anatabinenicotine exposure is synergistic with dss treatmentif the interaction term has the same sign as the pure dss effect eg both are positive as in the schema in contrast the relationship isantagonistic if the interaction term has an opposite sign to the pure dss effect b gsa results for the top reactome pathway categories theheatmap displays the gsa scores normalized rowwise to their maximum absolute values as well as their competitive q1 statistical significancethe fdrs were calculated only among the top reactome pathway categories which are sufficiently distinct in their gene content to assumeindependence of their enrichment results c hierarchical representation of the gsa results for all pathways contained in the top reactomeimmune system category and for the twofactor ana 20dss interaction the color map corresponds to the normalized gsa scores containedin the interval [ ] whereas their statistical significance competitive q1 p values ¤ is indicated by black circles around the nodes theactual labels of the nodes ie the reactome pathway names can be found in online resource the treelike structure of the reactomepathway collection enables topdown investigation within the relevant pathway categories by identifying increasingly specific biologicalprocesses ie involving fewer and fewer genes along the longest paths connecting the statistically significant pathways d npa results for thetlril1rtnfr network model the bar plot displays the npa values for the selected contrasts and their statistical significance is indicated bythe three colored asterisks note that by definition the positive npa values depend on the square of the input genelevel fold changes andtherefore might amplify the differences between the contrasts without preserving the additive relationships among them nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkgexpressionvaluesfrom theto validate the observations obtained by microarraytranscriptomics we quantified the expression of six leadingedge genesgenes of the gene set with the highestimmunedifferentialsystem extracellular matrix anization and signaltransduction reactome categories using realtime quantitative pcr qpcr selected genes included il33 signaltransduction and immune system categoriesil6signal transduction and immune system categoriesmmp13 extracellular matrix anization categoryserpine1 signal transduction and extracellular matrixanization categories thbs1 thrombospondin signal transduction and extracellular matrix anizationcategories and timp1 tissue inhibitor of metalloproteinase extracellular matrix anization and immunesystem categories qpcr results showed a clear decreasein dssinduced expression of il33 il6 mmp13 serpine1thbs1 and timp1 expression in the presence of anatabineat mgkg thereby confirming the findings obtainedfrom the microarray data fig 0cruiz castro of inflammation page of fig validation of microarray transcriptomic results using qpcr a boxandwhisker plots for the distribution of the qpcr expression levels δcqof the selected genes the boxes represent the quartiles while the whiskers extend to the most extreme data point which is no more than times the interquartile range from the box the horizontal brackets indicate the statistical significance of the corresponding comparisons mean pvalue smaller than respectively b scatter plots comparing the mouse colon differential expression values obtained bymicroarray horizontal axis and qpcr vertical axis the following similarity metrics were indicated beta is the slope of the best interceptfreelinear fit between microarray and qpcr values r2 is the coefficient of determination measuring the goodnessoffit and pval is the pvalueassociated to the null hypothesis beta nic nicotine mgkg ana anatabine mgkganatabine decreases dssinduced proinflammatorycytokine production and promotes il10 expressionnext we sought to evaluate the impact of nicotine andanatabine on the expression of colonic inflammatorycytokines by mapin line with the previous dataanatabine but not nicotine significantly reduced theabundance of dssassociated inflammatory cytokines including il6 kc tnfα il1α and gcsf whereas itincreased the levels of the antiinflammatory cytokineil10 at a daily dose of mgkg fig interestinglyanatabine also increased the abundance of il21 andshowed a clear tendency towards increasing colonic il 0cruiz castro of inflammation page of fig map results for colon biopsies statistical assessment of the differences observed in the abundance of selected cytokines between thestudy experimental groups and water control fold changes in the treatment groups nicotine and anatabine at and mgkg dss relative towater control are illustrated with colors ranging from blue decrease to red increase statistically significant differences on the basis of raw pvalues no adjustment has been made for multiple testing grey cells highlight missing estimates on the observed differences due to lackof cytokine quantifications nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkg1 levels fig taken together the map data confirmthe antiinflammatory effects of anatabine in dssinduced colitisdiscussionour study shows that oral administration of anatabinebut not nicotine reduces the clinical manifestations ofdssinduced colitis in a mouse model in line with thesefindings anatabine demonstrated a global downregulatory effect on dssinduced gene expression changes inthe colon whereas the effects of nicotine were morelimited in particular the results of gene expression profiling further supported the reduction of inflammatorysignaling processes upon anatabine treatment includingsuppression of il6 signaling as shown by gsa findingsand tlr signaling as shown by the results of networkperturbation analysis and gsa map also showed asignificant decrease in the abundance of il6 kc tnfαil1α and gcsf and an increase in the expression of il and the antiinflammatory cytokine il10several studies have reported on the antiinflammatoryeffects of nicotine on the dss mouse model of uc subcutaneous administration of nicotine was shown toameliorate tissue injury in dss colitis and il6 expression in cd4t cells via α7nachrs nicotine wasalso shown to decrease the activation of stat3 throughinduction of mir124 in gutinfiltrated lymphocytes andintestinal epithelial cells strikingly alsharari 0cruiz castro of inflammation page of observed that oral but not subcutaneous injection ofnicotine ameliorated intestinalinflammation and colonic tnfα expression in dsstreated mice in spiteof the reported beneficial effects our results do not support a protective effect of orally administered nicotineon dss colitis of note we found that nicotine significantly reduced dssassociated intestinal bleeding whichwas the only clinical parameter affected by this tobaccoalkaloid the vasoconstrictor effects of nicotine are wellstablished in the gut mucosa nicotine decreasesblood flow and cigarette smoking decreases rectalblood flow to normal levels in patients with uc however how changes in blood flow affect the pathophysiology of uc is still unclear the possible therapeuticuse of nicotine to induce remission in uc patients hasbeen evaluated in five clinical studies [] and twometaanalyses [ ] these studies have demonstrated avariable efficacy of nicotine therapy in induction of remission with several studies showing no effect [ ]moreover a high frequency of adverse events increasedthe withdrawal rate in the nicotine group in some studiesthus limiting the therapeutic benefit of nicotine nucleotidebindingto the best of our knowledge the present study isthe first to assess the impact of anatabine on experimental colitis gene expression analysis of distal colonbiopsy specimens highlighted the antiinflammatoryproperties of anatabine in multiple functional categories including immune responses signal transduction and extracellular matrix anization which inturn encompassed several tlr and cytokine signalingpathways genes contributing to the downregulation oftlr cascades included tlr2 tlr4 and tlr6 and anumber of downstream signaling factors shared byseveral tlrs including myd88 ripk2 irak3 irak4and nod1oligomerizationdomaincontaining protein as well as the nuclearfactors elk1 fos cyclic adenosine monophosphatecamp response elementbinding protein creb1activating transcription factor atf1 and atf2 seeonline resource of the respective gene lists for thecytokine signaling pathways the contributing genes included il6 and il6 receptor α ifnγ il4 cxcl10il22 receptor α2 il2 receptor α tnf receptor superfamily member 1a il17α and il17 receptor α jak1jak2 stat3 and stat4 members of the nfκbsignaling pathway also contributed to the overall reducincluding nfκb p65tion in inflammatory cascadesp105 and p100 subunits iκbα and the nfκb activating protein tab3 in agreement with the results oftranscriptional analysis map findings showed a significant decrease in dssassociated il6 kc tnfα il1αand gcsf protein expression and an increase in il10expression in the presence of anatabine strikinglywhile tlr downstream factors modulated by anatabineare shared by most tlrs the majority of cytokineassociated signaling molecules are specific for eachpathway the seemingly pleiotropic regulatory effects ofanatabine suggest that this alkaloid reduces inflammation by inhibiting the expression of several factors involved in different proinflammatory signaling cascadesprevious studies using in vitro and in vivo diseasemodels have demonstrated the antiinflammatory effectsof anatabine [ ] paris showed that this pyridine alkaloid reduced the plasma levels of il1 il6and il17a as well as the expression of il1 infγ andtnfα in the spleen of experimental autoimmune encephalomyelitis mice these authors also showedthat anatabine suppressed stat3 and nfκb phosphorylation in the spleen and brain of these mice anatabine also prevented lps and tnfαinduced nfκb andstat3 phosphorylation in shsy5y and hek cellshuman microglia and human blood mononuclear cells additionally anatabine prevented lpsinduced il1 expression in human whole blood as well as il1il6 and tnfα production in the plasma kidney andspleen in the lps mouse model phosphorylatedstat3 tnfα and il6 were also downregulated in thepresence of anatabine in a transgenic mouse model ofalzheimers disease our results on the effects of anatabine in the dssmouse model of uc are also in line with the findings ofa substantial number of studies demonstrating the protective effects of natural alkaloids in several animalmodels of colitis [ ] intraperitoneal administrationof the minor tobacco alkaloid and nicotinic receptoragonist anabaseine was shown to reduce tissue damagemyeloperoxidase activity and colonic tnfα expressionin a tnbs mouse model of colitis these mice alsoshowed reduced nfκb activation in lamina propriamononuclear cells while mice administered a nicotinicreceptor antagonist presented worse colitis symptomsthan those treated with tnbs alone the algaealkaloid caulerpin reduces dss colitis by suppressingnfκb activation and subsequently inhibiting the colonicproduction of tnfα ifnγ il6 ifnγ and il17 oral administration of berberine also ameliorates dssinduced colitis and downregulates the expression oftnfα ifnγ kc and il17 in colonic macrophages the plantderived alkaloid nmethylcytisine andthe tea alkaloid theophylline mitigate colitis by downregulating tnfα il1 and il6 expression in dss andacetic acid models of colitis respectively [ ] induction ofthe antiinflammatory cytokine il10 in thepresence of natural alkaloids has also been reportedthus indirubin ameliorates dssinduced colitis by suppressing the expression of colonic tnfα ifnγ and il2and upregulating il10 additionally indole alkaloids caulerpin and isatin have been shown to increase 0cruiz castro of inflammation page of the expression of il10 in dss and tnbs models of ibdrespectively [ ] of note our results show anincrease in the abundance of il21 and a tendencytowards increase in colonic il1 levels in the presenceof anatabine although il21 expression is increased inmany chronic inflammatory disorders genetic deficiencyof il21 is associated with ibd and inhibition of il21in the early phases of some inflammatory disorders exacerbates disease development suggesting the dual role ofil21 in the control of immune responses il21also promotes il22 expression in mucosal tcellsthrough a mechanism involving stat3 retinoidrelatedorphan receptor γt and aryl hydrocarbon receptorthereby helping protect immunodeficient mice from dsscolitis interestingly il21 has been recently shownto induce il1 production in dendritic cells through astat3dependent but nfκbindependent mechanismthereby suggesting a link between il21 and il1 mounting evidence suggests that alkaloidsin particular isoquinoline alkaloids present in traditional medicineherbsexertthroughregulation of nfκb and stat3 signaling pathways forexample sanguinarine and cavidine suppress the expression of nfκb p65 subunit thereby reducing colonictnfα and i | Colon_Cancer |
mgat5 knockout ko in hek293 cells induces metabolic changes resulting in increased intracellular udpglcnac increasedglycolysis enhanced spare respiratory capacity and higher citrate ï¬ux from the mitochondria mgat5 ko cells express constitutively high mica mainly regulated onthe transcriptional level through opening of the chromatin at the mica promoter mica expression in mgat5 ko cells is dependent on citrate turnover and histoneacetylation blocking citrate ï¬ux inhibits mica expression in numerous cancer cell lines and we propose that this is a central metabolic regulation of mica andimmune surveillanceintroductionnatural killer nk and cd8 t cells monitor autologouscells for markers of tumorigenesis and stress these immunecells express the nkg2d receptor that recognizes nkg2dligands nkg2dls upregulated on the surface of transformedcells nkg2dl expression is in many ways a doubleedged sword upregulation of nkg2dls on cancer cellsenhance nk cell ltration and promote cancer cytotoxicity conversely numerous cancer cells maintain chronicnkg2dl expression and evade immune elimination bydownmodulating and impairing nkg2d receptor signalingcancer cells that block nkg2dl surface expression toevade immune recognition and clearance can be treated withstressinducers such as histone deacetylase inhibitors hdacisheatshock or shortchain fatty acids scfas that upregulatenkg2dls to date studies have primarily focused ondelineating transient nkg2dl induction whereas not much isknown about regulation of their constitutive expressionmetabolic reprogramming is a central hallmark of cancercancer cells use aerobic glycolysis that was initially believedto be a result of dysfunctional mitochondria howeverlater advances have shown that cancer cells often use aerobicglycolysis alongside mitochondrial oxidative phosphorylationoxphos mitochondria are not merely the powerhouseof the cell but also provide metabolites for anabolic pathwaysnecessary for cell growth citrate can be exported from thetricarboxylic acid tca cycle for biosynthetic purposes inthe cytosol citrate is cleaved by atp citrate lyase acly togenerate acetylcoa and oxaloacetate oaa citrateis an inhibitor of glycolysis thus to maintain high aerobicglycolysis cancer cells require low cytoplasmic citrate moreover conversion of citrate by acly is a critical regulatorof gene transcription by producing acetylcoa for histoneacetylation several of these cancerassociated metabolicfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionproperties are shared with other highly proliferating cells suchas activated t cellsexpression of nkg2dls is associated with hyperproliferation and thus with highly active metabolism two studies havelinked nkg2dl expression to active glycolysis whereasone study reports that inhibition of glycolysis increased basalnkg2dl expression in breast cancer cell lines thesestudies emphasize a link to proliferative cell metabolism andsuggest that the role of glycolysis in nkg2dl regulation iscontextspeciï¬cnkg2dls fall into two groups the ul16 binding protein ulbp16 and the mhc class i chainrelated proteins aand b mica and micb surface expression of each nkg2dlis regulated individually and at all levels of protein biogenesis we have previously shown that surface expression ofspeciï¬c mica alleles depends on nglycosylation nacetylglucosaminyltransferase v mgat5 is an oncoproteincatalyzing the formation of 16branched nglycans thatpromote surface retention of glycoproteins but it is notknown if mgat5 regulates surface expression of mica growthfactor receptors are examples of mgat5 substrates and mgat5overexpression is associated with growth adhesion invasionand metastasis of cancer inhibition of mgat5 reducestumor growth enhances the antitumor responses by cd4 tcells and macrophages and promotes th1 diï¬erentiation in this study we examine the metabolic regulation of thenkg2dl mica we discover that mica was increased aftermgat5 knockout ko in a metabolically dependent way anduse this as a model to investigate the regulatory mechanismsof constitutive mica expression we ï¬nd that glycolysis andmitochondrial export of citrate promotes constitutive micatranscription in mgat5 ko cells a regulation that was alsoshown in several micaexpressing cancer cells in particularincreased mica transcription was associated with alteredchromatin accessibility of the mica promoter our ï¬ndingssuggest that citrate drives a metabolic stress that modulateschromatin accessibility to facilitate basal mica transcription andthereby regulate immune surveillancematerials and methodsanimalsfemale nmri mice to 10weeks old taconic lille skensveddenmark were used and all studies were performed inaccordance with the danish act on animal experimentationwhich implements directive 201063eu on the protection ofanimals in scientiï¬c research the studies were approved by theanimal experimentation inspectorate ministry of environmentand food denmark license no healthmonitoring was carried out in accordance with federation forlaboratory animal science associations guidelinesreagents pharmacological inhibitorsand dna constructspharmacologicalfrom sigmaaldrich werecompoundsnacetyldglucosamine glcnac a3286 pugnac a7229carbonylcyanide2dg d61345aminoimidazole4carboxamide2deoxydglucosetriï¬uoromethoxyphenylhydrazone fccp c2920 uk5099pz0160 bis25phenylacetamido134thiadiazol2ylethylsulï¬de bptes sml0601 potassium hydroxycitrate tribasicmonohydrate hc sodium dihydrogencitrate sodium acetate s5636 oxaloacetic acid oaa o41266mercaptopurine monohydrate 6mp azaserinea4142ribonucleotideaicar a9978 nacetylcysteine nac a9165 sodiumpropionate p1880 sodium butyrate b5887 dmso d2438pbs d8537 etomoxir sodium salt was purchased fromcayman chemicals ann arbor mi united states bristol bms303141 wasunited kingdom the gfpmycmica and mica vectors containingthe coding sequences of mica or mica alleledownstream of a generic leader a gfp cassette and a myctag were provided by dr m wills university of cambridgecambridge united kingdom pgl3basic pgl3bluciferase vector was purchased from promega promegamadison wi united states e1751 micaï¬reï¬y luciferasepromoter vectors and sv40renilla luciferase promoter vectorwere provided by prof c ocallaghan university of oxfordoxford united kingdom from tocris biosciencepuriï¬cation of peripheral bloodlymphocyteshuman peripheral blood mononuclear cells pbmcs wereisolated by histopaque1077 sigmaaldrich st louis mounited states separation from buï¬y coats obtainedfrom healthy blood donors the capital region blood bankcopenhagen university hospital copenhagen denmark toobtain peripheral blood lymphocytes pbls pbmcs weredepleted from monocytes by incubation with dynabeadsinvitrogen carlsbad ca united states as previouslydescribed pbls were activated in rpmi1640 withoutglucose gibco gaithersburg md united states supplemented with dialyzed fetal bovine serumfbs f9665 mm penicillinstreptomycin p4333 mmlglutamine g7513 mm sodium pyruvate s8636 and either mm dglucose g8769 or mm dgalactose g6404all purchased from sigmaaldrich pbls were activated withcd3cd28 beads invitrogen 11132d and 20uml hil2peprotech rocky hill nj united states for dayson day pbls were treated with ngml fr901228 nationalcancer institute bethesda md united states for hline pc3 and the keratinocytederived cellcell line cultivation and proliferationhuman embryonic kidneyderived hek293 cells the prostatecancer celllinehacat were purchased from american type culture collectionatcc manassas va united states nkg2d reporter cellct312 and the 2b4 parental cellline were kindly providedby chiwen chang trowsdale lab cambridge universitylines mdamb231 and mcf7 werethe breast cancer cellprovided by dr jos moreira departmentfor veterinaryfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressiondisease university of copenhagen denmark and henrikleï¬ers the state hospital copenhagen denmark respectivelythe cervical cancer cellline hela was provided by jesperjurlander the state hospital copenhagen denmark themelanoma cells skmel28 fm55m1 fm78 and fm86 andthe human colon adenocarcinoma cell lines ht29 and sw480were provided by dr per thor straten herlev universityhospital denmark hek293 mdamb231 and mcf7 cellswere cultured in dmem with glutamax gibco hela hacat pc3 fm55m1 fm78 fm86 skmel28 andsw480 were cultured in rpmi1640 sigmaaldrich r5886and ht29 were cultured in mccoys 5a medium sigmaaldrich m8403 media were supplemented with fbs and mm penicillinstreptomycin mm lglutamine was addedto rpmi1640 and mccoys 5a for longterm cell culture inglucosegalactose cells were cultured in dmem medium withoutglucose gibco supplemented with dialyzedfbs mm penicillinstreptomycin mm sodium pyruvate and mm glucosegalactose all cells were kept at culture conditions¦c and co2 and were passaged every daysfor proliferation assay wt and mgat5 ko cells wereseeded in or cellswell for each experimentcells were counted in triplicate wells after and h usingthe biorad tc20 automated cell counter biorad herculesca united statesgene editingmgat5 ko cells were generated by zinc ï¬nger nucleasetargeting in hek293 cells and subsequent cloning and selectionwas performed as described previously hek293cells were transfected with mrna sigmaaldrich or µgof endotoxin free plasmid dna using nucleofection on anamaxa nucleofector lonza copenhagen denmark mgat5ko clones were selected by loss of reactivity with lphaand clones were conï¬rmed to have mgat5 mutations usingpcr and sequencinglentiviralmediated gene transfer was performed with anmgat5 encoding vector constructed by inserting the mgat5sequence generated as a bluntend pcr product from a vectorfrom hw university of copenhagen copenhagen denmarkinto an entry vector system using the pentr directionaltopo cloning kit invitrogen k243520k350020 followingmanufacturers protocol topo clonal reaction entry vectorswere transformed into macht1 chemically competent e coliusing heatshock and soc medium followed by selectionpcr inserts were conï¬rmed by sequencing at euroï¬ns mwgoperons luxembourg colonies were ampliï¬ed and plasmidswere puriï¬ed with nucleobond xtra midi kit machereynagelduren germany mgat5 sequences were insertedinto plx302 lentiviral destination vector with lr clonase iienzyme mix invitrogen after proteinase k treatmentconstructs were transformed into dh5α using heatshock andsoc medium selected clones were ampliï¬ed and dna waspuriï¬ed using nucleobond xtra midi kit destination vectorswere checked for insertion using bsrgi digestion at ¦cmgat5coding lentiviral ps were packaged in hek293tcells transfected with a mix of µg pspax2 vector packagingvector µg pcmvvsvg envelope vector µg plx302vector carrying mgat5 and µl cacl2 to a ï¬nal volume of µl the dna mixture was complexed with µl 2x hbsunder constant air ï¬ow and the transfection mix was addeddropwise to hek293t cells in antibioticfree mediumcell culture medium was harvested days after transfection andviral p preparations were prepared by centrifugation at g for min lentiviral ps were added to cells andincubated for h cells were cultivated in puromycin µgmlselection medium for weeks functional mgat5 expressionwas validated by lpha bindingtransient transfectiontransient transfections were performed as described previouslyusing amaxa nucleofector device lonza dna wasintroduced to cells in µl nucleofector solution vlonza vca1003 and pulsed using the nucleofector programq001 for gfpmyctagged mica and micaconstructs cells were transfected with µg dna and analyzedthe next day transfection with shrnas or luciferase promoterconstructs was carried out by calciumphosphate transfectionbrieï¬y dnarna were prepared in µl cacl2 25mand adjusted to a ï¬nal volume of µl dna mixture wascomplexed with µl 2x hbs hepes nacl na2hpo4and added dropwise to cells scrambled sirnacontrol siidh1 and siidh2 ontarget plus smart poolswere purchased from ge healthcare dharmacon lafayetteco united statesfunctional assaysfor nkg2d downmodulation pbls were isolated as describedabove followed by depletion of cd4 cells using cd4 antibodyebioscience san diego ca united states anddynabeads mouse panigg invitrogen cd4depletedpbls were cultured in rpmi1640 sigmaaldrich r5886supplemented with human serum sigmaaldrich h3667 mm penicillinstreptomycin mm lglutamine and ngmlhil15 peprotech for days to enrich for nkcd8t cells nkg2d downmodulation assay was performed aspreviously described nkg2d ligands on eï¬ector cellshek293 wt or mgat5 ko cells were incubated with blockingnkg2dfc rd systems minneapolis mn united states1299nk or control igg1fc rd systems 110hg µgmlfor min at ¦c eï¬ector cells and target cells nkcd8t cells were mixed at indicated eï¬ectortarget ratios and spundown min g to allow conjugate formation after h cocultivation nkcd8 t cells were analyzed for nkg2d surfaceexpression by ï¬ow cytometry using accuri c6 ï¬ow cytometerbd bioscience franklin lakes nj united statesfor the reporter cell assay the nkg2dreporter cell line2b4ct312 and the parental control 2b4 cell line target cells were mixed with eï¬ector cells wt or mgat5 ko cellsthat were either blocked with nkg2dfc or control igg1fc asdescribed above eï¬ector and target cells were cocultivated atdiï¬erent et ratios for h gfp expression of target cellswas assessed with accuri c6 ï¬ow cytometer for in vivo assaytarget cells were labeled with vybrant did celllabeling solutionfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioninvitrogen v22887 according to manufacturers protocol andinjected intraperitoneally together with wt or mgat5 kocells in a ratio of each mice were usedper group target cells were harvested after approximately hwith peritoneal lavage and nkg2d activation of didpositivereporter cells were assessed as gfp expression with accuric6 ï¬ow cytometerwas assessed by accurate mass and retention time amrt plusfragment identiï¬cation at two collision energies and evdetailed acquisition methodology has been described previously udpglcnacudpgalnac detected peak screened byexpected calculated mass could be of either compound as thesetwo sugars could not be separated chromatographically hencehas been reported as a putative metabolite pending conï¬rmationlactate and dntp measurementsconcentrations of llactate was measured enzymatically withrandox colorimetric assay according to manufacturers protocolrandox crumlin united kingdom lc2389 reaction andanalysis was performed on an advia chemistry systemsiemens munich germanydntp levels were determined in cells harvested withtrypsinization and pelleted by centrifugation for g for min followed by resuspension of cell pellets in methanolfrozen in liquid nitrogen and boiled at ¦c for min sampleswere evaporated until dryness in a speedvac and whole cell levelsof dttp datp dctp and dgtp were determined using the dnapolymerase assay previously described lchrms metabolite proï¬lingto determine intracellular metabolite levels cell pellets from cells were resuspended in µl of cold methanolafter min sonication samples were prepared by svortex followed by min equilibration at room temperatureafter centrifugation at g for min at ¦c µl supernatants were collected transferred to ultrafreemccentrifugal ï¬lter devices merck millipore ltd cork irelandand centrifuged at g for min at ¦c from this µlwas transferred to lc vials and µl of each sample was pooledto a mixed qc samplelchrms was performed on a nity ii ultrahigh performance liquid chromatography uhplc systemcoupled to a ifunnel quadrupoletime of ï¬ight qtofmass spectrometer equipped with a dual ajs electrosprayionization source agilent technologies santa clara caunited states polar metabolites were separated on a sequantzichilic merck darmstadt germany column mm mm µm p size coupled to a guardcolumn mm mm µm p size and an inlineï¬lter mobile phases consisted of formic acid in water withsolvent a and formic acid in acetonitrile with solvent bthe elution gradient used was as follows isocratic step at bfor min b to b in min and maintained at bfor min then decreasing to b at min and maintainedfor min then returned to initial conditions over min and thecolumn was equilibrated at initial conditions for min the ï¬owrate was mlmin injection volume was µl and the columnoven was maintained at ¦c the acquisition was obtainedwith a mass range of mz for where full scan highresolution data is acquired at three alternating collision energies ev ev and ev positive and negative raw lchrmsï¬les were independently processed with an inhouse developedpcdl library for polar metabolites using proï¬nder version b06agilent technologies identiï¬cation of reported compoundsextracellular flux analysisthe seahorse xfe96 extracellular ï¬ux analyzeragilenttechnologies was used to measure ocr and ecar on hek293cells cells were seeded at the density cellswell ¼ hbefore the experiment one hour prior to assay run cells wererinsed and switched to xf media agilent technologies with mm sodium pyruvate and mm glucose or galactose andincubated at ¦c co2free incubator for the mitochondrialstress tests ocr was measured under basal conditions andduring sequential injection of µm oligomycin sigmaaldrich µm fccp sigmaaldrich c2920 and µmrotenone rot sigmaaldrich r8875 µm antimycina aa sigmaaldrich a8674 reported basal respiration iscalculated from the third measuring point with ocr after rotand aa subtracted atpcoupled respiration display ocr afteroligomycin subtracted from the third measuring point andmaximal respiration is ocr after fccp with ocr after rotand aa subtractedfor measuring the eï¬ect of hc ocr was assessed h after aninjection of mm hc13c6glucose tracing experiment cells were incubated for h in dmem medium withoutglucose supplemented with fbs mm sodium pyruvateand mm uniformly labeled [u13c]glucose cambridgeisotope laboratories tewksbury ma united states clm incubation medium samples were collected and cleared bycentrifugation g for min cells were washed and detachedsterically intracellular metabolites were extracted in ethanoland centrifuged at g for min ¦c to separatethe soluble extract supernatant from the insoluble componentspellet cell extracts and medium samples were lyophilizedand reconstituted in water for subsequent biochemical analysesextract samples were adjusted to ph with hcl and evaporatedto dryness under nitrogen ï¬ow analytes were extracted into ananic phase ethanolbenzene followed by derivatizationwith dmf86 mtbstfa with a modiï¬ed procedure from standards containing unlabeled metabolites of interest andcell extracts were separated and analyzed in a gas chromatographagilent technologies 7820a chromatograph jw gc columnhp5ms parts no 19091s433 coupled to a mass spectrometeragilent technologies 5977e the isotopic enrichment of themetabolites of interest was corrected for natural abundance of 13cusing the unlabeled standards and calculated according to data are presented as labeling of m x where m is the massof the unlabeled molecule and x is the number of labeled catomsin a given metabolite frontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionwestern blottingproteins were extracted using ripa buï¬er thermo scientiï¬cwaltham ma united states and proteinasephosphataseinhibitor cocktail thermo scientiï¬c for minon ice lysates were sonicated times for s and clearedby centrifugation at rpm for min at ¦c proteinextracts were denatured at ¦c for min in nupage samplebuï¬er and dtt sigmaaldrich proteins were resolvedusing sdspage gels invitrogen and transferred tonitrocellulose membranes invitrogen ib301001 using the iblotdevice invitrogen for total protein stain membranes werewashed in ddh2o and stained with revert protein stainsolution licor biosciences lincoln ne united states according to manufacturers protocol membranes wereblocked in tbst blocking buï¬er licor biosciences probed with primary antibodies in tbs w tween and bsa overnight on a shaker at ¦c and washed intbs tween secondary antibody was from licorlicor biosciences and signals were visualizedby the odyssey fc imaging system licor biosciencesoglcnacylation was detected with rl2 oglcnacylationantibody abcam cambridge united kingdom ab2739 atpcitrate lyase acly was detected with rabbit acly antibodycell signaling and acly phosphorylation with rabbitphosphoacly ser455 antibody cell signaling flow cytometryadherent cells were detached in pbs w mm edta invitrogen or by pipetting cell surface staining was done aspreviously described and cells were analyzed on accuric6 ï¬ow cytometer bd bioscience antibodies used for thisstudy were mica rd systems fab1300a ulbp256 rdsystems fab1298p nkg2d rd systems fab139a ulbp1rd systems fab1380p ulpb3 rd systems fab1517aulbp4 rd systems fab6285a micab bd bioscience icam1 leinco technologies c170 mouse igg1antimyctag merck millipore micb rd systemsmab1599 or igg2b isotype control rd systems mab004detected with secondary antimouse igg biolegend san diegoca united states binding of ï¬uorescently labeledaf647lpha invitrogen l32457 and fitcepha vectorlaboratories burlingame ca united states fl1121 was usedto measure surface levels of complex nglycans all isotypecontrols were purchased from bd biosciencefor staining with mitochondrial probes neutral lipid stainsor 2nbdg uptake cells seeded the day prior toexperiment were washed once in pbs and incubated for minat ¦c and co2 in warm growth medium containing nmtetramethylrhodamine methyl ester perchlorate tmrm sigmaaldrich t5428 nm mitotracker green fm invitrogenm7514 or for h in growth medium with µm 2nbdginvitrogen n13195 bodipy invitrogen d3922 wasdiluted in warm serumfree medium in a dilution andshaken vigorously to solubilize the lipids immediately beforeloading into the cells for min cells were washed twice inpbs fbs and detached sterically prior to analysisthe soluble nkg2dfc receptor 1299nk rd systemsand igg1fc 110hg rd systems were labeled with zenonalexa fluor against human igg1 z25408 invitrogen priorto staining of melanoma cellsin forwardsidescatter plotsdata were acquired with an accuri c6 instrument usingaccuri c6 software and analyzed in flowlogic v721 inivaitechnologies mentone vic australia by gating on viablecellsfollowed bysingle cell gating by areaheightscatter plots fscafsch geometric mean ï¬uorescent intensity mfi values aredisplayed in ï¬gures as mfi or with corresponding isotype controlsubtracted as 01mfifscsscreal time pcr analysistotal rna was extracted by phase separation in trizolchlorophorm and puriï¬ed on directzol spincolumns zymoresearch irvine ca united states according to manufacturersprotocol cdna was generated using superscript cdnasynthesis kit invitrogen under standard pcr conditionsfollowing primersequences were used for quantitativertpcr with brilliant sybr green qpcr master mixkit mica mica_f tggcagacattccatgtttctgmica_r ctcgtcccaactgggtgttg ulbp2 ulbp2_f cagagcaactgcgtgacatt ulbp2_r ggccacaaccttgtcattctidh1 idh1_f ctatgatggtgacgtgcagtcg idh1_r cctctgcttctactgtcttgccidh2 idh2_f agatggcagtggtgtcaaggagidh2_r ctggatggcatactggaagcag glut1 glut1_fctgctcatcaaccgcaac glut1_r cttcttctcccgcatcatct glut2 glut2_f tacattgcggacttctgtgg glut2_r agactttcctttggtttctgg glut3glut3_f cagcgagacccagagatg glut3_r ttggaaagagccgattgtag glut4 glut4_f tgggcttcttcatcttcacc glut4_r gtgctgggtttcacctcctrplp0_fcctcgtggaagtgacatcgt rplp0_r cattcccccggatatgaggc realtime qpcr was performed on biorad cfx96 realtime thermal cycler c1000 touch and alltranscripts were normalized to housekeeping rplp0 transcripthousekeepingand rplp0asgeneluciferase reporter assaycells were transiently transfected using calciumphosphatetransfection as described above with ï¬reï¬y luciferase promotervectors µg and an sv40promoter driven renilla luciferasevector µg cells were harvested and snap frozen hpost transfection pellets were lysed in dualglo luciferasereagent promega e2920 and ï¬reï¬y luciferase activity wasanalyzed by luminometer microbeta ii perkinelmer walthamma united states renilla luciferase activity was recorded bythe instrument after subsequent addition of volume dualglo stop glo promega e2920 to correct for transfectioneï¬ciency ï¬reï¬y luciferase signals were normalized to sv40renilla luciferase signals of corresponding sampleatacseqatacseq was performed as described previously foreach cellline cells were harvested from separatefrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioncultures and used to prepare tagmented chromatin replicatesof wt and replicates of mgat5 ko cell lines samplestotal quality of pcrampliï¬ed sequencing libraries was assessedusing a tapestation instrument with high sensitivity dnascreentapes agilent libraries were sequenced as paired endreads on a single lane of an illumina hiseq4000 ï¬ow cellresulting reads were aligned to the grch37hg19 referencegenome using rsubread and alignments were ï¬ltered toremove low quality duplicate and mitochondrial reads peakswere called using macs2 on merged reads from allsamples and diï¬erential peak accessibility between cell lines wasdetermined using edger with a threshold false discoveryrate of transcription factor binding motifs enriched indiï¬erentially accessible peaks were identiï¬ed using homer h3k4me3 chipseq data were downloaded from encode1 andare available under accession encff756ehfquantiï¬cation and statistical analysisresults are presented as mean ± sem diï¬erences were analyzedfor statistical signiï¬cance using prism or graphpad softwarela jolla ca united states statistical analysis was performed asstated in ï¬gure legends using unpaired ttest in 1a 1c 1e 3ef3h 5c 7a 7ef paired ttest in 4fg 7d multiple ttest in 1b1d 3d 4ab one sample ttest in 2ac 3c 4c 4e 7g twowayanova in 3a 5df 5hi 6a 6e 7hi or oneway anova in5g level of statistical signiï¬cance was determined by p p and p p resultsmgat5 knockout increases nkg2dlexpression and activates nkg2d in vitroand in vivoregulation of constitutive mica expression remains largelyunknown surface expression of certain mica alleles dependson nlinked glycosylation we questioned whetherthe cancerassociated glycosyltransferase mgat5 is required formica expression to assess the role of mgat5 in regulationof nkg2dl surface expression mgat5 ko clones weregenerated in hek293 cells remarkably mgat5 ko resultedin a permanently increased surface expression of the nkg2dlsmica micb and ulbp256 compared with parental wildtypewt cells figure 1a to conï¬rm mgat5 ko we measuredbinding of leukoagglutinin from p vulgaris lpha that bindsspeciï¬cally to mgat5modiï¬ed nglycans as expected lphabinding was reduced whereas binding of erythroagglutininfrom p vulgaris epha that interacts with mgat3modiï¬ednglycans was unaï¬ected thus verifying functional knockoutof mgat5 figure 1a modiï¬cation of mgat5 expressiontherefore associated with substantial changes in constitutiveexpression of several nkg2dlsto verify the functionality of mgat5 koinduced nkg2dlswe tested nkg2d activation in a reporter cell line expressing1httpswwwencodeprojecthuman nkg2d coupled to dap10cd3ζ signaling and nuclearfactor of activated t cells nfatcontrolled gfp ultimatelyexpressing gfp in response to nkg2d activation nkg2dgfp activation was higher after cocultivation with mgat5ko cells than with wt cells figure 1b corresponding to theincreased nkg2dl expression in mgat5 ko cells figure 1athe reporter cells without nkg2d supplementary figure s1aremained inactivated indicating that the activation was nkg2dmediated figure 1b moreover blocking nkg2dls withsoluble nkg2dfc receptor impaired the activation furthervalidating nkg2d speciï¬city supplementary figure s1bto test if mgat5 ko cells could activate nkg2d in vivowe adoptively injected nkg2d reporter cells together with wtor mgat5 ko cells into the peritoneum of nmri mice andmeasured gfp expression in reporter cells in line with ourin vitro data we observed a signiï¬cant increase in nkg2dgfpactivation by mgat5 ko cells compared with wt cells theresponse was nkg2dspeciï¬c since the control reporter cellswere unaï¬ected figure 1c these data verify that mgat5 koinduced nkg2dls maintain their functional integrity in vivonkg2d is downmodulated upon activation to furtherexamine the functionality of nkg2dl expression causedby mgat5 ko we assessed nkg2d downregulation afterreceptor activation nkg2d was further downregulated oncd4depleted peripheral blood lymphocytes pbls after cocultivation with mgat5 ko cells than with wt cells and thisdownregulation was abolished by blocking nkg2dls with asoluble nkg2dfc receptor figure 1d combined these dataindicate that ko of mgat5 upregulates mica and ulbp256resulting in nkg2d activation in vitro and in vivoto ensure that the mica upregulation was a result of mgat5ko we stably transfected mgat5 into wt and mgat5 kocells lpha binding was restored within days after transfectionconï¬rming expression of functional mgat5 interestingly ittook multiple passages for mica expression to decrease to wtlevels figure 1e and supplementary figure s1c suggestingthat mica is regulated in response to a longterm adaptation toaltered mgat5 expressionudpglcnac upregulates micaexpressionlongterm mgat5 deï¬ciency willlikely result in aberrantnglycosylation and an accumulation of the mgat5 donorsubstrate udpnacetylglucosamine udpglcnac to addressif mica was regulated by a change in nglycosylation inmgat5 ko cells we assessed the posttranslational regulationof mica by measuring surface expression of transgenicallyexpressed gfpmyctagged mica under a cytomegaloviruscmv promoter the mica alleles mica and micaare distinctly regulated posttranslationally and althoughmica was upregulated in mgat5 ko cells the regulationwas minor and unlikely to account for the profound changein endogenously expressed mica figures 1a 2a micatranscripts on the other hand were highly increased in mgat5ko cells figure 2b as well as ulbp2 mrna supplementaryfigure s2a suggesting that nkg2dls are transcriptionallyfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionfigure mgat5 knockout increases nkg2dl expression and activates nkg2d in vitro and in vivo a surface expression of nkg2d ligands and binding ofï¬uorescently labeled lpha mgat5 modiï¬cations or epha mgat3 modiï¬cations on hek293 wildtype wt and hek293 mgat5 knockout ko cells or isotypecontrol staining iso analyzed by ï¬ow cytometry data are presented as histograms representative of at least three independent experiments and in bar graphsshowing mean ï¬uorescence intensity mfi b in vitro nkg2d activation measured as gfp expression in nkg2d negative reporter cells control and nkg2dexpressing nkg2d reporter cells target cells cocultivated with wt or ko cells effector cells for h at indicated effectortarget et ratios c nkg2dactivation in vivo measured on reporter cells as in b after activation by wt or ko at a ratio in peritoneum of nmri mice for approximately h gfp expressionin didlabeled reporter cells signiï¬es nkg2d activation and is shown as gfp mfi values of cells from foursix mice per group d nkg2d downmodulation wasassessed on nkcd8 t cells target cells after cocultivation for h with wt or ko cells effector cells at indicated effectortarget ratios et nkg2dls on targetcells were blocked with nkg2dfc bl or unblocked with igg1fc un the graph depicts surface expression of nkg2d presented relative to surface nkg2dexpression on target cells alone e mica surface expression left and lphaepha surface binding right after lentiviral introduction of mgat5 into wt or kocells mfi values from antibody staining were corrected for isotype background staining 01mfi statistical analysis was performed by unpaired ttests in ace andmultiple ttest with fdr comparing wt and ko in bd p p p and p regulated in mgat5 ko cells notably we found that themgat5 substrate udpglcnac although indistinguishablefrom udpnacetylgalactosamine udpgalnac tended tobe higher in mgat5 | Colon_Cancer |
" camp responsive element binding protein creb5 is a transcriptional activator in eukaryotic cells that canregulate gene expression previously we found that creb5 was involved in the occurrence and development of colorectalcancer crc using bioinformatics analysis however the biological roles and underlying regulatory mechanism of creb5 incrc remain unclearmethods realtime pcr western blotting and immunohistochemistry were used to examine creb5 expression in vitroexperiments including migration assay woundhealing assay chicken chorioallantoic membrane assay and human umbilicalvein endothelial cells tube formation assay were used to investigate the effects of creb5 on crc cell migration and tumorangiogenesis ability additionally an orthotopic implantation assay was performed in nude mice to confirm the effects ofcreb5 in vivo furthermore gene set enrichment analysis was performed to explore the potential mechanism of creb5 incrcresults we found that creb5 expression was highly upregulated in crc creb5 overexpression was positively correlatedwith advanced who stages and tnm stages and shorter survival in crc patients moreover creb5 overexpressionpromoted while creb5 silencing reduced the invasiveness and metastatic capacity of crc cells both in vitro and in vivofurthermore creb5 directly interacted with the met promoter and activated the hepatocyte growth factormet signallingpathway importantly inhibition of met reduced the invasion and metastasis of creb5overexpressing crc cells suggestingthat creb5 promotes metastasis mainly through activation of met signalling our study demonstrates a crucial role for creb5 in crc metastasis by directly upregulating met expressioncreb5 may be both a potential prognostic marker and a therapeutic target to effectively overcome metastasis in crckeywords colorectal cancer creb5 invasion metastasis met correspondence llifimmucom liaowt2002gmailcom shuyang wang junfeng qiu and lei liu contributed equally to this work1department of pathology nanfang hospital southern medical universityguangzhou guangdong chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang of experimental clinical cancer research page of colorectal cancer crc is one of the most commoncancers ranking third in morbidity and tumorrelatedmortality among both men and women worldwidemoreover approximately of crc patients have metastases at the time of diagnosis metastasis is theleading cause of death among crc patients although systemic treatment of metastatic crc hasimproved the 5year survival rate is only andbecause ofthis poor prognosis understanding theunderlying mechanism of the metastatic process in crcis criticalfor both early detection of metastases andmore effective treatment the gene camp responsive element binding protein creb5 which is located on chromosome 7p151encodes a transcription activator in eukaryotic cells creb5 belongs to the atfcreb family the membersof which are characterized by a high affinity for campresponse elements cres the targets of the atfcreb family include transcriptional regulators including chromatinmodifying enzymes coactivators and corepressors genes involved in mitochondrial homeostasisand protein import and genes associated with proliferation and cell cycle entry metabolism proteases transporters and chaperones as an atfcreb familymember the creb5 protein contains several importantfunctional domainsincluding nterminal zinc fingerand cterminal bzip domains the latter of which includes a dna binding region and leucine zipper creb5 is a transcription factor that specifically binds tocre as a homodimer or a heterodimer with cjun orcrebp1 and functions as a credependenttransactivator creb5 is physiologically required for embryonic development in mice recent studies revealedthe roles of creb5 in the development and progressionof cancers examination of tcga pan cancer datasetsrevealed frequent creb5 amplification and overexpression in kidney cancers sarcomas lymphomas and lungadenocarcinomas as well as glioblastomas and gliomas experimentalinvestigations showed that creb5was upregulated in ovarian cancers hepatocellularcarcinoma and prostate cancer high creb5expression correlated with a poor prognosis in epithelialovarian cancer and hepatocellular carcinoma creb5 overexpression increased the proliferation of hepatocellular carcinoma moreover overexpression oramplification of creb5 promoted proliferation and mediated resistance to ar inhibition in metastatic castrationresistant prostate cancers in silico analysis showedthat the creb5 regulatory network was involved in crcmetastasis in addition qrtpcr assay revealed thatcreb5 mrna was upregulated in crc tissues and cells in vitro assays revealed that overexpression of creb5resulted in enhanced proliferation and migration andapoptosis inhibition in crc cells these findings suggest that creb5 may play an essential role in the progression of crc howeverthe specific function andmolecular mechanism of creb5 in crc metastasis remain largely unclearactivation of the hgfmet signalling pathway hasbeen reported to lead to the occurrence and metastasisof a variety of tumorsincluding crc breast cancerovarian cancer lung cancer and liver cancer as atyrosine kinase receptor met can be activated bydimerization multimerization and phosphorylation afterbinding to its ligand hepatocyte growth factor hgf activation of hgfmet can initiate downstreamsignalling pathways that drive malignant progression inmany types of tumors met is considered an essentialfactor for early invasion and metastasis of crc and canbe regarded as an important prognostic indicator in the present study we found that creb5 promotes crc invasion and metastasis by increasing metexpression to activate hgfmet signalling these results uncover a new molecular mechanism for cancermetastasis and suggest that creb5 may be a promisingtarget for crc treatmentmaterials and methodspatients and specimensa total of pathological specimens were obtainedfrom colon cancer patients between and atthe department of pathology nanfang hospital southern medical university the medical records of these patients provided information on sex age and thefollowing essential factors tumor pathological characteristics pathologic stage t stage dukes stage lymph nodemetastasis and distant metastasis ten pairs of fresh biopsies collected from crc patients and matched noncancerous mucosaltissue were obtained from theoperating room of nanfang hospital the fresh biopsieswere stored in liquid nitrogen before usein addition a tissue microarray no co802 containing colon cancer tissue specimens and adjacentnoncancerous tissue specimens was purchased fromailina biotechnology company approval for the use ofclinical materials for research purposes was obtainedfrom the southern medical university institutionalboard guangzhou china all samples were collectedand analysed with the prior written informed consent ofthe patientscell culturesthe human crc celllines sw480 ht29 hct15hct116 sw620 ls174t sw837 lovo dld1 andrko were purchased from the american type culturecollection sw620 ht29 and lovo cells were culturedin dmem medium gibco supplemented with 0cwang of experimental clinical cancer research page of foetal bovine serum fbs gibco sw480 hct116hct15 ls174t sw837 and dld1 cells were culturedin rpmi medium gibco with fbs gibcoplasmidscreb5 constructs were generated by cloning pcramplifiedfulllength human creb5 cdna into psinef2puro thefollowing primers were used for cloning including enzymesforward primer ² cgcgaattcatgatttatgaggaatccaa3² ecor i reverse primer ²ccggctagcttaaagaatcggattcaggt3² nhe i for deletion ofcreb5 short hairpin rna shrna sequences creb5²aacaagtcatccagcataa3² creb5shrna1shrna2 ²ggaatatctcgatgcataa3² were separately cloned into a gv248 vector psinef2puro and gv248were purchased from addgene incthe intensity of staining was graded according to thefollowing criteria no staining weak staining lightyellow moderate staining yellow brown and strong staining brown the staining index was calculated as the staining intensity score à the proportion ofpositive tumor cells using this method of assessmentwe evaluated the expression of creb5 in benign breastepithelium and malignant lesions by determining thestaining index with scores of and cutoff values for creb5 were selected on the basis of ameasure of heterogeneity with the logrank statisticaltest with respectto overall survival optimal cutoffvalues were identified a staining index ¥ was used todefine tumors with high creb5 expression and anindex ¤ was used to define tumors with low creb5expressionrna isolation reverse transcription rt and realtimepcrtotal rna samples from cultured cells and primarytumor tissues were extracted using trizol reagent invitrogen usa according to the manufacturers instruction realtime rtpcr was performed at least threetimes in triplicate using sybr green mix toyobojapan and the abi prism sequence detectionsystem applied biosystems usa the data were normalized to the geometric mean of the housekeeping genegapdh and calculated using the 2δδct method primerexpress was used to design the realtime pcr primersand primer sequences for amplification are shown insupplementary table s2luciferase reporter assaygenomic dna extracted from sw480 cells was used as atemplate to amplify met promoter fragments met promoter fragments were obtained by pcr and constructedinto pgl3basic plasmid using a fast ligation kit followingmanufacturers instructions sangon b620511 thesequences of the pcr primers are listed in supplementarytable s4 cells at confluence in a 24well plate weretransfected using lipofectamine fortyeight hoursafter transfection luciferase activity was measured usingthe dualluciferase reporter assay system promega corpmadison wi usa and normalized to renilla luciferasegene expression all the experiments were performed intriplicatewestern blotting analysiswe carried out western blotting as previously described using anticreb5 abcam ab168928 antimetcell signaling technology antipmetcellsignaling technology antiakt cell signalingtechnology antipaktcell signaling technology antierkcell signaling technology antiperk cell signaling technology and antisnail cell signaling technology antiantiαtubulin antibodybodies mouse monoclonalsigma was used as the internal controlimmunohistochemistryimmunohistochemistry ihc staining was performed aspreviously described using creb5 antibody abcamusa ab168928 the degree of ihc staining wasreviewed and scored independently by two observersbased on both the proportion of positively stained tumorcells and the intensity of staining [ ] the proportion of tumor cells was scored as follows no positivetumor cells positive tumor cells positive tumor cells and positive tumor cellschromatin immunoprecipitation chip assaychip assays were carried out as previously described precleared lysates were incubated with creb5 antibody abcam ab168928 or normal mouse immunoglobulin g cst as a negative control overnightat °c with rotation the human met promoter wasamplified by pcr all chip assays were performed threetimes and the sequences of the pcr primers are listedin supplementary table s3migration assaya boyden chamber with an 8μmpore filter membranewas used for the in vitro migration and invasion assaybriefly cells à in culture medium containing fbs were seeded in the upper chamber and culturemedium with fbs was added in the lower chamberas a chemoattractant the upper side of the filter wasfirst coated with matrigel bd biosciences sanjose ca usa after incubation for h cells on theupper side of the filter were removed with cotton swabscells that migrated to the lower surface of the filter werefixed in paraformaldehyde and stained with giemsa 0cwang of experimental clinical cancer research page of the migratory cells were counted random Ãfields per well three independent experiments wereperformed and the data are presented as the mean ±semwoundhealing assaycells were seeded in 6well plates and incubated underpermissive conditions until confluence after serumstarvation for h wounds were created in the confluent cells using a pipette tip wound healing within thescrape line was then observed and photographed at indicated time points each experiment was repeated at leastthree timeschicken chorioallantoic membrane assaya chicken chorioallantoic membrane cam assay wasperformed on the sixth day of development of fertilizedchicken eggs as previously described human umbilical vein endothelial cell tube formationassayfirst μl of matrigel was pipetted into each well of24well plates and polymerized for min at °c human umbilical vein endothelial cells huvecs à in μl of conditional medium were added to eachwell and incubated at °c in co2 for h imageswere obtained under a brightfield microscope and thecapillary tubes were quantified by the counting lengthorthotopic mouse metastatic model to 6weekold balbc athymic nude mice nunuwere obtained from the animal center of southernmedical university guangzhou china all mice werehoused in a sterile environment cells à permouse were orthotopically inoculated into the caecumof anaesthetized nude mice the mice were sacrificedwithin weeks after surgery individual ans were excised and metastases were observed by histological analysis tissues were then fixed with formaldehyde andparaffinembedded and then 5mm sections were cutand stained with haematoxylin and eosin he thenumbers of gross metastatic foci were determined usinga dissection microscope all the mice used in this studywere maintained under specific pathogenfree conditions and all animal experiments were conducted in accordance with standard procedures and approved by theinstitutional use committee for animal carestatistical analysisall statistical analyses were carried out using spss version pearson correlation analysis was used for expression correlation analysis the survival curves of crcpatients in low and highcreb5 expression groups wereanalysed by the kaplanmeier method and the logranktest was used to compare differences p was considered significantresultscreb5 is upregulated in crc and associated with a poorprognosisthe expression of creb5 was analysed in differenttypes of malignant tumors in the public database oncomine wwwconominecom revealing that creb5was upregulated in crc tissues in of crc datasetssupplementary fig s1a additionally a gene set enrichment analysis gsea plot showed significant enrichment of tumorrelated genes and crcrelated genesets in the highcreb5 expression group supplementary fig s1b realtime pcr and western blotting analyses showed that creb5 was differentially expressed incrc cell lines supplementary fig s1cd in additioncreb5 was significantly upregulated in ten crc tissuescompared with adjacent normal intestinal epithelial tissues fig 1a and b ihc showed that creb5 proteinwas weakly expressed in normal tissue but markedly increased in adenocarcinoma cells and was mainly localized in the nucleifig 1c kaplanmeier survivalanalysis showed that crc patients with higher creb5protein expression levels had a poorer prognosis fig1d in addition creb5 expression levels were significantly correlated with the t classification lymph nodemetastasis and distant metastasis p supplementary table s1 creb5 expression was substantiallyhigher in tumors from patients with distant metastasismoreover high creb5 expression was also positivelycorrelated with who stages p supplementarytable s1 these data suggested that creb5 expressionis significantly correlated with advanced stages of crccreb5 activates met signallinggseas of creb5regulated gene signatures revealed thathigher creb5 expression was positively correlated withenrichment of an met signalling pathway signaturegse17538 fig 2a to validate this result we establishedstable creb5overexpressing and creb5knockdowncrc cell lines fig 2b upregulation of creb5 significantly increased while knockdown of creb5 decreasedthe expression of total met at both translational andtranscriptional levels fig 2c and d in addition creb5overexpression markedly increased but creb5 downregulation significantly attenuated the expression of phosphorylated met perk pakt and snail fig 2c moreovermet expression was increased in a dosedependent manner at both translational and transcriptional levels by transiently transfecting sw480 cells with a creb5 expressionvector fig 2e 0cwang of experimental clinical cancer research page of fig creb5 is upregulated in crc and associated with a poor prognosis a and b realtime pcr and western blotting analysis of creb5 expression inpaired human colon cancer tissues and adjacent noncancerous tissues p quantity one software was used to quantify the protein expressionlevels c ihc representative images of creb5 expression in normal intestinal epithelium and crc tissues scale bar μm d the paraffin samples of crc patients were divided into a lowcreb5 expression group n and a highcreb5 expression group n based on ihc results thekaplanmeier method was used to analyse survival curves and the logrank test was used to compare differences p fig creb5 activates the met signalling pathway a gsea of gse17538 in met signalling pathways es p b stable overexpressionand interference cell lines were detected by western blotting and realtime pcr c the expression of met and downstream signalling moleculesin creb5knockdown or creb5overexpressing cells was observed by western blotting d creb5 had an effect on met by realtime pcr in theindicated cells e after transient transfection of different amounts of the creb5overexpression plasmid in sw480 cells the protein and mrnalevels of met were detected by western blotting and realtime pcr respectively p 0cwang of experimental clinical cancer research page of creb5 associates with the met promotergiven that creb5 is a transcriptional factor and upregulatesmet at the transcriptional level we performed a luciferasereporter assay to investigate whether creb5 can increasemet promoter activity a 27kb fragment of the fulllengthmet promoter region was subcloned into a luciferase vector met promoter activity wasincreased by cotransfection with a creb5 expression vector in sw480 cellsbut decreased in hct116 cells expressing creb5 shrna ina dosedependent manner compared with empty vectorsfig 3a to determine the effective regions of the met promoter that creb5 may affect we transfected met promotertruncations fig 3b into hct116 cells expressing eithercreb5 shrna or a scramble control sequence as shown infig 3c luciferase activity was increased in cells carrying thefulllength met promoter and truncations and bp upstream of the transcription start site but not incells carrying truncations to bp or to bp knockdown of creb5 expression bycotransfection of creb5 shrna significantly decreased metpromoter activity fig 3c furthermore we performed chromatin immunoprecipitation chip assays and identified thatthe to 223bp region of the met promoter whichcontains an ap1 motif was a creb5 protein binding sitefig 3d these data identify met as a direct transcriptionaltarget of creb5downregulation of creb5 represses invasiveness andreduces the metastatic potential of crc cellsnext we investigated the role of creb5 in invasivenessand metastasis in crc cells silencing of creb5 significantly compromised the migratory and invasive abilitiesof crc cells fig 4a and b supplementary fig s2a andb the tubule formation and chicken cam assays revealed that knockdown of creb5 strongly inhibited theformation of tubules by huvecs and inhibited angiogenesis in cams fig 4c and d supplementary figs2c orthotopic inoculation assay showed that knockdown of creb5 inhibited liver metastases fig 4eknockdown of creb5 also obviously extended the overall survivaltime of nude mice inoculated with thecrc cell lines fig 4f these results indicate that silencing creb5 inhibits the invasiveness and metastasis of crc cellsinhibition of met attenuates the invasion and metastasisof crc cells by creb5 in vivo and in vitroto determine the functional relationship between creb5and met in the invasion and metastasis of crc weknocked down met using two met shrnas or suppressed met activation using the met inhibitor crizotinib emd silencing or inhibition of met insignificantlysw480creb5or ht29creb5cellsfig creb5 regulates met and binds directly to the met promoter a the met promoter sequence was cloned into pgl3basic vector containing theluciferase reporter gene and then transfected into crc cells with the indicated treatments b schematic diagram of the full and truncated met promoterc the fulllength met promoter or its truncations were cloned into pgl3basic vector containing the luciferase reporter gene and then transfected intohct116 cells with creb5 shrna or empty vector d chip analysis of creb5 binding to the met promoter in sw480 cells p 0cwang of experimental clinical cancer research page of fig downregulation of creb5 inhibits the invasion and metastasis of crc cells in vivo and in vitro a and b woundhealing assay and transwellmigration assay were performed to evaluate the invasive and migratory abilities of crc cells with different treatments in vitro c huvec tubeformation after stimulation with the indicated conditioned medium d representative images of the cam assay histograms show the formationof secondary and tertiary blood vessels after stimulation with the indicated conditional medium scale bar mm e orthotopic transplantationwith the indicated hct116 cells in nude mice n in each group was performed and representative gross images of the livers and intestinesare shown the arrows indicated the tumors liver sections were stained with haematoxylin and eosin he scale bar μm f the kaplanmeiermethod was used to analyse survival curves in the specified treatment groups and the logrank test was used to compare differences p decreased the expression of phosphorylated met perkand pakt as well as snail supplementary fig s3a inaddition the invasive and migratory abilities of sw480creb5 or ht29creb5 cells were partially diminished byinhibition of met fig 5a and b supplementary fig s3band c moreover upregulation of creb5 expression enhanced the capacity of crc cells to induce tube formationand angiogenesis in cams in contrast angiogenesis ability was partially diminished by met inhibition fig 5cand d supplementary fig s3d orthotopic inoculationassay showed that creb5 significantly promoted liver metastases and decreased the overall survival of mice fig 5eand f conversely inhibition of met significantly attenuated the formation of metastatic foci by sw480creb5cells and extended the survival time of mice inoculatedwith sw480creb5 cells fig 5e and fcreb5 expression positively correlates with metexpression in crcto assess a potential link between creb5 and met expression in human crc we analysed tcga crc dataand identified a strong positive correlation between highexpression levels of creb5 and met p r fig 6a in addition analyses of fresh crc tissuesshowed that creb5 expression was positively correlatedwith met expression at both mrna p r and protein levels p r fig 6b and c 0cwang of experimental clinical cancer research page of fig overexpression of creb5 promotes the invasion and metastasis of crc cells but inhibition of met weakens these effects a and b theinvasive and migratory abilities of crc cells in vitro with different treatments were evaluated by woundhealing assay and transwell migrationassay c huvec tube formation after stimulation with the indicated conditional medium d representative images of the cam assay histogramsshow the formation of secondary and tertiary blood vessels after stimulation with the indicated conditional medium scale bar mm eorthotopic transplantation with the indicated sw480 cells in nude mice n in each group was conducted and representative gross images ofthe livers and intestines are shown the arrows indicate the tumors liver sections were stained by he scale bar μm f the kaplanmeiermethod was used to analyse the survival curves of different treatment groups and the logrank test was used to compare differences p p p furthermore ihc revealed that creb5 expression waspositively correlated with met fig 6d p discussionmetastasis of crc is a multistep process requiring the accumulation of genetic andor epigenetic alterations andabnormal expression of genes involved in signal transduction pathwaysincluding oncogenic mutation of krasand activation of the erkmapk pathway wntβcatenin signalling and tgfβ signalling ap1 dnabinding sequences act as crucial response elements fortranscriptional activation by the raserk pathway creb5 is a credependent transactivator downstream ofthe raserk signalling pathway since it interacts with cjun which is one of the ap1 subunits to form a homodimer or a heterodimer along with foxd1 and atf3creb5 formed a transcription factor regulatory networkthat negatively regulates mapk signalling which is suppressed by fzd3 in melanoma previous studies haverevealed that creb5 mrna was upregulated in crc asdemonstrated by bioinformatics analysis or qrtpcrexamination in human cancer tissues [ ] in thecurrent study we demonstrated that creb5 was highlyupregulated in crc at both the mrna and protein levelsoverexpression of creb5 was significantly associatedwith aggressive cellular characteristics of crc eg an 0cwang of experimental clinical cancer research page of fig creb5 positively correlated with met expression in crc a correlation analysis of creb5 and met in tcga crc data r p band c correlation analysis of creb5 and met at the mrna r p and protein levels r p in fresh crc tissues dthe expression of creb5 and met protein in specimens including colon cancer tissue specimens and adjacent normal tissue specimens wasdetected by ihc representative ihc images left and correlation analysis right of creb5 and met expression scale bar μm high expressionof creb5 n high expression of met n low expression of creb5 n low expression of met n p advanced who stage and an advanced tnm stage andpoorer patient outcomes suggesting that creb5 might bean oncogene and a prognostic marker of crc progressionconsistent with our results upregulation of creb5 hasbeen reported to be responsible for poorer outcomes inpatients with epithelial ovarian cancer and hepatocellular carcinoma in prostate cancers creb5 overexpression occursthrough both copy number gain and increased gene expression however examination of tcga colorectalcancer datasets via cbioportal revealed rare amplificationof creb5 suggesting that overexpression of creb5 iscontrolled attranscriptional and posttranscriptionallevels creb5 has been shown to be a downstream target of lncrna snhg5mir1323p and circularrna circvapamir125a in addition fzd3 inhibits transcriptional networks controlled by creb5 however the alternative mechanisms involved inupregulation ofthe creb5 gene and activation ofcreb5mediated signalling require further investigationthe effects of creb5 overexpression on promotingcell proliferation and migration have been demonstratedusing in vitro assays in human hepatocellular carcinomacells and crc cell lines [ ] in the current studywe showed that creb5 overexpression promoted whilecreb5 silencing reduced the invasiveness and metastaticcapacity of crc cells both in vitro and in vivo mechanistically creb5 directly interacted with the met promoter and activated the hgfmet signalling pathwayimportantlyinhibition of met reduced the invasion 0cwang of experimental clinical cancer research page of and metastasis of creb5overexpressing crc cells suggesting that creb5 promotes metastasis mainly throughactivation of met signalling our data provide solid evidence that upregulation of creb5 plays an essential rolein crc metastasis recently overexpression or amplification of creb5 was reported to promote proliferationand mediate resistance to ar inhibition in metastaticcastrationresistant prostate cancers these datasuggest that creb5 may function as a multitaskingregulator in cancer progression and clinical outcomessignallingtransportimmunegrowth factorscreb family members can be phosphorylated via various intracellular signal transduction pathways such asprotein kinase a pka calmodulindependent proteinkinasecamk mitogenactivated protein kinasesmapks and other kinases upon phosphorylationcreb recruits crebbinding protein cbp and binds tothe cres of the promoters of its target genes target genes containing consensus sites for creb bindinginclude those related to metabolism transcription neuropeptidesneurotransmitters cell cyclecellsurvivalregulationdna repairreproductiondevelopmentandstructure specifically knockdown of creb1creb5increased tumor necrosis factor alpha tnfα levelsenhanced the expression of phosphonfκb p65 andnfκb p65 and induced immunosuppression in monocytes in prostate cancer creb5 could improve resistance to enzalutamide with the help of foxa1 andselectively enhance the interaction of ar with targetgenes critical for survival however little is knownabout the downstream targets of creb5 involved in theprogression of crc our results showed that creb5 directly interacted with the met promoter and activatedthe hgfmet signalling pathway which in turn increased the expression of downstream erk and pi3ksignalling cascades meanwhile the expression of snailan essential emt transcription factor was also upregulated via the creb5hgfmet axistranscriptional factor that interacts with the met promoter at the ap1 motif and activates met expressionin our data suggest that creb5 has an essential role in crc metastasis by regulating the protooncogene met interfering with creb5 may representan alternative therapeutic target to prevent or reducemetastasis in crcsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s13046020016730additional file table s1 the relationship between creb5 expressionand clinicopathological parameters table s2 primer sequences used forrealtime pcr ² to ² table s3 sequence of primers for chip assaytable s4 sequence of primers for luciferase reporter assayadditional file figure s1 bioinformatics analysis of creb5expression the expression of creb5 in crc and other malignant tumorswas analyzed by oncomine database the inclusion criteria were that thedifference of creb5 expression between tumor tissue and normal tissuewas more than times and the arrangement of gene position was lessthan with p the outliers in the red and blue boxes representthe number of data sets with high and low expression of creb5respectively the right table of a represents the copa score of creb5 in crc data sets b the two crc chips gse17538 n andgse35896 n from the public database of geo was analyzed bygsea the plot showed significant enrichment of tumorrelated gene setkegg_pathway_in_cancer and colorectal cancerrelated gene setkegg_colorectal_cancer in the creb5 high expression group cand d realtime pcr and western blotting analysis of creb5 endogenous expression in indicated crc cellsadditional file figure s2 representative images of woundhealingassay a transwell migration assay b and huvec tube formation assayc with i | Colon_Cancer |
"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 |
coronaviruses covs belong to a family of envelopedviruses with a positive sense singlestranded rna genomecovs cause illness ranging from upper respiratory tractinfections urtis resembling the common cold to lowerrespiratory tract infections lrtis such as bronchitis pneumonia and even severe acute respiratory syndrome sarswith most serious disease outcomes in the elderly immunocompromised patients and infants [ ] hcovoc43oc43 hcov229e 229e hcovnl63 nl63 andhcovhku1 hku1 were the ï¬rst documented humancovs hcovs which usually cause urtis and less frequently are associated with ltri diseases in the lastdecades two human coronaviruses created great concernfor the world medical community due to significant diseaseand mortality [ ] in severe acute respiratorysyndromecoronavirus sarscov was characterized byacute atypical pneumonia and diï¬use alveolar damagedad in roughly patients and with almost deathsrepresenting a nearly mortality rate more recentlyin a new human coronavirus designated as middle eastrespiratory syndromecoronavirus merscov was identiï¬ed and the global ongoing outbreak of mers with over oï¬cial cases and deaths represented approximately case fatality rate to date in humans over the last few months a new strain of human coronavirus sarscov2 also known as 2019ncov hascaught the worlds seven continents attention with its rapidglobal spread aï¬ecting at least countries and territoriesinfecting more than and claiming more than lives worldwide the coronavirus pandemichas promoted isolation and uncertainly fear and panicworldwide in additionlikely lead to changes inpolitical and economic power in ways that can be determined only later it willit is important to note that there are many similaritiesamong diï¬erent coronavirus species but not in all aspects 0coxidative medicine and cellular longevitydepending on the molecular mechanism of viral inhibitionpromoted by an antiviral agent the analysis of the data andcomparison between animal and human covs must be donevery carefully in fact it is important to note that there arediï¬erences between human and animal cov receptorswhich will likely result in diï¬erent aï¬nities or unlikely interactions of an antiviral agent with the diï¬erent cov receptors howeverif the antiviral agent interferes with thereplication andor assembly of the covs there is a higherprobability of obtaining similar antiviral activity results inhuman cov tests [ ] following this line our searchin specialized literature was focused mainly on studies thatinvestigated the anticoronavirus eï¬ects of natural antioxidants by inhibiting proteases for viral replication materials and methodsthe present study was carried out based on a search of the literature of natural antioxidants and coronavirus the searchperformed in the pubmed database included studies published until march and used the following keywordscoronavirusstressmerscov sarscov 229e nl63 oc43 hku1merscov virus infection and middle east respiratorysyndrome virus the scientiï¬c publications were selectedfrom studies published in the english languageantioxidants ï¬avonoids oxidative pathogenic mechanism of coronavirusinduced cell damagethe high mortality rate associated with the three pathogenichcovs has been mainly attributed to the development ofdigestive and respiratory tract injuries observed followinginfection acute atypical pneumonia and diï¬use alveolardamage that progress to deposition of ï¬brous tissue denudedairways haemorrhage and elevated macrophage ltrationare sometimes accompanied by watery diarrhoea dehydration and vomiting [ ]despite the molecular mechanisms of coronavirusinduced intestine and lung pathogenesis not fully elucidatedand still unclear studies have suggested that lateterm diseaseprogression is unrelated to viremia it is now believed morelikely to be associated with the immunopathological mechanism [ ] viral clearance and subsequent recovery frominfection require activation of an eï¬ective host immuneresponse however many immune eï¬ector cells may alsocause injury to host tissues together with ammatoryand immune response signaling the presence of oxidativecompounds such as reactive oxygen species ros playsimportant roles in the pathogenic mechanism of cell damageinduced by covs through oxidative stress oxidative stress is deï¬ned as an interruption andorderegulation of the signaling and redox system that can becaused by an imbalance in the production of oxidant andantioxidant species among the main oxidant agentsros and reactive nitrogen species rns stand out in orderto counterbalance the oxidant species there is an antioxidantsystem formed by enzymes and nonenzymatic molecules however during pathological events such as viral infections there may be an increase in the production of oxidantspecies not neutralized by the antioxidant system resultingin oxidative stress that promotes cellular damage throughprotein denaturation changes in the functions of nucleicacids lipid peroxidation and cell death []in addition during viral infection oxidative stress contributes to viral pathogenesis through stimulating ammation loss of immune function and increased viral replicationthat may occur due to the activation of the nuclear factorkappa b nfκb transcription pathway [] currentevidence suggests that cytokine dysregulationalso calledcytokine stormcontributes to severe disease caused by thepathogenic covs [ ] the exact mechanisms are notclear yet but research on uenza a virus shows that infection causes a rapid ux of ammatory cells this isfollowed by an increase in reactive oxygen species productionand cytokine expression and release which ultimately leadsto acute lung injury in general rna viruses promotechanges in the bodys antioxidant defense system aï¬ectingenzymes such as superoxide dismutase sod and catalasecat in addition to reducing the levels of antioxidantmolecules such as ascorbic acid carotenoids and reducedglutathione gsh [] wu reported that glucose6phosphate dehydrogenasean important antioxidantenzyme that produces nadph knockdown cells were moresusceptible to infection by hcov229e than normal cells interestingly ye and colleagues have reported that theinhibition of ros production alleviates ammation causedby uenza a virus infections in an experimental model of sarsinduced acute lunginjury in mice it was noted that phospholipid oxidationdue to oxidative stress is one of the main triggering factorsof acute lung injury this happens through the activation ofthe innate immune response culminating in the activationof pulmonary macrophages via tlr4triftraf6nfκbsignaling furthermore hypoxia caused by acute lunginjury can cause myocardial injury due to the production ofros aggravating infections caused by coronavirus disease covid19 mitochondria have an essential function in energy generation and for this reason their function and integrity arestrictly regulated in order to respond to varying energyrequirements and environmental conditions mitochondria are known to function as the control point in apoptoticpathways releasing proapoptotic factors mainly ros whichfunction as a signaling molecule that may result in cell death[ ] some studies have shown a relationship betweencoronavirus infection and dysfunctional or damaged mitochondria leading to the release of ros and other proapoptotic substances [ ] in a recent study xu reportedthat ros and p53 play key roles in regulating many kindsof the cell process during coronavirus infection in vero cellsaccording to the authors coronavirus infection appears toinduce a timedependent ros accumulation which in turnis linked to regulatory mechanisms of p53 activation andapoptosis in infected cells antioxidant substances promote improvement in casesof disease caused by coronaviruses such as apolipoproteinda lipocalin that promoted a neuroprotective eï¬ect against 0coxidative medicine and cellular longevityencephalitis induced by human coronavirus oc43in micethis protective eï¬ect occurred through the reduction of oxidative stress cerebral lipid peroxidation and regulation ofammation [ ] also the treatment with antioxidantssuch as pyrrolidine dithiocarbamate or nacetylcysteine significantly inhibits moreover melatonin promotes downregulation of acute lungoxidative injury due to its antiammatory and antioxidantactions making it a possible compound in the treatment ofcovid19 based on these studies compounds thathave antioxidant actions can be helpful in the treatment ofinfections promoted by coronaviruscoronavirusinduced apoptosisin general antioxidant properties of polyphenolic compounds such as some ï¬avonoids have been associated withthe presence of aromatic phenolic rings that promote theelectron donation and hydrogen atom transfer to free radicals acting as free radical scavengers reducing agents andquenchers of single oxygen formation thus the aimof this study was to investigate the antioxidant capacity andantiviral activity of natural antioxidants against coronavirusthe compounds are illustrated in figure occurrence and antioxidant properties ofanticoronavirus compoundsquercetin can be found in plants such as rubus fruticosus land lagerstroemia speciosa l pers [ ] also quercetinshows antioxidant activity at a concentration of μmoll inhepg2 cells inhibiting oxidative stress promoted by h2o2 promotes an increase in sod cat and glutathioneperoxidase gpx and reduces lipid peroxidation in rats withchronic prostatitischronic pelvic pain syndrome moreover quercetin improves sepsisinduced acute lung injury inrats by reducing lipid peroxidation and ammation andincreasing sod and cat levels in addition quercetin glycosides with antioxidant activity such as quercetin 3βglucoside have already been isolated from plants such as passiï¬ora subpeltata ortega andchamomilla suaveolens pursh rydb [ ] the administration of quercetin 3βglucoside mgkg poinstreptozotocininduced diabetic rats promotes an increasein the levels of antioxidant enzymes sod cat andgpx and nonenzymatic antioxidants vitamins c and eand gsh and a reduction of lipid peroxidation quercetin 3βgalactoside hyperoside is found mainly in plantsof the hypericum genus such as hypericum perforatum l[ ] moreover it showed cardioprotective activity inhigh glucoseinduced injury of myocardial cells throughdecreased apoptosis and ros production and increasedsod levels quercetin 7ramnoside is also found inplants of the hypericum genus such as hypericum japonicum thunb ex murray this ï¬avonoid shows hepatoprotective activity against carbon tetrachloride in mice bydecreasing lipid peroxidation and increasing cat andgsh levels in addition to presenting values of μmand22diphenyl1picrylhydrazyldpph and ²azinobis3ethylbenzthiazoline6sulphonic acid abts assays respectively μm intheepigallocatechin gallate is present in parkia roxburghii gdon and is one of the main metabolites found in green teaand liubao tea camellia sinensis lo kuntze also gallocatechin gallate can be found in this plant [] literaturedata reveal that the administration ip of mg100 g ofepigallocatechin gallate in rats with streptozotocininduceddiabetes mellitus promotes a reduction in oxidative stressthrough reductions in parameters such as indirect nitricoxide synthesis and status total oxidative as well as anincrease in levels of cat and total antioxidant capacity ofplasma furthermore it promotes cardioprotection byantioxidant mechanisms green tea has a high antioxidant capacity due to the highlevels of catechins present he and collaborators compared the antioxidant activities of catechins and reported thatepigallocatechin gallate has greater antioxidant activity viaradical scavenging activity μm with values of ± ± and ± compared to its epimergallocatechin gallate with values of ± ± and ± in the dpph abts and ferric reducingantioxidant power frap respectively amentoï¬avone is a biï¬avonoid present in leaves ofginkgo biloba l garcinia brasiliensis l and nandinadomestica l [] this biï¬avonoid has a high antioxidantcapacity demonstrated in scavenging tests ofdpph abts superoxide and hydroxyl radicals moreover amentoï¬avone prevents acute lung injury induced bysepsis in rats by decreasing thiobarbituric acid reactive substance tbars levels and by increasing levels of sod andgsh apigenin is mainly present in ï¬owers and leaves beingabundantly found in apium graveolens l petroselinum crispum mill fuss and matricaria chamomilla l sánchezmarzo and collaborators evaluated the antioxidantcapacity of apigenin using the trolox equivalent antioxidantcapacity teac oxygen radical absorbance capacityorac and frap assays the results show that apigeninhas good antioxidant activity with values of ± μmol teammol ± μmol teammol and ± μmol fe2mmol respectively in addition oraladministration of apigenin mgkgday for days in anexperimental model of cardiotoxicity induced by doxorubicin in rats promoted cardioprotection by reducing levels ofmalondialdehyde mda increasing sod levels and preventing cardiomyocyte apoptosis luteolin is present in foods such as carrot cabbage teaand apple and is found in ugni molinae turcz [ ] datashow that luteolin μgml increases the levels of gsh theexpression of gsh synthetase and the activity of sod andcat in human colon cancer cells ht29 furthermoreluteolin attenuates the sepsisinduced acute lunginjury in mice by reducing lipid peroxidation and increasingsod and cat activity in addition to suppressing the nfκbpathway herbacetin is ubiquitous in plants of the genus rhodiolasuch as rhodiola rosea l herbacetin glycosides are alsopresent in the roots of r sachalinensis a bor and show antioxidant activity veeramani reported that theadministration of herbacetin mgkg po in mice with 0coxidative medicine and cellular longevityohohohohhoohooohohhooohoh oquercetinohohoooohohohquercetin 3ð½galactosideohohooohoohoohohhoohohohoh oquercetin 7ramnosideohooohohoohoohoohohohquercetin 3ð½glucosidehooohooohohohohohohepigallocatechin gallateohgallocatechin gallateohhooohoh ohooluteolinhooohoooohoohohoohrhoifolinhohohoohohohohohohoohoohooh oamentoflavoneohhooohohohoherbacetinhohoooohooohohohoapigeninooohooohhopectolinarinooopsoralidinhooohohohohcatechinohhoohoooh ohelichrysetinohohomyricetinohohohhohoohoohoisobavachalconeohhooohscutellareinohresveratrolfigure chemical structures of bioactive antioxidants against coronavirusobesityassociated insulin resistance promotes an increasein the activity of the enzyme glucose6phosphate dehydrogenase which is directly related to the production ofnadph pectolinarin is present in plants of the genus cirsiumsuch as cirsium setidens nakai and cirsium japonicum dcthe administration of pectolinarin and mgkg pofor two weeks in rats promotes antioxidant eï¬ects in hepatic 0coxidative medicine and cellular longevitytable antioxidant properties of natural inhibitors of coronaviruscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencetype of cells μmoll mgkg po mgkg poinhibiting oxidative stress promoted byh2o2promoted an increase in sod cat andgpx and reduced lipid peroxidationreduces lipid peroxidation and increasessod and cat levelsincreases levels of sod cat gpx mgkg povitamins c and e and gsh and reduces nmoll mgkgic50 μm dpphec50 μm abtsrats with streptozotocininduced diabetes mellitus mg100 g iprats with streptozotocinnicotinamideinduceddiabetes mellitus mgkg po μm mgkg μgml ± μmol teammol ± μmol teammol and ± μmol fe2mmolrespectively mgkg pomodelshepg2 cellsrats with chronicprostatitischronic pelvicpain syndromesepsisinduced acute lunginjury in ratsstreptozotocininduceddiabetic ratshigh glucoseinducedinjury of myocardial cellsccl4induced liver damagemodel in micedpphabtsquercetinquercetin 3βglucosidequercetin 3βgalactosidequercetin ramnosideepigallocatechingallateepigallocatechingallatedpphabtsfrapgallocatechingallateamentoï¬avoneacute lung injury inducedby sepsis in ratsdpph abts superoxideand hydroxyl radicalsteacoracfrapcardiotoxicity induced bydoxorubicin in ratshuman colon cancer cellsht29acute lung injury inducedby sepsis in micemice with obesityassociated insulinresistance inductionhepatic injury induced bydgalactosamine in ratsoracapigeninluteolinherbacetinpectolinarinrhoifolincatechinlipid peroxidationdecreases apoptosis and ros productionand increases sod levelsdecreases lipid peroxidation and increasescat and gsh levelsscavenging of free radicalsreduces indirect nitric oxide synthesis andtotal oxidative statusincreased levels of cat and totalantioxidant capacity of plasmaincreased levels of cat sod and gshreduced levels of superoxide and proteincarbonyl pco and prevented dnadamage ± ± and ± ± and ± respectively ± respectivelydecreases tbars levels and increaseslevels of sod and gshscavenging of free radicalsscavenging of free radicalsreduces levels of mda increases sodlevels and prevents cardiomyocyteapoptosis μgmlincreases levels of gsh expression of gshsynthetase and the activity of sod and mgkg ip mgkg po and mgkg poapproximately troloxequivalents μmcatreduces lipid peroxidation increases theactivity of sod and cat and suppressesthe nfκb pathwayincreases the activity of glucose6phosphate dehydrogenaseincreases levels of sod gsh glutathionereductase and glutathione stransferasescavenging of free radicalsscavenging of free radicals 0coxidative medicine and cellular longevitytable continuedtype of cellscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencemodelsabtsfrap ± mol troloxequivalentsmol ± mol trolox equivalentsmoldihydrorhodamine oxidation assayandic50 ± μmisobavachalconedpph sc50frapabts sc50psoralidinelectron spin resonancemyricetinhelichrysetinscutellareinresveratroldpphchinese hamster lungï¬broblast cells v794treated with h2o2oracdpphabtssuperoxide radicalsdpph sc50r2rats with obstructive lungdiseasehypercholesterolemicapoeko mouserespectively μm ± mmic50 μmequivalent to feso4 mm respectively·7h2o and μgml and μgml μgml ± trolox equivalents ± μm ± μm and ± μm respectivelyscavenging of free radicalsscavenging of free radicalsscavenging of free radicals and respectivelyprevents dna damage and lipidperoxidationincreases the activity of sod cat andgpxscavenging of free radicalsscavenging of free radicals μmoldmscavenging of free radicals mgkg mgkgincreases sod activity and reduces mdalevelsinhibits the activity and expression ofnadph oxidasesincreases sod gpx and cat levels injury induced by dgalactosamine by increasing levels ofsod gsh glutathione reductase and glutathione stransferase [ ]rhoifolin is found in citrus fruits such as citrus limettarisso studies have indicated that its radical peroxyl scavenging capacity is higher than trolox in orac assays approximately trolox equivalents μm [ ]meanwhile the catechin is a ï¬avonoid present inleaves of green tea wine and fruits [ ] grzesik investigated the antioxidant action of catechinthrough the abts scavenging activity and frap teststhe results show values of ± mol troloxequivalentsmol and ± mol trolox equivalentsmol respectively in addition catechin shows greaterprotective properties in the dihydrorhodamine oxida ± μm than gsh and ascorbiction assay ic50acid and μm respectively psoralidin is a prenylated coumestan which is found inplants of the fabaceae family such as psoralea corylifolia lxiao and collaborators investigating the antioxidant potential of compounds isolated from p corylifolia observed thatpsoralidin shows the best antioxidant activity by the methodof electron spin resonance spectroscopy with an ic50 value of μm the compound isobavachalcone has been isolated fromplants of the fabaceae and moraceae families [ ] isobavachalcone shows a strong antioxidant activity in dpphsc50 frap and abts sc50 assays with values of μm ± mm equivalent to feso4·7h2o and mm respectively in addition the compound has beenreported to inhibit the nfκb pathway in sephadexinducedlung injury in rats [ ]helichrysetin is a chalcone that is found in plants of thehelichrysum genus such as helichrysum odoratissimum l in a study investigating the antioxidant activity of natural and prenylated chalcones vogel found that helichrysetin is the substance that shows the highest antioxidantactivity in the orac test with values of ± troloxequivalents myricetin is widely found in the plant families myricaceae and anacardiaceae and is widely used as health foodsupplement due to its antioxidant properties [ ]bennett demonstrated that myricetin reacts withoxygencentered galvinoxyl radicals more than timeshigher than vitamin e dalphatocopherol furthermoremyricetin was able to scavenge and on the dpphassay μgml and μgml respectively interestinglythe compound prevents dna damage by lipid 0coxidative medicine and cellular longevity2h2o2 2gsh2o2h2h2o2vit evit cgshnadphnonenzymatic antioxidantsgpxsodcatsemyzne tnadixoitna2h2o gssgo2 h2o2o2 h2omdapcotbarsbio m arkers of oxidative stressgeneration of rosoxidative stresscell damagenaturalantioxidants sesadixohpdan2o2 nadphnadp 2o2 hfigure the main antioxidant mechanisms of natural compounds reported in this review dashed line inhibition full line activationperoxidation and increasing the activity of sod cat andgpx in chinese hamster lung ï¬broblast cells v794treated with h2o2 scutellarein is found in scutellaria barbata d don andpolygonum viscosum buchham [ ] liu investigated the antioxidant activity of scutellarein through thedpph abts and superoxide scavenging assays they notedthat the compound shows good antioxidant activity withvalues of ± μm ± μm and ± μmrespectively while the trolox a standard antioxidant compound presented ± μm ± μm and ± μm respectively resveratrol is found in grapes peanuts and blueberriesand can be isolated from veratrum grandiï¬orum o loes literature shows that resveratrol has good antioxidantvalues of μmoldmactivity with dpph sc50r2moreover it is able to reduce the production of ros by inhibiting the activity and expression of nadph oxidases byeliminating oxidant agentsincluding radical hydroxylsuperoxide hydrogen peroxide and peroxynitrite the treatment of resveratrol mgkg po in ratsreduces oxidative stress in obstructive lung disease byincreasing sod activity and reducing mda levels indicatinga decrease in lipid peroxidation table shows themain actions of natural antioxidants discussed in this studyand figure illustrates these activities effect of natural antioxidants incoronavirus infectionsthis review focused on studies reporting on the anticoronavirus activity of natural antioxidants based on exclusioncriteria data from nineteen compounds were discussedthe oxidative stress pathway could potentially be a keyelement in coronavirusinduced apoptosis and pathogenesis for this reason it is interesting to investigate the useof antioxidants as potential therapeutic toolseither as analternative or as an adjuvant to conventional therapiesinthe treatment of coronavirus infections among the antioxidant compounds evaluated as for coronavirus infectionsare the ï¬avonoids which are compounds widely found infruits vegetables and certain beverages in fact researchgroups have reported that antioxidant ï¬avonoids includingcatechin luteolin apigenin quercetin and quercetin rhamnosideinhibit ros accumulation and apoptosis ofcells infected with diï¬erent coronavirus including porcineepidemic diarrhoea coronavirus pedv and transmissiblegastroenteritis coronavirus tgev []as shown with the recent covid19 pandemic thesearch for alternative or new antiviral therapies for theremainstreatment of coronavirus diseasesimportantbased on the literature antioxidanttherapies oï¬er anattractive option 0coxidative medicine and cellular longevitytable natural antioxidants tested in in vitro coronavirus infection models and their main results and mechanism of actionantioxidanttype of cells testedconcentrationic50antiviral eï¬ectmechanism of actionreferencecatechintgevinfected st cellscatechin μminhibition of tgevinduced apoptosissuppression of the tgevinducedbcl2 reduction baxredistribution cytochrome crelease and caspase3 activation resveratrolmersinfected vero e6cellsresveratrol μmquercetinepigallocatechingallategallocatechingallate gcgquercetin rhamnosideq7rrecombinant 3clprowas expressed in pichiapastoris gs115quercetin μmepigallocatechingallate μmgallocatechingallate μmpedvinfectedvero cellsq7r μminhibition ofmersinducedreduction of the expression ofinfectionapoptosis andnucleocapsid n protein essential prolonged cellular survivalfor merscov replicationafter virus infectioninhibition of coronavirusreplicationreduction of the formationof a visible cytopathiceï¬ect cpe without dnafragmentationgcg displayed a binding energyof kcal mol1 to the active siteof 3clpro and the galloyl moietyat 3oh position was required for3clpro inhibition activitynot speciï¬city amentoï¬avoneapigeninluteolinquercetinquercetin3βgalactosideherbacetinrhoifolinpectolinarinsarscov 3clproinhibition usingï¬uorescence resonanceenergy transfer analysismolecular dockingsprfretbasedbioassays andmutagenesistryptophanbasedï¬uorescence methodherbacetinisobavachalconequercetin3βdglucosidehelichrysetintryptophanbasedï¬uorescence methodamentoï¬avone3βgalactoside μmapigenin μmluteolin μmquercetin μmquercetin μmherbacetin μmrhoifolin μmpectolinarin μmherbacetin μmisobavachalcone μmquercetin3βdglucoside μmhelichrysetin μminhibition of sarscovreplicationflavonoids exhibited sarscov3clpro inhibitory activity[ ]inhibition of merscovreplicationflavonoids exhibited merscov3clpro inhibitory activity isobavachalconepsoralidinlineweaverburk anddixon plotsmyricetinscutellareinsprfretbasedbioassaysisobavachalcone μmpsoralidin μmmyricetin μmscutellarein μminhibition of sarscovreplicationisobavachalcone and psoralidinexhibited sarscov papainlikeprotease inhibitory activitymyricetin and scutellareininhibition of sarscovpotently inhibit the sarscovreplicationhelicase protein in vitro byaï¬ecting the atpase activity the high number of deaths and clinical complicationsobserved in sars and merscov epidemics motivatedthe search for eï¬ective therapeutic agents this was necessitated when many of the tested conventional drugs andtherapies proved ineï¬ective in treating sarsantiviralcov infections for exampletreatment ofthe initial 0coxidative medicine and cellular longevitycell culture coronavirusnaturalantioxidants suppress bcl2 reductionsuppress bax redistributionsuppress cytochorome c releasesuppress caspase3 activationreduce formation of a visiblecytopathic effect without dnafragmentationreduce nucleocapsid n protein expressioninhibit 3clike proteaseinhibit 3clike proteaseinhibit papainlike proteaseinhibit helicase protein by affectedatpase activitytgevpedvmerscovsarscovfigure inhibitory actions of natural antioxidants against coronavirussarscov with antiviral agents such as ribavirin and corticosteroids did not achieve very satisfactory resultsmainly because corticosteroids exertimmunosuppressoreï¬ects on the humoral and cellular immune systems[ ] other drugs such as pentoxifylline were considered for the treatment of sars due to its interestingtherapeutic propertiesantiammatoryantiviral immunomodulatory and bronchodilatory eï¬ectshowever it too was not successful in the clinical treatment of sarscov infection includethatinhibition ofmany antioxidant compounds show antiviral activityagainst sarscov the antiviral activity has been mainlyattributed to thethe 3clike protease3clpro of sarscov a vital enzyme for sarscov replication as an example multiples studies havereported that quercetin and quercetinderived compoundssuch as quercetin 3βgalactoside display potent 3clproinhibitory e5ï¬ect and consequent reduction of sarscovreplication other antioxidants such as epigallocatechin gallate gallocatechin gallate amentoï¬avone apigeninluteolin herbacetin rhoifolin and pectolinarin are alsofound to eï¬ciently block the enzymatic activity of sarscov 3clpro [ ]moreover some natural antioxidants exhibit promisingantiviral activity against sarscov infection by interferingwith diï¬erent targets involved in sarscov replication inparticular the sarscov papainlike protease plpro andsarscov helicase protein kim reported that isobavachalcone and psoralidin inhibit plpro in a dosedependentmanner with ic50 ranging between and μm previously yu reported that myricetin and scutellareinpotently inhibit the sarscov helicase protein in vitro byaï¬ecting the atpase activity merscov is another zoonotic coronavirus transmitted between animals and human beings that causes severemorbidity and mortality no antiviral medicines with satisfactory eï¬cacy for the treatment of merscovinfectedpatients have been identiï¬ed to date similar to sarscov natural antioxidant libraries have been probed forpotentialinhibitory compounds against merscov 3clike protease jo showed that herbacetin isobavachalcone quercetin 3βdglucoside and helichrysetinfourcompounds with recognized antioxidant activity can blockthe enzymatic activity of merscov 3clpro using atryptophanbased ï¬uorescence method furthermore theexperimental and computational studies show that ï¬avonoland chalcone are favourite scaï¬olds to bind with the catalytic site of merscov 3clpro in a study performed by lin the antiviral activitiesof resveratrol were investigated in mersinfected vero e6cells the authors reported a significantinhibition ofmerscov infection and prolonged host cell survival aftervirus infection which they speculate was promoted by resveratrol in addition they also found that the expression ofthe nucleocapsid n protein which is essential for merscov replicationis decreased after resveratrol treatment it is important to mention that in vitro models of coronavirus infection also show antiviral activity of ï¬avonoidsextracted from ï¬owering cherry cultivars and black tea[ ] finally antioxidants such as resveratrol alsoare able to block infection produced by herpesvirus the discovery of antiviral compounds from a bioactivecompound against other viruses is an interesting strategy forobtaining new antiviral drugs table shows the mainactions of the natural antioxidants against the coronavirusand figure summarizes these activities 0c conclusionsin conclusion this review shows that antioxidant compounds prominently ï¬avonoids exhibit antiviral action inmodels of coronavirus infections in general the antiviralactivity might be attributed at least in part to the inhibitoryeï¬ect on the enzymatic activity of targets involved in coronavirus replication including sarscov 3clpro sarscovpapainlike protease plpro sarscov helicase proteinand merscov 3clpro in addition some studies provideevidence that the reduction of ros accumulation retardsthe coronavirusactivated apoptotic signaling thereforethe mechanisms of oxidative stress could be the key elementto be studied in coronavirus infectionsincluding thoserelated to ammatory processes arising from the action ofthis virus obviously further investigations are needed toelucidate other pharmacological mechanisms by which natural antioxidants play an antiviral eï¬ect despite the ï¬ndingsreported in this reviewthey cannot be generalized tocovid19 however the data provided support to the investigation of natural antioxidants as a potential therapeuticapproach in the treatment for covid19 and its severe clinical complications either as an alternative or as an adjuvantto conventional therapies and contribute to the search fornew prototypes in the development of drugs against coronavirus infectionsabbreviations229e3clproabtshuman coronavirus229e3clike protease²azinobis3ethylbenzthiazoline6sulphonic acidcoronavirusescatalasediï¬use alveolar damage22diphenyl1picrylhydrazylferric reducing antioxidant powerreduced glutathioneglutathione peroxidasehuman coronaviruseshuman coronavirushku1lower respiratory tract infectionsmalondialdehydecovscovid19 coronavirus disease catdaddpphfrapgshgpxhcovshku1lrtismdamerscov middle east respiratory syndromecoronavirusnfκbnuclear factor kappa bhuman coronavirusnl63nl63human coronavirusoc43oc43oxygen radical absorbance capacityoracprotein carbonylpcoporcine epidemic diarrhoea coronaviruspedvplpropapainlike proteasereactive nitrogen speciesrnsreactive oxygenated speciesrossarssevere acute respiratory syndromesarscov severe acute respirator | Colon_Cancer |
" the 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 |
"autophagy is an evolutionary cellular program that serves for thebreakdown of cytoplasmic components within lysosomes [] inidentified autophagy in's electron microscopic studies firstmammalian cells but the molecular pathways were not understooduntil the discovery of autophagy genes atgs in yeast by performinggenetic screening it is a cytoprotective rather than a selfdestructive process it is extensively accepted as a main regulator of innateand adaptive immune mechanisms the change in which completelyimpact the pathogenesis of disease and the processes that are influencedby autophagy includes the regulation of inflammation antigen presentation and bacterial clearance moreover autophagy aids in themaintenance of fundamental anelle populations such as mitochondria which is necessary for cellular bioenergetics and homeostasis homeostasis of the cell is been accomplished by maintaining the biosynthesis and turnoverthere are two broader protein degrading systems in eukaryoticcells first is the ubiquitinproteasome system responsible for the selective breakdown of most shortlived proteins and second is the lysosomalsystem the primary anelle called lysosome ineukaryotes is known for degradation through its acid hydrolases inunfavourable nutrient deprivation condition autophagy arbitrates aregulated phagocytosis via lysosomes autophagy is mediated byautophagosome that is thought to be an on selective degradation systemas it engulfs some of the cytoplasmic contents the ubiquitinproteasome system concedes only ubiquitinated proteins for degradation andit marks a remarkable contrast to autophagosome process autophagosomes a doublemembered vesicle that engulfs durableproteins impaired anelles intracellular pathogenic anisms andtransports it to the lysosomes that are fused to form autolysosome andthe inner vesicle along with its cargo is been degraded at the time ofstarvation the remaining macromolecules are again recycled to thecytosol for reuse the precise mechanism in cargo recognition isuncertain but this process involves ubiquitination autophagicprocess is separated into distinct steps which includes induction recognition selection of cargo formation of vesicle then occurs the fusion of autophagosomevacuole followed by the degradation of thecargo and release into the cytosol various atg proteins are indulged inthis process and it consists of the central autophagic machinery autophagy encompasses different process by which cells delivercytoplasmicaredegradation theylysosomalsubstratesfor corresponding author at department of biotechnology psg college of arts and science civil aerodrome post coimbatore indiaemail address rashmiffrajangmailcom rr rasmi all authors deserve contribution equally101016jlfs2020118308received june received in revised form august accepted august available online august elsevier inc all rights reserved 0cs vishnupriya life sciences macroautophagy chaperonemediated autophagy cma and microautophagy all the three processes of autophagy are morphologically distinct in microautophagy the lysosomal membrane invaginations or protrusions are needed to seize cargo once thecargo is captured the uptake directly happens at the limiting membrane of the lysosome that includes intact anelles cma uses chaperones to sequester cargo proteins that have a pentapeptide motifthese proteins are unfolded and translocated directly across the lysosomal membrane via lamp2a receptor macroautophagy involves requisition of cargo vesicle formation and its subsequenttransport to the lysosome in recent years deletion of the autophagy related genes atg invarious model anisms has proved that autophagy plays decisive rolein adaptive responses to stress cellular differentiation and development an oncogenic event may establish by the partial minimization inthe autophagic capacity atg6beclin one of the phylogeneticallypremeditated autophagy genes is often subdued at one locus in humancancers studies in mice have shown that beclin is a haplo insufficienttumor suppressor autophagic programmed cell death was primarily depicted in actively developing tissues several conjugationsystems comprising of the atg genes are available that take part inautophagasomal elongation one such system is the lc3 microtubule associated protein light chain and atg8 conjugation system lc3 is the mammalian conjugative protein ortholog of yeastprotein atg8 the lipid derivative phosphatidylethanolamine bindsto lc3 to form lc3ii an important molecular marker of autophagylc3ii remains on the mature autophagosome until it fuses with thelysosomes fig the beclin complex gives rise to an incipientautophagosome membrane and it assemble around cargo in a vesiclethat combines with a lysosome forming autolysosome that is degradedby acid hydrolases present in the lysosomes lung injury clinical implicationsthe primary an of gas exchange is the pair of lungs theymediate inspiration of oxygen and elimination of mono and dioxides ofcarbon lung also serves as an attractive target for the entry of thepathogens regulation of the pulmonary functions is mediated by intricate cells of endothelial and epithelial lining dendritic cells alveolarmacrophages and fibroblasts all these pulmonary cells are highlyheterogeneous in nature they together in association respond to thelung injury by provoking inflammatory and immune responses airway epithelial cells express pattern recognition receptors prrsalong with toll like receptors ctype lectins rigi and inflammasome components which are involved in the innate response against microbes microbial cell wall constituents likelipopolysaccharide are sensed by prrs and induce an inflammatoryresponse alveolar epithelium comprises type i and type ii alveolarcells apart from these the mucus layer and the physical barrier madeby the epithelium contribute to the first line of defense lung injury is described as any damage to the associated tissues and compartments of the pulmonary system the concept of lung injury influenced by the microenvironment can be demonstrated via series ofchanges in cell deformability and manifestation of intercellular adhesive molecules the major causes of lung injury are atelectasisalveolar instability volutrauma barotrauma infections and oxygentoxicity the pathogenicity of acute and chronic lung injury iscorrelated with the release of proteases free radicals and growth regulatory proteins by the alveolar macrophages neutrophil takescare of the extreme ranges of lungs searching for pathogens therebyfig mechanism of autophagy ulk1pi3kmtor signalling pathway binds to endoplasmic reticulum and activates dcedp1 that initiates beclin and atg512conjugation system which forms isolation membrane this is followed by the sequestration process that leads to autophagosomal formation the autophagosomefuses with lysosomes to form autolyososome that leads to degradation 0cs vishnupriya eliminating via phagocytosis they undergo transendothelial andtransepithelial migration primary host defense mechanism oflungs include the immunity conferred by surfactant proteins spnamely spa and spd they link both innate and adaptive immunity byregulating the responses of innate immune cells antigen presentingcells apcs and tcells they can act as potential biomarkers of lunginjury the implication of autophagy over lung injury is tortuousas its function varies highly with the cell types specific to the pulmonary disorders it provides both defensive and injurious outcomes of lung injury lung injury types and associated pathologies acute lung injury ali and acute respiratory distress syndromeardsacute lung injury and acute respiratory distress syndrome arecommon grievous diseases among critically illpatients and are evidential sources of mortality and morbidity they are expressed alongwith hypercapnia and hypoxemia ards is outlined as the most seriousform of ali ali is characterized by inflammation of lung surfacesresulting in the disruption of alveolarcapillary membrane followed bytransmigration of neutrophils significant event in the procession of aliand ards and outbreak of cytotoxic mediators immune responsemediated by several components like tcells macrophages naturalkiller nk cells chemokines and proinflammatory cytokines also has apredominant role in the progression of the acute injury and results inimmune reconstitution inflammatory syndrome iris immune cellsinvolved in the immune response like macrophages and monocytes arethought to be involved in the pathophysiology of iris elevatedlevels of cd14 cd16 monocyte population and an resulting increase in the proinflammatory cytokines il6 tnfα and c reactiveprotein crp are also observed in tuberculosis tbiris patients lung endothelial biomarkers like vwf and epithelial biomarkers likespd are the diagnostic targets of ali sepsis pancreatitis pneumoniatrauma transfusions aspiration and inhalation of toxic gases are someof the clinical factors of ali and ards histological patterns ofali and ards have the chances of demonstrating diffuse alveolar damage alveolar haemorrhage eosinophilic pneumonia and acute fibrinous pneumonia associated with ards may also result frompathogens like fungal viral bacterial and parasitic most commonpathogen strains include streptococcus pneumoniae respiratory virusesstaphylococcus aureus fungal pathogens like pneumocystis jirovecii andaspergillus fumigatus legionella pneumophila and enteric gramnegativeanisms among bacteria the virulence capability of pseudomonas aeruginosa is one of the prime determinant of the severity of thelung injury disease progress takes place in three degrees namelyacute phase exudative subacute phase proliferative and chronicphase fibrotic radiological features during the acute phasesignify twosided patchy groundglass densities relating to interstitialedema and hyaline membranes the important constituent of innateimmune system the pattern recognition receptors prrs are affiliatedwith the advancement of ali and ards the ligands of prrs includepathogenassociated molecular patterns pamps which induce inflammatory signalling events and the damageassociated molecularpatterns damps which induce neutrophilmediated tissue damageincreased incidences of mitochondrial damps result in high mortalityrates a common type of ali is the transfusionrelated acute lunginjury trali trali is defined as adult respiratory distress syndromeoccurring with transfusionsis manifested by pulmonary insufficiency severe hypoxia and dyspnea it is often reported as theneutrophil pmnmediated syndrome a particular study has reported that the potential risk factors for ali and ards may be associated with larger tidal volume vt and higher airway pressure pawduring one lung ventilation olv in postpneumonectomy aliardspatients and also in patients who developed aliards longtermeffects after acute lung injury are related to neurological impairmentsitlife sciences namely neuromuscular dysfunction neurocognitive dysfunction andneuropsychological dysfunction some of the minor impacts includeheterotopic ossification stiae and frozen joints chronic lung injury cli and bronchopulmonary dysplasia bpdchronic lung injury is characterized by a condition called pleuroparenchymal fibroelastosis ppfe in which a rapid multiplication ofsubpleural intestinal elastic fibres majorly in the upper lobes predominates along with other clinicopathological conditions following chronic lung injury cells undergoing apoptosis is cleared by aprocess called efferocytosis it is a type of phagocytosis confined only tothe cells that undergo apoptosis to maintain the cellturnover in pulmonary airways carried out mainly by the macrophages while immature dendritic cells mesenchymal cells and epithelial cells can alsoperform efferocytosis recently sarscov2 manifested as severerespiratory infection resulted in number of deaths worldwide commonrisk factors associated with death in sarscov2 patients identified arehypertension diabetes cardiovascular disease or chronic lung disease the most prevailing chronic lung injury ofinfants is thebronchopulmonary dysplasia bpd occurs when preterm infants suffering from various respiratory syndromes like meconium aspirationsyndrome and neonatal pneumonia are subjected to treatment withsupplemental oxygen and extensive mechanical ventilators it is proventhat the common risk factors of bpd increases with a fall in birthweight prematurity and gestational period apart from theabove other perinatal risk factors with which bpd is associated with areintrauterine growth restrictions race or ethnicity chorioamnionitis and genetic risk factors it is characterized bysaccular formation and elastic fibre aggregation of distal air spacesinflation and edema barotrauma and pulmonary oxygen toxicity arepathologies of bpd bpd is multifactorial in nature studiesdeclare that surviving patients of bpd have established pulmonarydysfunction incidence adults with bpd history are strictly prohibitedto cigarette smoking another form of bpd called the new bpdwhen surfactants are used as treatment new bpd is depicted by pulmonary hypertension and abnormalities in vasculopulmonary development glucocorticoids and tgfbeta are the efficient modulators thatinitiate bpd injury bpd infants have significantly lower levels ofvascular endothelial growth factor and platelet endothelial cell adhesion molecules chronic obstructive pulmonary disease copd and asthmacopd is a general illness worldwide it is defined as the completeirreversible state of airflow limitation characterized by weak inflammatory response of the lungs emphysema chronic obstructivebronchiolitis fibrosis blocking and narrowing of airways deprivationof lung parenchyma and elasticity immune cells like tlymphocyteswith cd8 dominance blymphocytes macrophages and neutrophilsare the regulators of copd acute provocation of copd is definedas the continuous deterioration from steady state facilitating an alteration in typical medication for rudimentary copd systemicinflammation responses are a result of leukotriene b4 tnfalpha interleukin il8 and proteases a decrease in the ratio of cd4 to cd8is a typical feature of pulmonary inflammatory responses additionalimpacts of copd are cardiovascular diseases nervous effects and osteoskeletal effects smoking is an important cause of systemicoxidative stresses immoderate inflammatory responses and emphysema as manifested as copd implications copd being an hetergoenousdisease patients with exacerbations are found to be associated withbacterial infections like moraxella catarrhalis streptococcus pneumoniaand haemophilus influenza apart from bacteria other microbes likevirus and fungi also comtribute to the lung microbiota and pathogenesisof lung microbiota through initiation of chronic inflammation it hasbeen found that bacteria and viruses fungi can promote local andsystemic inflammation that may contribute to the pathogenesis ofcopd specific biomarkers of copd observed are creactive 0cs vishnupriya protein crp il6 il10 and ccl18parc skeletal muscle losstakes place in copd wasting of the cell mass is evidenced to be theresult of tnfα participation in the pathogenesis of copd muscleglutamate reduction is linked with lactic acidosis in copd patientsending in muscle wastage similar to copd asthma is characterized by pulmonary obstruction and similar immune responses onlyvariation from copd is that the airway obstruction in asthma is reversible and it does not affect lung parenchyma dendritic cells are themodulators of th2 cells playing an important immune response ofasthma severe asthma is equal to the effects of copd illustrated by theincrease in neutrophils tumor necrosis factor tnf cxcl8 and decreased reception to corticosteroids while reversible copd is potentially to have subsequent asthma and copd ventilatorinduced lung injury vili and ventilatorassociated lunginjury valithe prevailing lung injury cases with ards when given ventilatorassistance mechanically in a clinical setup may develop additional lunginjuries ending up in ventilatorassociated lung injury vali similarlyin experimental models lung injury can be provoked by external application of injurious ventilation procedures contributing to ventilatorinduced lung injury vili thus vali can adversely aggravate thehealth of the ards patients at low lung volumes of ventilation atelectrauma occurs at high lung volumes of ventilation barotrauma andpulmonary edema occur biomarkers studied via experimental modelsof vali include several proinflammatory molecules like tnfα il1βil8 and antiinflammatory molecules like il10 il6 and stnfr1 respectively [] positive endrespiratory pressure peep and tidalvolume applied have direct influence on vali and are to be studiedspecifically during ali and edema it is reported that peep when provokes overinflation the extent of edema also increases mechanism ofsurfactant inactivation is seen in alveolar microvessels with an increasein fluid filtration further greater the lung volume higher is thetransmural pressure in them a retrospective cohort study revealedthat height and gender of the patients should be taken into considerations along with establishing limitations to large tidal volumes beforesetting up a ventilator experiments explain that decrease in thepeak pulmonary arterial pressure or respiratory frequency can lessenthe grimness effects of vali it is proven that hypercapnic acidosisaffords protection to vili addressable implications of vali arebarotrauma volutrauma atelectrauma biotrauma and oxytoxic effects cellular pathology includes physical disruption of cells and tissuesand activation of cytotoxic responses leucocytes are raised to likelyinteract with the endothelium as the increasing intraalveolar pressurefastens the transit time of them inferences for current medicalpractices involve lungprotective ventilation survival of patients can beencouraged by prone positioning future clinical practices involveprecisioned ventilationindividualized tidal volumes using drivingpressure individualized peep and extracorporeal strategies pulmonary fibrosis pf and cystic fibrosis cfidiopathicpulmonary fibrosis ipf is a degenerative interstitial fibrosing disease prevalent worldwide it is also termed as cryptogenicfibrosing alveolitis a typical honeycomblike structure of asymmetricairspaces covered by dense fibrosis is observed ipf is followed by interstitial pneumonia which is featured by deficit inflammation and withabsence of homogeneous participation of lung tissues studies havebeen made since ages which revealed several inherited forms of pulmonary fibrosis mutations in various genes were associated to pfnamely sftpc sftpa2 tert terc and muc5b incidence withrheumatoid arthritis and scleroderma are more likely to form pf thereis a chance of clearance of alveolar basement membrane and occurrenceof hyperplastic epithelial cells ipf ensures migration of fibroblastsinto the fibrinrich exudates thus a chemoattractant activity is createdin the airspaces after lung injury this process is proven to be regulatedthe proby lysophosphatidic acid experiment proves thatlife sciences inflammatory cytokines il1β directly regulates the initiation of acuteand chronic inflammation making it a valuable target of ipf cystic fibrosis is characterized as a most common autosomal recessivegenetic disorder caused by the mutation in cftr gene transmembraneprotein genecystic fibrosis transmembrane conductance receptor pathology of the disease involves bronchiolitis obstruction of pathways endobronchiolar infection impaired ciliary actions atelectasisfinally leading to secondary alveolar injury bacterial infections of saureus p aeruginosa and h influenzae causes cf it is the pulmonarymacrophages and polymorphonuclear neutrophils that forms the defense against infections while lymphocytemediated mucosal injury isobserved in cf patients cf patients have increased flow of tnfαil8 and il1β submucosal glands of the lungs are the crucialhosts of cftr genes as the number expressed are high hence cfcontributes to epithelial airway lining abnormalities with respect to thechanges taking place in the submucosal glands respectively the macromolecular secretions of the submucosal glands have changes in theircomposition viscosity and greatly impacts on the mucociliary clearance radiationinduced lung injury riliseveral complications of the lung malignancies require treatmentsinvolving radiation therapy rt extensive reports claim that rt maylead to a state of rili the threedimensional dosimetric predictors canbe emphasized for the risk of symptomatic rili the alveolarcapillary subunit forms the most radiosensitive complex of the lungs thusthe rili is also called as the diffuse alveolar damage radiation exposure provokes the production of reactive oxygen species creatinghigh toxicity levels in the lung parenchyma this may ultimately lead tolung fibrosis which develops one to six months after rt accompaniedby dyspnea in addition to fibrosis rili also develops radiationinduced pneumonitis gradually after months to years almost all thepatients undergoing rt have the chance of developing fibrosis radiationinduced pneumonitis is characterized by cough occasional fevernonspecific symptoms of dyspnea and chest pain with or without deformities in pulmonary functional tests while radiationinduced pulmonary fibrosis is characterized by cough differential levels of dyspnea chest pain or symptomless and stable scarring of the lung tissueswhen detected radiographically it has been stated that the incidence and occurrence of rili is directly influenced by dose and volume determinants of rt several studies provide information onrili that result in provoking inflammatory responses a biphasicmanifestation of cytokines is observed in the lung tissues once exposedto rt one such study carried out to assess the cytokine productionin c3hhen irradiated mice revealed a spatial change in the expression of proinflammatory cytokines among various cellular compartments of the lungs further it was noted that the bronchoalveolar lavage cells responded immediately while the interstitial cells contributedonly in the later stages during pneumonitis profiling of cytokinesnamely the interleukins interferons monocyte chemotactic protein1tumor necrosis factors macrophage inflammatory proteins and granulocyte colonystimulating factors can be performed to analyse the developmental risks of symptomatic radiationinduced lung injury in vitro studies experiments state that the application of melatonin andcarnosine compounds reduced the reactive oxygen species and inflammatory cytokines produced following rili regulators of autophagy in lung injurynumerous regulators of autophagy play a vital part in the development of lung injury with regard to autophagy the following are theimportant regulators fig the mechanism by which these autophagy regulators act is shown in the table lc3biithe microtubuleassociated protein light chain lc3 is the 0cs vishnupriya life sciences fig figure depicts the autophagy regulators in regard to lung injury various conditions like hypoxia starvation environmental stress and cigarette smoke leadsto autophagy that in turn causes lung injury activation of class ipi3kmtor pathway takes place under hypoxic and starvation conditions while environmentalstress results in provoking beclin 1vps34 pathway that induces the sequestration of the phagophore formation cigarette smoke induces ros accumulation which inturn causes egr1 e2f signalling to activate lc3ii along with sqtm1p62 and atg512 conjugation system the ros generated may also leads to apoptosis theautophagosome fuses with lysosomes and forms autophagolysosome that causes pulmonary arterial hypertension and lung injuryprinciple autophagic protein expressed on the doublemembraned autophagosome nearly eight homologues of lc3 proteins are studied inmammals the amino acid composition of these homologues classifiesthem into two subfamilies first one comprising of lc3a splicingvariants lc3b and lc3c taking part in autophagosomal membraneelongation while the second one comprising of gabarap gabarapl1 gabarapl2 and gabarapl3 taking part in the maturation ofautophagosome generally lc3b occurs in cytosol as lc3bi by theproteolytic cleavage of cterminal of lc3b which then unites withphosphatidylethanolamine to form lc3bii to assemble on the autophagosomal membrane conjugation of phosphatidylethanolamine withlc3bii can be revoked by the activity of atg4 thus it is clear thatoccurrence of lc3bii is crucial in the process of autophagy lc3bii levels are analysed through immunoblotting while limitationsare degradation of lc3bii by autophagy itself and the nonindication ofautophagic flux at distinct time points this can be overcome by comparison studies with lc3bi and using lysosomal protease inhibitors antilc3b antibodies can be applied to detect autophagy invarious cell types application of antilc3b to glioblastoma tissuesdemonstrated a positive detection of lc3b levels both in vitro and invivo suggesting a latent monitoring system of lc3b severalstudies state that hyperoxic conditions can result in ali and ards thisfurther triggers the morphological biomarker of autophagy lc3bii toaccumulate thereby determining the fate of cell clearance experimentsperformed in hyperoxiainduced human bronchial epithelial cells andcultured epithelial cells beas2b clearly stated that the expression oflc3bii was high mediated by the apoptotic regulators similarexperiment carried out in the hyperoxiainduced lung injury in c57bl mice inferred the involvement of apoptotic pathways in the activationof lc3bii interaction of lc3bii with fas proteins was observedmarking the importance of lc3bii in the management of ali pulmonary hypertension is a main cause of copd manifestation inlungs that affects vascular architecture when chronic hyperoxia wasinduced in the lung tissue extracts of patients with pulmonarytable regulators of autophagy in lung injury and their mechanismregulatorslc3biibeclin p62hif1bnip3mtormechanismelongation of autophagosome and its maturation requires atg8map1lc3 protein lc3i combines with phosphatidylethanolamine forming lc3ii essential for autophagosome formationthe initiation of the isolation membrane that forms autophagosome after the sequestration process is regulated by beclin and pi3kp62 along with sqstm1 is the receptor for polyubiquitinated substrates it helps in the transportation of cargo into the autophagosome by bindingwith lc3ii for degradationduring hypoxic conditions hif1 induces bnip3 that in turn brings about cell survival through autophagy and provoke cell death by apoptosisthe mammalian homologue atg13 is phosphorylated by mtor that binds ulk1 proteins fip200 phosphorylates ulk and initiates the isolationmembrane formation in autophagyreference no 0cs vishnupriya hypertension the upregulation of lc3b prevailed indicating the regulatory role of lc3b in vascular cell proliferation and mediatingadaptive cellular responses smoking results in various implications of lung injury in due course a multimeric protein complexcomprising of lc3bcaveolin1fas occurs under basal state extensivestudies reveal that smoking triggers lc3b to initiate the dissociation ofcaveolin1 from fas protein thus facilitating apoptotic pathways accordingly emphysema a destructive expression of copd isworsened by cigarette smoking in vitro studies in lung tissues of miceon exposure to cigarette smoking ensured the driving role of lc3b inregulating apoptotic mechanisms and finally developing emphysemarespectively all these experiments prove the comprehensive bridgebetween autophagy and apoptosis highly regulated by the expressionlc3b lc3bi and lc3bii bind to microtubule associated protein1b map1b wherein overexpression of map1b decreases the levels oflc3bii protein kinase c is known to cease autophagy by interferingwith autophagosomal formation both in vitro and in vivo studiesconfirmed that the lc3b phosphorylation by protein kinase c takesplace consistently in lungs the emergence of autophagy either asa protective role or maladaptive response due to sepsis was studied in acecal ligation and puncture clp induced septic mice it was ensuredautophagy to be a protective response yet an overexpression of lc3bii in the later stages of sepsis leads to ali describing a maladaptive role lung injury can be provoked by ischemiareperfusion in whichautophagy is stated as the safeguarding mechanism by moderatelymaintaining the level of lc3bii also the ischemiareperfusioninduced lung injury is positively governed by the erk12 signallingpathway that regulates the cellular expression of lc3bii respectively nanoparticles of zinc oxide on exposure to lungs may induceali further zinc oxide nanoparticles resulted in the raise of autophagosomal structures followed by the accumulation of lc3bii proteinsthus zno nanoparticlesinduced ali is autophagy dependent 3methyladenine a classical autophagy inhibitor reduced the manifestationof lc3bii and lowered the release of zinc particles thereby stoppingzno nanoparticlesrelated toxicity of lungs similarly the artificially synthesized polyamidoamine dendrimers pamam used as aneffective drugdelivery system may sometimes result in pamam nanoparticlesinduced ali the levels of lc3bii biomarkers were highindicating the autophagic responses beclin beclin was first identified by beth levine as becn1atg6 inchromosome 17q21 in the year and it is the major autophagyregulating gene it is a coiledcoil protein of molecular weight kda comprising of amino acids and acts together with bcl2an antiapoptotic protein beclin is an indispensable autophagypromoting gene that is homologue to the mammalian yeastatg6 gene which regulates cell survival of different types and is involved in the constitution of autophagosomes the initiation ofthe anization of autophagosomes is regulated by class iii phsophoinositide 3kinase pi3k and autophagy related gene beclin beclin has got a novel bcl2 homology region3 bh3 domainthe bh3 domain in beclin1 can bind to bcl family proteins that initiateapoptotic signalling and prevents the beclin 1mediated autophagy byremoving beclin from hvps34 either phosphorylation or ubiquitination of beclin or bcl2 can disunite bcl2 from beclin andincrease the activity of vps34 kinase which brings about increase infunction during autophagy autophagyassociated protein beclin1 binds to lc3i that adapts to its membranebound form lc3iiand it cooperates with the ubiquitinbinding protein p62sequestosome sqstm1 the first an that fails during sepsis is lungs thefamiliar complications of sepsis are ali and ards ali activated various autophagy related proteins like lc3ii beclin and lysosomerelated protein lamp2 and rab7 expressions in sepsisinduced ali to evaluate the function of autophagy in severe sepsis an experiment is carried out using endotoxemia that frequently uses septiclife sciences shock and clp which is a clinical polymicrobial sepsis model herebecn1 mice was susceptible to clpinduced sepsis in cysticfibrosis transmembrane conductance regulator cftr autophagy bybeclin overexpression cystamine or antioxidants and the restorationof beclin recovers the localization of beclin to endoplasmic reticulum and regresses the cf airway phenotype both in vitro and in vivoin scnn1btransgenic and cftr f508del homozygous mice and also inhuman cf nasal biopsies in lpsinduced ali there are threedistinct complexes of beclin1vps34 have been identified the firstcomplex contains beclin1 vps34 vps15 and atg14l second complexcontains beclin1 vps34 vps15 and ultraviolet irradiation resistanceassociated gene uvarag and the final complex contains beclin1vps34 vps15 uvrag and rubicon among these complex the onethat contains atg14l is concerned in the formation of autophagosomewhile others are in the autophagosome and endosome maturationbeclin forms the bridge in the recruitment of inducers and suppressorsof autophagy and simultaneously behaves as a key modulator in autophagosome formation mesenchymal stem cells mscs increases the translation level of beclin but not its transcription ratemscs might alleviate lpsali via downregulation of mir142a5p thatpermits pulmonary epithelial cells pecs to proceed with beclin mediated cell autophagy in lung disease the bacterial stu | Colon_Cancer |
" natural orifice specimen extraction surgery is a novel technique of minimally invasive surgery thepurpose of this study was to compare the safety of laparoscopic anterior resection with natural orifice specimenextraction noselar and abdominal incision specimen extraction aiselar for sigmoid or rectum tumorsmethods medline pubmed embase central cochrane central register of controlled trials scopus andclinicaltrials databases were systematically searched for related s up to august the primary outcomesincluded postoperative complications overall postoperative complication incisionrelated complicationanastomotic fistula and severe complication and pathologic results lymph nodes harvested proximal resectionmargin and distal resection edge the statistical analysis was performed on stata softwareresults ten studies comprising patients were used for metaanalysis compared with aiselar noselar hadmore advantages in terms of overall postoperative complication odds ratio or ci to p incisionrelated complication or ci to p distal resection edge weighted meandifference wmd cm ci to cm p recovery of gastrointestinal function wmd day ci to day p pain scores in postoperative day wmd ci to p additional analgesics usage or ci to p and hospital stay wmd day ci to day p while the operation time of noselar was prolonged wmd min ci to min p the anastomotic fistula severe complication lymph nodes harvested proximalresection margin intraoperative blood loss and longterm outcomes in noselar were comparable with aiselars the safety of noselar was demonstrated and it could be an alternative to conventional surgery inlaparoscopic anterior resection for sigmoid and rectal tumors however further randomized and multicenter trials are requiredkeywords natural orifice specimen extraction nose laparoscopic anterior resection rectal tumor sigmoid tumor metaanalysis correspondence guangenyang163com1department of colorectal surgery hangzhou third hospital hangzhou zhejiang peoples republic of chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0che world of surgical oncology page of introductionover the past three decades laparoscopic surgery hasevolved incessantly especially in the field of colorectalsurgery it has been widely accepted by surgeons and patients in light of the better perioperative outcomes andanalogical longterm effectiveness compared with opensurgery for colorectal cancers []for conventional laparoscopic colorectal operation asmall laparotomy in the abdomen is required for specimen harvested and colorectal anastomosis because ofthe miniincision it causes many undesirable outcomessuch as incision pain wound infection scar and even incision hernia and the advantages of laparoscopic surgeryare reduced [] to minimize those drawbacks anovel surgical variant known as natural orifice specimenextraction nose surgery with the features of naturalorifice specimen extraction and totalintraperitonealanastomosis has been introduced and is becoming ahotspot [] some studies have reported the oncology and safety outcomes between nose surgery andconventional laparoscopic surgery are comparable [] and the nose surgery therefore is supposed tohave a progress of minimally invasive surgeryrecently some metaanalysis studies had comparednatural orifice specimen extraction nose with abdominal incision specimen extraction aise in laparoscopiccolorectum resection for the colorectal disease [ ]however colorectum resection comprises right hemicolectomyleft hemicolectomy and anterior resection procedures among those surgeries were largely different and the surgical procedures and the excision extension between sigmoid and rectum resection aresimilar in addition several studies compared laparoscopic anterior resection with natural orifice specimenextraction noselar with abdominal incision specimen extraction aiselar for sigmoid or rectum tureleased [] hence wemors wereconducted this metaanalysis to evaluate the safety ofnoselar in sigmoid and rectal tumorsrecentlymethodsstudy design and inclusion criteriathis metaanalysis followed the preferred reportingitems for systematic reviews and metaanalysis prisma statements the inclusion and selection criteria were determined before starting a literature searchonly when studies with fulltext on sigmoid or rectaltumors that compared noselar and aiselar andreported at least one of the endpoints of focus were retrieved and analyzed the most comprehensive researchwas recruited when overlapping researches was conducted by the same team no language restriction wasapplied conference s case reportsreviewsrobotic surgery and singleportwere not consideredlaparoscopic surgeryselection criteria conformed to the framework ofpico participant intervention comparison and outcome patients diagnosed with sigmoid or rectal tumorsbenign and malignant tumors requiring surgery wereincludedinterventions consisted of noselar andaiselar noselar was compared with aiselarin all eligible studies primary endpoint outcomes werepostoperative complications overall postoperative complication abdominal incisionrelated complication anastomotic leak and severe complication claviendindoclassification ¥ iii and pathologic results retrievedlymph nodes proximal resection edge distal resectionedge secondary outcomes included operation timeblood loss pain score numeric rating scale score additional analgesics gastrointestinalfunction recoveryhospitalization duration 5year diseasefree survivaldfs and 5year overall survival os search strategythe following databases had been searched up to august medline pubmed central cochrane central register of controlled trials embase scopus andclinicaltrials for a more accurate search the followingkeywords andor mesh terms were used sigmoid neoplasms rectal neoplasms colorectal neoplasmslaparoscopynatural orifice specimen extractiontransvaginal specimen extraction transanal specimenextraction and transrectal specimen extraction thespecific search strategies among databases existed differences the search strategy of pubmed was presented inadditional text reference s of the eligible studies were reviewed to find the potentially relevant studiesstudy selection and quality assessmentretrieved studies were independently assessed for relevance by reviewers changjian wang and jinmingchen by screening the title and in order to enhance sensitivity studies were removed only when bothreviewers excluded the study subsequently a fulltextassessment was performed on the initial screening included studies the risk of bias was assessed by thenewcastleottawa scale nos for observational studies and studies achieving five or more stars were eligible cochrane collaborations tool for assessing risk forbias was used for randomized controlled trials [ ]all discrepancies were discussed before a final decisionwas madedata collection and statistical analysisdata from the recruited studies were extracted by tworeviewers changjian wang and jinming chen andused formeananalysis outcome valuesfurther 0che world of surgical oncology page of standard deviation and median interquartile rangewere extracted from each study considering potentialheterogeneity among studies we pooled the results byusing a randomeffects model the weighted mean difference wmd and confidence intervals cis wereapplied for continuous variables and the odds ratioor and cis were used for dichotomous variablesthe continuous outcomes were adopted the inverse variance method and dichotomous outcomes were adoptedthe mantelhaenszel statistical method when a studymerely offered the outcomes with median and interquartile range an estimation based on formulas designed byhozo was performed if a study did not provide the hazard ratio hr and cis of 5year dfsorand os the methods presented by tierney wereused for data extraction from survival curves thechisquare test and isquared value were used for measuring heterogeneity and i2 p was definedas significant heterogeneity sensitivity analyses basedon nos score ¥ and the sample size of noselargroup ¥ were conducted to assess the potentialsource of heterogeneity and the robustness of the resultspublication bias was examined with a funnel plot andharbord test p was considered statistically significant the statistical analysis was performed onstata softwareresultsliterature selection and characteristicsthe initial database search identified s ofwhich were removed based on the title and assessment the rest of the literature were evaluated byfulltext assessment and studies were excludedcharacteristics of the excluded studies were presented inadditional table ten studies were finally included forfurther qualitative and quantitative synthesis [ ] all of these were retrospective studies the process of the search and selection wasshown in fig a total of patients were recruitedin those studies with patients in the noselargroup and patients in the aiselar group themain characteristics of studies and patients were presented in table and details were shown in additionalfig flow chart of studies included in the metaanalysis 0che world of surgical oncology page of sunasunasunasunasunasunarosunaiangavsunasunasunarorororomutcermutceridomgsimutceridomgsimutceridomgsimutceridomgsiralesaipurnsonerocsetisinemcepsnoitcartxenoitacolromutleamefeamlrednegmutceridomgsimutcermutcermutcerralesonpuralesaipuraeyaegaralesonpuralesaipuralesonpugnitnuocisetapcitrapngisedydutsinogerraeyydutsisedutsdeducnliehtfoscitsiretcarahcinamelbatevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorternapajlateadasihianhclateuhianhclategnianhclategnahzianhclateuohzianhclategnxiianhclateuliydutsaissurydutsevitcepsorternawatihbaruaslateevitcepsorterecnarflatetsonedevitcepsorterydutsydutsianhclategnawrnleacsawattoeltsacwensonnoitcartxenemicepsnoisicnilianmodbahtiwnoitceserroiretnacipocsorapalralesainoitcartxenemicepsecifirolarutanhtiwnoitceserroiretnacipocsorapalralesonisnoitaverbbaegnarinademronoitavedidradnats±naemsadrocertondetropera 0che world of surgical oncology page of euavlpytienegoretehieuapvldmwstluserldeoopicrororalesairalesonstneitapfoonfoonisedutsnoitacilpmocsemoctuollafostluserldeoopehtelbatsemoctuoyramirpsemoctuoevitarepotsopllarevonoitacilpmocnoitacilpmocdetaerlnoisicnilautsifcitomotsananoitacilpmocerevesdetsevrahsedonhpmylsemoctuoilcgoohtapingramnoitceserliamxorpegdenoitceserlatsidnoitcnufsemoctuoyradnocesemitnoitarepossoldooblevitarepoartnilanitsetnortsagifoyrevocerdopinapevitarepotsopegasusciseganallanoitiddayatslatipsohsoraeyevfisfdraeyevfiecnereffidnaemdethgewdmwinoitcartxenemicepsnoisicnilianmodbahtiwnoitceserroiretnacipocsorapalralesainoitcartxenemicepsecifirolarutanhtiwnoitceserroiretnacipocsorapalralesonisnoitaverbbaliavvruseerfesaesidsfdliavvrusllarevosoyadevitarepotsopdopoitarsddoro 0che world of surgical oncology page of table the results of the pooled outcomes were summarized in table primary outcomesall included studies reported the overall postoperativecomplication the pooled data revealed that the postoperative complication in of patients whotreated with noselar and of patientswho treated with aiselar the or was ci to p with low heterogeneity i2 fig 2a among the studies eight studies reportedthe incisionrelated complication in of patients who underwent noselar and of patients who underwent aiselar the or was ci to p with no heterogeneity i2 fig 2b nine studies reported the anastomoticfistula of which ng reported zero events in bothgroups the remaining eight studies recorded anastomotic fistula in of patients sufferednoselar and of patients suffered aiselar or was ci to p withno heterogeneity i2 fig 2c a severe complication was defined based on the claviendindo classification two of the included studies recorded severecomplication claviendindo classification ¥ iii and thesevere complication in of patients withnoselar and of patients with aiselar or was ci to p withsignificant heterogeneity i2 fig 2da total of nine studies reported lymph node harvestthere was no significant difference in lymph node harvestbetween the two groups wmd ci to p no significant heterogeneity was observed i2 fig 3a the mean number of dissectedlymph nodes in the noselar group was and theaiselar group was the proximal resection marginwas reported in studies and the wmd in the upper resection edge was cm ci to cm p with no heterogeneity i2 fig 3b the distalresection margin was reported in studies and the wmdin the inferior resection edge was cm ci to cm p with no heterogeneity i2 fig3c the length of the distal resection margin in the twogroups was cm noselar group and cmaiselar groupsecondary outcomesa total of nine studies recorded operation time and intraoperative blood loss the pooled data revealed thatthe wmd of operative duration was min ci to min p heterogeneity i2 fig 4a the wmd of blood loss was ml ci to ml p heterogeneity i2 fig 4bfig forest plot comparing postoperative complications in thenoselar group and aiselar group a overall postoperativecomplication b incisionrelated complication c anastomotic fistulaand d severe complication 0che world of surgical oncology page of fig forest plot comparing pathologic outcomes in the noselar group and aiselar group a lymph nodes harvested b proximal resectionmargin and c distal resection edgesix studies provided data about the recovery of gastrointestinal function the wmd of bowel movement was day ci to day p heterogeneity i2 fig 5a the postoperative painwas recorded in studies and studies hisada and wang recorded the postoperative pain periodand the remaining reported the pain scores in postoperative day pod the wmd of pain score in pod 0che world of surgical oncology page of fig forest plot comparing intraoperative outcomes in the noselar group and aiselar group a operation time and b blood loss was ci to p heterogeneity i2 fig 5b the additional analgesicusage rate was also reported in those studies and theor of additional analgesics usage was ci to p heterogeneity i2 fig 5c theduration of hospital stay was reported in nine studiesthe wmd of hospital stay was day ci to day p heterogeneity i2 fig 5dfiveyear diseasefree survival dfs and 5year overallsurvival os were available in two studies the hazardratio hr in the 5year dfs was ci to p heterogeneity i2 fig 6a and thehr in the 5year os was ci to p heterogeneity i2 fig 6bsensitivity analysissensitivity analysis results based on the nos score ¥ and the sample size of noselar group ¥ were presented in additional table it showed a slight inconsistency in distal resection edge operation time andrecovery of gastrointestinal function and all the otheroutcomes showed a similar trend of results between thetwo groupspublication biasa funnel plot of overall postoperative complication wasperformed to detect publication bias it showed that allthe inclusive studies were within the confidenceinterval and no publication bias was found fig inaddition a harbord test confirmed there was no publication bias p discussionas a technique used for clinical treatment the safety ofnoselar should be efficiently proved morbidity isone of the most efficient indicators for assessing thesafety of an emerging technique postoperative complications may not only lead to failures of surgery but alsothreaten lives the overall postoperative complicationrate in noselar was lower than that in aiselar severe morbidity claviendindo ¥ iiiamong the two techniques was not a significant difference the operation involving digestive tract reconstruction anastomotic leakage is a potential risk once itoccurs reoperation is usually inevitable the incidence of anastomotic leakage in noselar wassimilar with that in aiselar in addition theincidence ofincisionrelated complications in noselar was significantly lower than that in theaiselar group obviously the reduction ofcomplications in noselar has largely attributed tothe decrease of incisionrelated complications althoughthe complications in noselar were reduced the riskof bacteria contamination in the peritoneal cavity shouldnot be neglected costantino had reported the peritoneal contamination in the nose group was higherthan that of the conventional group hence measures such as bowel preparation prophylactic antibioticsperitonealtransluminalwound retractor and abdominal drains are recommended to avoid the contamination of the peritonealcavity irrigationtransanallavagethe postoperative pathology results to some extentalso reflect the safety of a surgery this metaanalysisshowed lymph nodes harvested between the two groupswas comparable and it also conformed to the minimumrequirement of the guideline retrieved more than nodes in our metaanalysis the proximal marginin the noselar group was similar with the conventional group however the distal margin in the nosegroup was longer than that of the aiselar group the 0che world of surgical oncology page of potential cause of this difference was the use of transanalspecimen eversion and extraabdominal resection technique in the nose group [ ] because of thisprocedure the distal rectal resection is performed extraabdominally under direct vision moreover circumferential resection margin crm between the two groupshave no difference [ ] in addition accordingto our metaanalysis the longterm outcomes 5yeardfs and 5year os were comparable all of those indicated the nose technique was a safety procedure in thetreatment of sigmoid and rectal cancers nevertheless aconcern about tumor seeding was raised during the procedure of enterotomy and specimen extraction it is necessary to apply several measures such as the use ofprotection devices sterile specimen bags and avoidingoverpulling and compression during specimen extraction as a minimally invasive surgery noselar had moreadvantages in alleviating patients distress the reductionof pain scores in postoperative day pod was observed and this reduction could be attributed to thetrauma in noselar being further reduced owingto less pain the need for additional analgesics was alsoreduced in addition accelerating postoperative recoverywas also observed the recovery of gastrointestinal function and hospital duration in patients who sufferednoselar was much shorter besides some scholarsmay doubt if there have alterations in sexual urinary ordefecation function in the groups according to the included studies there were no differences in functionaloutcomes such as sexual urinary or defecation betweentwo groups [ ] even though a small part of patientsexperienced function alteration and the alternation wasreversible [ ] those all demonstrated thatnoselar was a safety surgery and to some extent ithad advantages in postoperative recoverynevertheless our study has several limitations firstlyintersphincteric resections were mixed with coloanalanastomoses with sigmoid cancer in our studies although there exist some differences we mixed the twotechniques and mainly considered there existing common procedures between sigmoid and rectum resectionin laparoscopic anterior resection and some studies didnot record the methods of outcome evaluation such asblood loss evaluation to some extent it reduces thecomparability of outcomes secondly the present metaanalysis relied solely on retrospective studies and someoriginal studies not presented how patients were selectedto be candidates for one technique or another the quality of all included studies was regarded as fair or good however this type of study cannot be comparedwith a randomized controlled trial and potential biascannot be ruled out thirdly this study only recruitedone multicenter research and some outcomes includedfig forest plot comparing postoperative recovery in the noselar group and aiselar group a recovery of gastrointestinalfunction b postoperative pain pod c additional analgesicsusage and d hospital stay 0che world of surgical oncology page of fig forest plot comparing longterm outcomes in the noselar group and aiselar group a 5year dfs and b 5year oslimited studies so further multicenter randomized controlled and more comprehensive studies containing adequate outcomes are needed fourthly the results ofsome pooled results among studies existed heterogeneity the sensitivity analysis could not be detected as thecause of heterogeneity although some results existedheterogeneity the major results were homogeneity andthe heterogeneity of outcomes such as operation duration blood loss and hospital stay can be explained byclinical heterogeneity such as the difference of patientssurgeons patient management and differences in surgical proficiency in nose technology in addition the results of the major parameters were robust all in all theresults of this analysis are convincedaccording to our metaanalysisthe advantages ofnose are reduced overall complications especiallyincisionrelated complications increased distal resectionedge enhanced recovery of gastrointestinal function reduced postoperative pain reduced additional analgesicsusage and shortened hospital stay and without an auxiliary patients operated by the nose technique achievebetter aesthetics however the operative time is prolonged although the nose technique has many advantagesthere are many requirements that should befollowed before the application of this technique in colorectal surgery firstly the nose should be operated byexperienced surgeons with conventionallaparoscopiccolorectal surgery secondly the indication of noseshould follow the indication of conventionallaparoscopic colorectal resection the depth of tumor invasionshould be within t3 and body mass index bmi shouldbe less than kgm2 for transanalnose and less than kgm2 for transvaginalnose transanal nose suitsfor male or female patients and the tumor diameter isfig funnel plot of the overall postoperative complications 0che world of surgical oncology page of recommended less than cm while transvaginal noseis only applied for female patients and the tumor diameter is limited within cm and the emergent conditionssuch as bowel obstruction perforation and massivebleeding are excluded all in all as surgeons follow appropriate indicationsthe noselar for sigmoid or rectal tumors is a safesurgery and the longterm outcomes between twooperations have no difference and the benefits of thenoselar in shortterm outcomes are noticeablethese findings promote enthusiasm in support ofnose surgery as an alternative approach forthetreatment of sigmoid and rectal tumorssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s1295702001982wadditional file additional file text the search strategy of pubmedadditional file additional table characteristics of the excludedstudiesadditional file additional table summary of the included studiesadditional file additional table the results of sensitivity analysisadditional file additional table quality assessment based on thenos for retrospective studiesabbreviationsnoselar laparoscopic anterior resection with natural orifice specimenextraction aiselar laparoscopic anterior resection with abdominal incisionspecimen extraction wmd weighted mean difference or odds rationos newcastleottawa scale dfs diseasefree survival os overall survivalcis confidence intervals hr hazard ratio pod postoperative day acknowledgementswe wish to thank the timely help given by junfeng hu in statistic analysisand mengdan zhou in language editingauthors contributionsguangen yang and zhong shen contributed to the conception and designof the study jun he performed the literature search and the writing of themanuscript changjian wang and jinming chen performed the data extraction haibo yao performed the data analysis qinyan yang and jianmingqiu participated in the writing of the manuscript guangen yang and haibo yao helped to revise the intellectual content the authors read and approved the final version of the manuscriptfundingthis work was funded by the zhejiang natural science foundation grantno lq19h160013 and the zhejiang medical health science and technologyproject grant no and the hangzhou health science andtechnology project grant no 2017a26availability of data and materialsall the data analyzed in this study was obtained from the included originals or related authorsethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of colorectal surgery hangzhou third hospital hangzhou zhejiang peoples republic of china 2departments ofgastroenterology pancreatic surgery zhejiang provincial peoples hospitalhangzhou zhejiang peoples republic of chinareceived february accepted july references wang cl qu g xu hw the short and longterm outcomes oflaparoscopic versus open surgery for colorectal cancer a metaanalysisint j color dis ishibe a ota m fujii s suwa y suzuki s suwa h momiyama m watanabej watanabe k taguri m midterm followup of a randomized trial ofopen surgery versus laparoscopic surgery in elderly patients withcolorectal cancer surg endosc allaix me giraudo g mistrangelo m arezzo a morino m laparoscopicversus open resection for colon cancer 10year outcomes of aprospective clinical trial surg endosc lee l aboukhalil m liberman s boutros m fried gm feldman lsincidence of incisional hernia in the specimen extraction site forlaparoscopic colorectal surgery systematic review and metaanalysissurg endosc hennessey db burke jp nidhonochu t shields c winter dc mealy k riskfactors for surgical site infection following colorectal resection a multiinstitutional study int j color dis goto s hasegawa s hata h yamaguchi t hida k nishitai r yamanokuchis nomura a yamanaka t sakai y differences in surgical site infectionbetween laparoscopic colon and rectal surgeries subanalysis of amulticenter randomized controlled trial japanmultinational trialanization prev int j color dis park js choi gs lim kh jang ys kim hj park sy jun sh clinical outcomeof laparoscopic right hemicolectomy with transvaginal resectionanastomosis and retrieval of specimen dis colon rectum ooi bs quah hm fu cw eu kw laparoscopic high anterior resection withnatural orifice specimen extraction nose for early rectal cancer techcoloproctol franklin mj kelley h kelley m brestan l portillo g torres j transvaginalextraction of the specimen after total laparoscopic right hemicolectomywith intracorporeal anastomosis surg laparosc endosc percutan tech wolthuis am de buck voa d'hoore a laparoscopic natural orificespecimen extractioncolectomy a systematic review world j gastroenterol xingmao z haitao z jianwei l huirong h junjie h zhixiang z totallylaparoscopic resection with natural orifice specimen extraction nose hasmore advantages comparing with laparoscopicassisted resection forselected patients with sigmoid colon or rectal cancer int j color dis leung al cheung hy fok bk chung cc li mk tang cn prospectiverandomized trial of hybrid notes colectomy versus conventionallaparoscopic colectomy for leftsided colonic tumors world j surg hisada m katsumata k ishizaki t enomoto m matsudo t kasuya ktsuchida a complete laparoscopic resection of the rectum using naturalorifice specimen extraction world j gastroenterol ma b huang xz gao p zhao jh song yx sun jx chen xw wang znlaparoscopic resection with natural orifice specimen extraction versusconventional laparoscopy for colorectal disease a metaanalysis int j colordis liu rj zhang cd fan yc pei jp zhang c dai dq safety and oncologicaloutcomes of laparoscopic nose surgery compared with conventionallaparoscopic surgery for colorectal diseases a metaanalysis front oncol zhou s wang x zhao c pei w zhou h liu q liang j zhou z wang xcomparison of shortterm and survival outcomes for transanal natural 0che world of surgical oncology page of orifice specimen extraction with conventional minilaparotomy afterlaparoscopic anterior resection for colorectal cancer cancer manag res xing j zhang c yang x wang h wang h yu e fu c comparison of shortterm outcomes of transrectal specimen extraction during laparoscopicsigmoid radical resection versus conventional laparoscopically assistedprocedure zhonghua wei chang wai ke za zhi wang r wei z liu q li w xiao l han hf yang s transanal versustransabdominal specimen extraction in laparoscopic rectal cancer surgery aretrospective analysis from china wideochir inne tech maloinwazyjne ng hi sun wq zhao xm jin l shen xx zhang zt wang j outcomes oftransanal natural orifice specimen extraction combined with laparoscopicanterior resection for sigmoid and rectal carcinoma an observational studymedicine baltimore 20189738e12347liu z efetov s guan x zhou h tulina i wang g tsarkov p wang x amulticenter study evaluating natural orifice specimen extraction surgery forrectal cancer j surg res hu jh li xw wang cy zhang jj ge z li bh lin xh shortterm efficacy ofnatural orifice specimen extraction surgery for low rectal cancer world jclin cases moher d liberati a tetzlaff j altman dg preferred reporting items forsystematic reviews and metaanalyses the prisma statement ann internmed w64 hawker ga mian s kendzerska t french m measures of adult pain visualanalog scale for pain vas pain numeric rating scale for pain nrs painmcgill pain questionnaire mpq shortform mcgill pain questionnaire sfmpq chronic pain grade scale cpgs short form36 bodily pain scalesf36 bps and measure of intermittent and constant osteoarthritis painicoap arthritis care res 201163suppl 11s240 higgins jp altman dg gotzsche pc juni p moher d oxman ad savovic jschulz kf weeks l sterne ja the cochrane collaborations tool forassessing risk of bias in randomised trials bmj 2011343d5928 ga wells bsdo the newcastleottawa scale nos for assessing the qualityof nonrandomised studies in metaanalyses hozo sp djulbegovic b hozo i estimating the mean and variance from themedian range and the size of a sample bmc med res methodol tierney jf stewart la ghersi d burdett s sydes mr practical methods forincorporating summary timetoevent data into metaanalysis trials denost q adam jp pontallier a celerier b laurent c rullier elaparoscopic total mesorectal excision with coloanal anastomosis for rectalcancer ann surg saurabh b chang sc ke tw huang yc kato t wang hm tzuliang cwfingerhut a natural orifice specimen extraction with single stapling colorectalanastomosis for laparoscopic anterior resection feasibility outcomes andtechnical considerations dis colon rectum dindo d demartines n clavien pa classification of surgical complicationsa new proposal with evaluation in a cohort of patients and results of asurvey ann surg almazrou am suradkar k mauro cm kiran rp characterization ofreadmission by day of rehospitalization after colorectal surgery dis colonrectum costantino fa diana m wall j leroy j mutter d marescaux j prospectiveevaluation of peritoneal fluid contamination | Colon_Cancer |
"immunotherapy is revolutionising cancer treatment and has already emerged as standard treatment for patients with recurrent or metastatic gastric cancer gc recent research has been focused on identifying robust predictive biomarkers for gc treated with immune checkpoint inhibitors icis the expression of programmed cell death protein ligand1 pd l1 is considered a manifestation of immune response evasion and several studies have already reported the potential of pd l1 expression as a predictive parameter for various human malignancies meanwhile based on comprehensive molecular characterisation of gc testing for epstein barr virus and microsatellite instability is a potential predictive biomarker culminating evidence suggests that novel biomarkers such as the tumour mutational burden and gene expression signature could indicate the success of treatment with icis however the exact roles of these biomarkers in gc treated with icis remain unclear therefore this study reviews recent scientific data on current and emerging biomarkers for icis in gc which have potential to improve treatment outcomesintroductionthe prognosis for metastatic or recurrent gastric cancer gc remains very poor making it the third leading cause of cancer related death worldwide1 however the recent development of immune checkpoint inhibitors icis targeting the programmed cell death protein1 pd1 and programmed cell death protein ligand1 pd l1 pathways has produced improved outcomes for gc and already been successfully incorporated into clinical practice2 in particular checkpoint inhibition with anti pd1 monoclonal antibodies such as pembrolizumab and nivolumab has led to durable and significant responses in a minority of gcs3 as a result interest has increased in selecting the right patient population to benefit such icis along with further exploration of immunotherapy notably various tumour related and host related factors with a critical impact on systemic immune functions may influence the response to icis6 moreover a significant proportion of patients do not respond to these therapies and there can also be a threat of unpredictable immune related adverse events aes and even severe toxicity7 therefore identifying more robust predictive biomarkers is critical to optimise treatment with icis while avoiding unnecessary treatment of patients who could develop life threatening or life altering aesgc is a heterogeneous and complex disease9 thus various approaches such as molecular classification have already been proposed for the subhistological exploration of gc as a potential tool for more effective therapeutic strategies the cancer genome atlas research network tcga suggested a comprehensive molecular characterisation of gcs using various platforms and proposed four distinct subtypes epstein barr virus ebv positive microsatellite unstable microsatellite instability msi genomically stable and tumours with chromosomal instability10 more recently the asian cancer research group acrg described four subtypes msi microsatellite stable mssepithelial to mesenchymal transition mssand msstp53 negative11 tp53 positive while the above subtypings show some overlapping molecular aberrations msi was identified as a subtype by both tcga and acrg2 msi is a molecular marker of a defective function of the dna mismatch repair mmr system that recognises and repairs the erroneous insertion deletion and mis incorporation of bases that can arise during dna replication and recombination as well as repairing some forms of dna damage12 it is also well known that msi high msi h tumours exhibit a high tumour mutational burden tmb neoantigen load and immune infiltration making them respond well to icis13 meanwhile the distinct characteristics of ebv positive gc is the overexpression of pd l1 and pd l214 the driving features of pd l1 positivity in ebv positive gc can also be effectively targeted with immunotherapy similar to the msi h subtype interestingly in a recent phase ii trial by kim kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0copen accesspembrolizumab showed a promising efficacy in patients with msi h and ebv positive tumours15 accordingly based on a better molecular characterisation of gc this review focuses on the current and emerging biomarkers for icis that would facilitate precision medicineclinical evidences of icis in gcseveral phase ii and iii trials have recently investigated the pd1 and pd l1 blockade in gc as summarised in table pembrolizumab is an igg4 human antibody targeting pd1 thereby interfering with the interaction between pd1 and pd l120 in the phase ib keynote trial patients with pd l1 positive gc or gastro oesophageal junction gej cancer were enrolled in both asian and non asian countries21 the objective response rate orr was and durable responses were seen with a median duration of responses of weeks the subsequent phase ii multicohort keynote059 trial cohort enrolled patients with recurrent or metastatic gc and gej cancer who received two pretreated lines of chemotherapy4 here the orr was and median overall survival os was months following these two trials the randomised phase iii keynote061 trial compared pembrolizumb monotherapy with paclitaxel in patients with pd l1 positive gc that had progressed on first line flouropyrimidine and platinum doublet chemotherapy17 while patients with a pd l1 status were initially unselected pd l1 positive patients with a combined positive score cps of or higher were included in the latter part of the study the cps is the number of pd l1 staining cells including tumour cells lymphocytes and macrophages divided by the total number of viable tumour cells while a tumour proportion score tps is the percentage of viable tumour cells showing partial or complete membrane staining relative to all viable tumour cells present in the sample22 approximately of patients were found to have pd l1 positive tumours using the cps pembrolizumab did not meet its primary end point of a longer os and progression free survival pfs in patients with pd l1 positive tumours notwithstanding it is worth noting that patients who expressed pd l1 cps of or higher exhibited a better benefit from treatment with pembrolizumab in post hoc analyses although these subgroup analyses should be interpreted with cautionsubsequently the keynote062 was a large randomised first line clinical trial of patients with advanced gc or gej cancer who were randomly assigned to one of three arms pembrolizumab at mg every weeks for up to years pembrolizumab plus chemotherapy cisplatin and fluorouracil or capecitabine or placebo plus chemotherapy23 pembrolizumab was non inferior to chemotherapy for os in patients with cps or higher no survival benefit was observed with the addition of pembrolizumab to chemotherapy compared with chemotherapy alone in this studynivolumab also an igg4 antibody is very similar in structure to pembrolizumab except that nivolumab binds to the n terminal loop on the pd1 molecule while pembrolizumab binds to the cd loop24 the phase iii attraction2 ono453812 trial compared nivolumab with a placebo in asian patients with unresectable or recurrent gc that was refractory to or intolerant of at least two previous standard chemotherapy regimens5 the results showed a significantly prolonged os for the nivolumab group with a year os rate of vs more recently the long term survival was reported showing year survival rates of for nivolumab and for placebo25 nivolumab also showed a significant advantage compared with the placebo in terms of pfs and the radiological objective response therefore these results provide randomised evidence that nivolumab is a valid approach to improving the clinical outcomes for patients with gc in a third line and subsequent line setting however the overall clinical value of attraction02 was partially limited by several important issues26 the patient population was only asian and pd l1 positivity reported at a low frequency of as this was assessed as tps rather than cps was not associated with the survival outcomes plus there was no comparative data on quality of life a phase iii checkmate032 study also reported that nivolumab and nivolumab plus ipilimumab provide a durable antitumour activity in heavily pretreated western patients with chemotherapy refractory gc gej cancer and oesophageal cancer19 in particular the clinical benefit of nivolumab monotherapy was consistent with that reported for asian patients in the attraction02 study yet similar to the attraction02 study there was no association of pd l1 positivity according to tps with survival outcomes therefore ongoing randomised controlled trials of icis including pembrolizumab and nivolumab in earlier line treatment need to unify assessment of pd l1 expression and create more accurate profiles of aes in gcavelumab is an igg1 antibody which binds to the front beta sheet of pd l1 and possesses pd1pd l1 blockade activity with antibody dependent cell mediated cytotoxicity adcc28 there are some differences between pd1 inhibition and pd l1 inhibition as pd1 targeting therapeutic antibodies including pembrolizumab and nivolumab block the pd1pd l1 or pd1pd l2 interaction to restore tumor specific t cell reactivity without mediating adcc24 the javelin gastric trial the ï¬rst study to compare avelumab with standard chemotherapy in third line treatment for gc did not achieve its primary end point of improving os or the secondary end points of pfs and orr18 this negative finding may be attributed to the usage of the active comparator in the control arm in addition there are possible reasons including the different drug biding sites heterogeneity of tumour biology and methodology of pd l1 testing notwithstanding fewer patients experienced aes with avelumab than with chemotherapy although researchers found no evidence of clinical beneï¬t compared with commonly used chemotherapy in any of the examined subgroups including the tumour pd l1 expression status kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0csecnerefersodehcaer tonsfp rristnopdne yramirp ichpargoegrrinogerlaboglso sfplaboglsorrrrinasainasanretsewsolabogl tneitaprebmunrecnac cirtsagn i srotibhniii tnopkcehc enummi lgnitauave seduts iii idna ii esahpdetcees l l ebatsmra tnemtaertsutats ldpgnittesesahpeman ydutstegratstnegabamuzilorbmepdetceesnul eni ldrihtretal robmuzilorbmepevitisop dnoceslexatilcapenilbamuovnilobecapldetceesnul eni ldrihtretal roiiiiiiii trohoc etonyekdpbamuzilorbmepetonyekdpbamuzilorbmepi notcarttaonodpbamuovnlixopac roxosbamuovnil bamumilipibamuovnilbamuovnil bamumilipibamuovnilretal rodetceesnuleni ltsrifiiiiii notcarttadp trapdetceesnul eni ldrihtiiietamkcehcdpbamuovnlibamuovnlii¡ecohc snacsyhpiibamuevaldetceesnuleni ldrihtiii cirtsagnlevaj i ldpbamuevalevitisop ldp erew stneitapgnoma open access dna s xos etar esnopser rri lavvrus eer fnossergorpi sfp dnagii lnetorphtaed llec demmargorp ldp inetorphtaed llec demmargorp dpi lavvrus llarevo so nitaplil axodna enbacepac iixopacnitaplilaxoskeew yreve iipovni gkg mbamum ilipl i supgkg mbamuovn il skeew yreve povniii gkg m bamumilipl i supgkg mbamuovn illecyc tnemtaert kee w a fohcae dna syadno mg m nacetoniri ro dna syad no mg m lexatilcap¡kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0copen accessthese results might support the potential of avelumab for combination or maintenance therapy notwithstanding the recent javelin gastric trial with maintenance avelumab therapy following initial chemotherapy in gc produced disappointing results29 although there was more efficacy seen with avelumab in an exploratory analysis of pd l1 positive patients with a cps ¥ vs months no significant difference with respect to os was observed between the avelumab maintenance and continued chemotherapy groups vs months p0177as discussed above several clinical trials are currently ongoing with various strategies30 the phase iii attraction04 nct02746796 trial is evaluating nivolumab plus standard chemotherapy oxaliplatin plus either s1 or capecitabine versus chemotherapy alone in asian patients16 the phase ii component part of this study showed chemotherapy plus nivolumab led to an orr of and median pfs of months while the subsequent phase iii trial part is evaluating the survival outcomes another phase iii trial checkmate649 nct02872116 has completed recruitment with over patients randomising patients to oxaliplatin based chemotherapy alone or in combination with nivolumab or nivolumab plus ipilimumab32 the third arm of this study terminated recruitment early due to early safety signalantiangiogenic treatment such as ramucirumab and bevacizumab is also generating recent interest as several studies have suggested that simultaneously blocking angiogenesis and pd1 pathways induces synergistic antitumour effects especially involving the control of the tumour microenvironment tme33 researchers including the current authors noted that ramucirumab in combination with pembrolizumab jvdf showed a manageable safety profile with favourable antitumour activity in patients with previously treated gc35 consequently the phase iii nivoram nct02999295 trial has evaluated the safety and tolerability of the addition of ramucirumab to nivolumab in patients with gc as second line therapy7 as an alternative antiangiogenic and multitargeted kinase inhibitor a phase i trial of regorafenib plus nivolumab is also exploring in gc nct0340687136 this trial demonstrated an orr of in gc and icis pretreated patients with gc achieved a partial response pr in patients interestingly of the seven patients who had received prior anti pd1pd l1 treatment three achieved an objective responseanother interesting phase ii trial investigating the role of pembrolizumab plus trastuzumab combined with chemotherapy in patients with previously untreated human epidermal growth factor receptor positive tumours is currently ongoing nct0295453637 preliminary results from this study showed of patients experienced reduction in target lesions after one dose of pembrolizumabtrastuzumab before oxaliplatincapecitabine was introduced in second cycle the high orr of was coupled with median pfs of months this has led to the current phase iii keynote811 study randomising patients to chemotherapy plus trastuzumab with or without pembrolizumb nct0361532638 thus these therapeutic strategies including combination treatment represent a true opportunity in the contemporary treatment of gc and may produce further success when considering integrative genomic databiomarkers for icis in gcthe identification and validation of reliable biomarkers are important to facilitate precise patient selection and increase the clinical benefit from icis an overview of the predictive roles of biomarkers obtained from tissue or blood and their characterisation in the management of gc is briefly summarised in table pdl1 expressiontesting for pd l1 expression by ihc is the current standard in most solid tumours and several studies have already assessed the clinical outcomes according to the pd l1 expression status in gc nevertheless different antibodies are being used for ihc to assess with different performance and different scoring criteria for pd l1 expression a recent multicentre study blueprint pd l1 ihc comparison project attempted to compare the performance of each ihc pd l1 assay in lung cancer39 three assays including 22c3 for pembrolizumab for nivolumab and sp263 for durvalumab were found to be comparable to each other in the staining of tumour tissue whereas sp142 for atezolizumab was found to be less sensitive however further study is required to carefully validate these assays in gcas noted above in the keynote059 trial pd l1 expression was assessed using the pd l1 ihc 22c3 pharmdx assay and measured using a cps4 this trial demonstrated a higher orr vs in patients with high pd l1 expression defined as cps ¥ both pfs and os were also more prolonged in this group40 although pembrolizumab in a second line trial keynote061 did not significantly prolong os greater benefits were seen in tumours with higher pd l1 expression cps ¥ orr25 cps ¥ orr16 cps orr217 however the attraction02 and javelin gastric trials showed no clinical improvement for pd l1 positive tumours as they used tps rather than cps18 these differences may also have been due to the use of different cut off points and scoring systems a lack of standardisation of the assays and testing platforms the heterogeneous nature of pd l1 expression in tumours intratumouralintertumoural heterogeneity and intraobserverinterobserver variability41 for instance the attraction02 study retrospectively evaluated pd l1 expression using a pd l1 ihc pharmdx assay defined as the tps while pd l1 expression was prospectively assessed in tumour cells tumour associated lymphocytes and macrophages using a 22c3 pharmdx assay in keynote06117 as such pd l1 positivity was only reported in about patients using tps with nivolumab whereas it is generally kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0ctable overview of candidate biomarkers associating with response to immune checkpoint inhibitors in gastric cancerbiomarkerssample source methodspd l1tumourihctreatmentrecent results in gastric cancerreferencespembrolizumab expression of pd l1 ¥ associated with open accessbetter clinical efficacy orr mrdpembrolizumab expression of pd l1 cps of or higher associated with better clinical efficacy os orrpembrolizumab expression of pd l1 associated with higher response rateebv positivitymsi htumourin situ hybridisationpembrolizumab ebv positivity associated with higher tumour msi testing or ihcnivolumbresponse ratemsi h associated with clinical efficacy orr dcr ospembrolizumab msi h associated with better clinical efficacy orrpembrolizumab msi h associated with better clinical efficacy orr ospembrolizumab msi h associated with better clinical tmbtilstumour or bloodtumourwes or targeted sequencingimage analysing software or manually countedtoripalimabicisefficacy orrtmb associated with better clinical efficacy orr ospresence of tils associated with better clinical efficacy in various solid tumours but very limited data in gcgeptumour multigene profilingpembrolizumab ifn gamma gene signature associated with better clinical efficacy orr pfspembrolizumab t cell inflamed gene signature gut microbiotastoolnlrbloodculture or molecular technique sequencingmetagenomicscomplete blood counticisicisnivolumabassociated with better clinical efficacy orr pfsvarious species associated with enhancement and iraes of icis in various solid tumoursincreased nlr correlated with dcr and osdecreased change of nlr associated with better survivalsixty seven patients had tumours from the stomachcps combined positive score dcr disease control rate ebv epstein barr virus gc gastric cancer gep gene expression profiling icis immune checkpoint inhibitors ifn interferon ihc immunohistochemistry iraes immune related adverse events mrd median response duration msi h microsatellite instability nlr neutrophil to lymphocyte ratio orr overall response rate os overall survival pd l1 programmed cell death protein ligand pfs progression free survival tils tumour infiltrating lymphocytes tmb tumour mutational burdenbetween and using cps ¥ as the cut off more recently using the checkmate032 study data tumours were re scored using cps and there was better correlation between nivolumab treatment and survival even at the cps ¥ level44 thus one of the coprimary patient population in the first line checkmate649 study that has recently completed recruitment is including a subpopulation of patients with pd l1 cps ¥ebv positivityebv status is also emerging as a potential biomarker for personalised treatment strategies in gc14 in situ hybridisation detection of ebv encoded small rna in tumour cells is generally recommended to identify ebv associated gc ebvagc30 the incidence of ebvagc varies from to in different regions with an average of worldwide45 nevertheless this subgroup is associated with better prognosis thus less frequently found in advanced or metastatic setting in particular ebv positive tumours frequently display pd l12 overexpression and occasional immune cell signalling activation10 several research groups found that the level of pd l1 expression ranging from approximately to of ebvagc with kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0copen accessvariable results between studies20 pd l1 positivity has also been significantly associated with a poorer prognosis than pd l1 negativity in ebvagc furthermore ebv triggers a significantly higher infiltration of cd8 t cells in tme46 in previous studies of patients with ebv positive cancer the current authors showed that high levels of tumour infiltrating lymphocytes tils were associated with a favourable prognosis while intratumoural pd l1 positivity with a worse prognosis47in the phase i javelin solid tumour trial where avelumab was shown to be beneficial for a patient with metastatic gc it is worth noting that ebv positive tumours with a low mutation burden and msi tumours with a high mutation burden had statistically significantly higher tumour lymphocytic infiltration when compared with mss tumours48 the strength of immune mediated signalling signatures in ebv positive tumours also represents a t cell inflamed tme10 these findings support the concept that icis can be used in patients with gc with ebv by suppressing the pd1 pathway in tumour cells and allowing immune activation a recent phase ii trial by kim demonstrated improved efficacy associated with pembrolizumab in patients with ebv positive tumours15 this study enrolled patients with pretreated gc in a subgroup analysis pembrolizumab monotherapy as salvage treatment showed that all six ebv positive patients with gc attained pr orr100 with a median duration of months however in another study out of patients considered ebv positive were treated with toripalimab50 only one pr was observed with two stable and one progressive diseases patients with pr was also pd l1 positive these contrasting results with pembrolizumab could be due to toripalimab rather than ebvagc as a predictive biomarker for icimicrosatellite instabilityhighmmr deficiency is generally characterised by a failure to repair dna replication associated errors leading to the accumulation of mutations in microsatellite regions of the genome51 these phenomena are known as msi52 currently two different methods have been validated for detecting msi h53 the mmr status is assessed by ihc staining to measure the expression levels of the proteins involved in dna mmr and a polymerase chain reaction based exam also tests the length of repetitive dna that are known as microsatellite in the normal and tumour tissues while there are discrepancies between the ihc of mmr protein expression and msi test results the overall concordance in the two tests is high52 the incidence of msi in gc varies between countries being relatively high in approximately of western patients51 msi h gc is commonly associated with intestinal type female sex older age lack of lymph node metastases and onset in the distal stomach54 to date multiple retrospective studies and limited prospective studies have reported on a positive association between msi h and a better prognosis in resectable gc55 for example the magic study reported that patients with msi h tumours have superior survival compared with patients with mssmsi low msi l tumours when treated with surgery alone and conversely have inferior survival to patients with mssmsi l tumours when treated with perioperative chemotherapy plus surgery56 however similar to ebv status patients with msi h had better prognosis thus only of patients with metastatic gc would be mmr deficient the prognostic and predictive values of the msi status on the survival of patients with metastatic gc remain a subject of debate52theoretically in the presence of mmr deficiency undetected dna replication errors leading to a tumour with a high mutational burden reproduce various neoantigens that stimulate t cell activation and tumour infiltration by immune cells keynote012 trial reported that msi h tumours showed a partial response in two out of four patients regardless of pd l1 expression21 a subgroup analysis of keynote059 revealed an orr of for patients with msi h gc4 in keynote061 and more recently reported keynote062 there was a substantially enhanced survival benefit in patients treated with pembrolizumab compared with chemotherapy17 similar to the results for ebv positive tumours the clinical study by kim also showed that msi h tumours responded particularly well to pembrolizumab monotherapy orr85715 in the checkmate032 trial that assessed the efficacy of another pd1 monoclonal antibody nivolumab the orr was for the msi h group vs for the msi l group or mss group19 therefore this evidence highlights the potential of msi h as a predictor of the response to icis in gcof note whereas msi hmmr deficiency is the most consistent predictor of efficacy to icis in gc a substantial portion of msi h gc still has unsatisfactory outcomes even with icis the degree of msi and resultant mutation load in part might explain the variable response to pd1 blockade in mmr deficient tumours57 tumours sensitive to pd1 antibodies showed a loss or a reduction in tumour allele frequency of missense non synonymous single nucleotide variant and indel mutations after pd1 treatment suggestive of immune editing of tumour cellstmb and neoantigentmb may be a potential biomarker of outcomes with icis in multiple solid tumour types41 generally cells have a number of repair pathways to maintain their genome stability13 the mutational load acquired by defective dna repair pathways frequently alters protein function and expression resulting in the formation of neoantigens that serve a source of antitumour immune response therefore it is reasonable to hypothesise that tumours with a high mutational load are more likely to produce neoantigens and increase immunogenicity58 in turn this course of reaction induces a more intensified immune response resulting in tumours becoming more sensitive to treatment with icis41 although tumour specific neoantigens with high clonality are more predictable and beneficial for the response to icis accurate measurement kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0cof these neoantigens is known to be expensive and time consuming59 in this situation tmb could be a good approach for indirectly evaluating the neoantigen load tmb is defined by the total number of somatic non synonymous mutations per coding area of the tumour dna58 several studies have already demonstrated the predictive impact of tmb in lung cancer and melanoma one early study by yarchoan observed a significant correlation between tmb and orr for anti pd1 or anti pd l1 therapy60 rizvi also reported that patients with tmb 50th percentile exhibited an improved durable clinical benefit rate and pfs versus those with lower tmb61 this benefit was also seen in the checkmate227 study that included patients with advanced non small cell lung cancer nsclc who received a combination of nivolumab and ipilimumab as the first line metastatic setting62 a significantly prolonged pfs was reported for the patients with higher tmb treated with the combination treatment irrespective of the expression of pd l1 likewise a large scale study across multiple cancer types found a significant association between tmb and the clinical outcome63 these findings can also provide a novel strategy for subgroups with high tmb considering that the measurement of the mutational load is a critical factor for therapeutic successhowever for patients with gc there is still insufficient evaluation and conflicting results on the utility of tmb as a biomarker of the response to icis65 interestingly wang performed a tmb analysis of patients with chemorefractory gc who were treated with toripalimab as a monotherapy in a prospective phase ibii clinical trial50 in this study tmb high tmb h patients responded signiï¬cantly better than the tmb low patients orr vs p0017 a survival benefit has also been demonstrated for patients with high tmb os vs months p0038 similar correlation was also found between tmb h according to circulating tumour dna and better survival when treated with pembrolizumab in gc15 in light of recent approaches this close relationship between tmb and clinical outcomes also points to the possibility of tmb as a predictive biomarker in patients with gc however in the study with regorafenib plus nivolumab due to small sample size tmb did not correlate with response or pfs to this combination36despite its identiï¬ed significant predictive role there are still many challenges in precisely estimating tmb first it is difficult to apply the protocol including whole exome sequencing or targeted sequencing panels using next generation sequencing to clinical practice due to various problems such as the sample amount cost sensitivity coverage and analysis time8 second a standardised cut off value for tmb has not yet been clearly established since many studies have reported a wide range of cutoffs for different tumour types58 thus given the variety of tmb cutoffs assays related with tmb in clinical studies should be interpreted cautiously moreover the availability of tumour sampling to detect tmb is commonly limited and tmb may present temporal open accessvariability a novel blood based tmb approach could be considered as an alternative method as the advantages of repeating sampling during treatment could provide information of a dynamic immune reaction8 plus this approach is less invasive and enables investigators to document the evolution of tmb interestingly in a study by gandara et al blood based tmb was correlated with tissue based tmb and showed a longer pfs in patients with metastatic nsclc who received treatment with atezolizumab68 in summary tmb could be a novel and independent biomarker that reflects the therapeutic effects of icis in gc however its accuracy in predicting the efficacy of icis varies among studies and still needs to be explored for gctumour infiltrating lymphocytesthe immune microenvironment of tumours is now recognised as an important determinant for understanding the relationship between a patients immune system and their cancer informing prognosis and guiding immunotherapy like icis69 tumour cells are typically surrounded by infiltrating inflammatory cells such as cytotoxic t cells helper t cell subsets regulatory t cells tumor associated macrophages dendritic cells and myeloid lineage leucocytes70 among these differentiated lymphocytes referred to as tils are considered a manifestation of the host immune response against tumour cells and seem to play an important role in various human malignancies71 several studies have already reported the potential of ti | Colon_Cancer |
" natural orifice specimen extraction surgery is a novel technique of minimally invasive surgery thepurpose of this study was to compare the safety of laparoscopic anterior resection with natural orifice specimenextraction noselar and abdominal incision specimen extraction aiselar for sigmoid or rectum tumorsmethods medline pubmed embase central cochrane central register of controlled trials scopus andclinicaltrials databases were systematically searched for related s up to august the primary outcomesincluded postoperative complications overall postoperative complication incisionrelated complicationanastomotic fistula and severe complication and pathologic results lymph nodes harvested proximal resectionmargin and distal resection edge the statistical analysis was performed on stata softwareresults ten studies comprising patients were used for metaanalysis compared with aiselar noselar hadmore advantages in terms of overall postoperative complication odds ratio or ci to p incisionrelated complication or ci to p distal resection edge weighted meandifference wmd cm ci to cm p recovery of gastrointestinal function wmd day ci to day p pain scores in postoperative day wmd ci to p additional analgesics usage or ci to p and hospital stay wmd day ci to day p while the operation time of noselar was prolonged wmd min ci to min p the anastomotic fistula severe complication lymph nodes harvested proximalresection margin intraoperative blood loss and longterm outcomes in noselar were comparable with aiselars the safety of noselar was demonstrated and it could be an alternative to conventional surgery inlaparoscopic anterior resection for sigmoid and rectal tumors however further randomized and multicenter trials are requiredkeywords natural orifice specimen extraction nose laparoscopic anterior resection rectal tumor sigmoid tumor metaanalysis correspondence guangenyang163com1department of colorectal surgery hangzhou third hospital hangzhou zhejiang peoples republic of chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0che world of surgical oncology page of introductionover the past three decades laparoscopic surgery hasevolved incessantly especially in the field of colorectalsurgery it has been widely accepted by surgeons and patients in light of the better perioperative outcomes andanalogical longterm effectiveness compared with opensurgery for colorectal cancers []for conventional laparoscopic colorectal operation asmall laparotomy in the abdomen is required for specimen harvested and colorectal anastomosis because ofthe miniincision it causes many undesirable outcomessuch as incision pain wound infection scar and even incision hernia and the advantages of laparoscopic surgeryare reduced [] to minimize those drawbacks anovel surgical variant known as natural orifice specimenextraction nose surgery with the features of naturalorifice specimen extraction and totalintraperitonealanastomosis has been introduced and is becoming ahotspot [] some studies have reported the oncology and safety outcomes between nose surgery andconventional laparoscopic surgery are comparable [] and the nose surgery therefore is supposed tohave a progress of minimally invasive surgeryrecently some metaanalysis studies had comparednatural orifice specimen extraction nose with abdominal incision specimen extraction aise in laparoscopiccolorectum resection for the colorectal disease [ ]however colorectum resection comprises right hemicolectomyleft hemicolectomy and anterior resection procedures among those surgeries were largely different and the surgical procedures and the excision extension between sigmoid and rectum resection aresimilar in addition several studies compared laparoscopic anterior resection with natural orifice specimenextraction noselar with abdominal incision specimen extraction aiselar for sigmoid or rectum tureleased [] hence wemors wereconducted this metaanalysis to evaluate the safety ofnoselar in sigmoid and rectal tumorsrecentlymethodsstudy design and inclusion criteriathis metaanalysis followed the preferred reportingitems for systematic reviews and metaanalysis prisma statements the inclusion and selection criteria were determined before starting a literature searchonly when studies with fulltext on sigmoid or rectaltumors that compared noselar and aiselar andreported at least one of the endpoints of focus were retrieved and analyzed the most comprehensive researchwas recruited when overlapping researches was conducted by the same team no language restriction wasapplied conference s case reportsreviewsrobotic surgery and singleportwere not consideredlaparoscopic surgeryselection criteria conformed to the framework ofpico participant intervention comparison and outcome patients diagnosed with sigmoid or rectal tumorsbenign and malignant tumors requiring surgery wereincludedinterventions consisted of noselar andaiselar noselar was compared with aiselarin all eligible studies primary endpoint outcomes werepostoperative complications overall postoperative complication abdominal incisionrelated complication anastomotic leak and severe complication claviendindoclassification ¥ iii and pathologic results retrievedlymph nodes proximal resection edge distal resectionedge secondary outcomes included operation timeblood loss pain score numeric rating scale score additional analgesics gastrointestinalfunction recoveryhospitalization duration 5year diseasefree survivaldfs and 5year overall survival os search strategythe following databases had been searched up to august medline pubmed central cochrane central register of controlled trials embase scopus andclinicaltrials for a more accurate search the followingkeywords andor mesh terms were used sigmoid neoplasms rectal neoplasms colorectal neoplasmslaparoscopynatural orifice specimen extractiontransvaginal specimen extraction transanal specimenextraction and transrectal specimen extraction thespecific search strategies among databases existed differences the search strategy of pubmed was presented inadditional text reference s of the eligible studies were reviewed to find the potentially relevant studiesstudy selection and quality assessmentretrieved studies were independently assessed for relevance by reviewers changjian wang and jinmingchen by screening the title and in order to enhance sensitivity studies were removed only when bothreviewers excluded the study subsequently a fulltextassessment was performed on the initial screening included studies the risk of bias was assessed by thenewcastleottawa scale nos for observational studies and studies achieving five or more stars were eligible cochrane collaborations tool for assessing risk forbias was used for randomized controlled trials [ ]all discrepancies were discussed before a final decisionwas madedata collection and statistical analysisdata from the recruited studies were extracted by tworeviewers changjian wang and jinming chen andused formeananalysis outcome valuesfurther 0che world of surgical oncology page of standard deviation and median interquartile rangewere extracted from each study considering potentialheterogeneity among studies we pooled the results byusing a randomeffects model the weighted mean difference wmd and confidence intervals cis wereapplied for continuous variables and the odds ratioor and cis were used for dichotomous variablesthe continuous outcomes were adopted the inverse variance method and dichotomous outcomes were adoptedthe mantelhaenszel statistical method when a studymerely offered the outcomes with median and interquartile range an estimation based on formulas designed byhozo was performed if a study did not provide the hazard ratio hr and cis of 5year dfsorand os the methods presented by tierney wereused for data extraction from survival curves thechisquare test and isquared value were used for measuring heterogeneity and i2 p was definedas significant heterogeneity sensitivity analyses basedon nos score ¥ and the sample size of noselargroup ¥ were conducted to assess the potentialsource of heterogeneity and the robustness of the resultspublication bias was examined with a funnel plot andharbord test p was considered statistically significant the statistical analysis was performed onstata softwareresultsliterature selection and characteristicsthe initial database search identified s ofwhich were removed based on the title and assessment the rest of the literature were evaluated byfulltext assessment and studies were excludedcharacteristics of the excluded studies were presented inadditional table ten studies were finally included forfurther qualitative and quantitative synthesis [ ] all of these were retrospective studies the process of the search and selection wasshown in fig a total of patients were recruitedin those studies with patients in the noselargroup and patients in the aiselar group themain characteristics of studies and patients were presented in table and details were shown in additionalfig flow chart of studies included in the metaanalysis 0che world of surgical oncology page of sunasunasunasunasunasunarosunaiangavsunasunasunarorororomutcermutceridomgsimutceridomgsimutceridomgsimutceridomgsiralesaipurnsonerocsetisinemcepsnoitcartxenoitacolromutleamefeamlrednegmutceridomgsimutcermutcermutcerralesonpuralesaipuraeyaegaralesonpuralesaipuralesonpugnitnuocisetapcitrapngisedydutsinogerraeyydutsisedutsdeducnliehtfoscitsiretcarahcinamelbatevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorternapajlateadasihianhclateuhianhclategnianhclategnahzianhclateuohzianhclategnxiianhclateuliydutsaissurydutsevitcepsorternawatihbaruaslateevitcepsorterecnarflatetsonedevitcepsorterydutsydutsianhclategnawrnleacsawattoeltsacwensonnoitcartxenemicepsnoisicnilianmodbahtiwnoitceserroiretnacipocsorapalralesainoitcartxenemicepsecifirolarutanhtiwnoitceserroiretnacipocsorapalralesonisnoitaverbbaegnarinademronoitavedidradnats±naemsadrocertondetropera 0che world of surgical oncology page of euavlpytienegoretehieuapvldmwstluserldeoopicrororalesairalesonstneitapfoonfoonisedutsnoitacilpmocsemoctuollafostluserldeoopehtelbatsemoctuoyramirpsemoctuoevitarepotsopllarevonoitacilpmocnoitacilpmocdetaerlnoisicnilautsifcitomotsananoitacilpmocerevesdetsevrahsedonhpmylsemoctuoilcgoohtapingramnoitceserliamxorpegdenoitceserlatsidnoitcnufsemoctuoyradnocesemitnoitarepossoldooblevitarepoartnilanitsetnortsagifoyrevocerdopinapevitarepotsopegasusciseganallanoitiddayatslatipsohsoraeyevfisfdraeyevfiecnereffidnaemdethgewdmwinoitcartxenemicepsnoisicnilianmodbahtiwnoitceserroiretnacipocsorapalralesainoitcartxenemicepsecifirolarutanhtiwnoitceserroiretnacipocsorapalralesonisnoitaverbbaliavvruseerfesaesidsfdliavvrusllarevosoyadevitarepotsopdopoitarsddoro 0che world of surgical oncology page of table the results of the pooled outcomes were summarized in table primary outcomesall included studies reported the overall postoperativecomplication the pooled data revealed that the postoperative complication in of patients whotreated with noselar and of patientswho treated with aiselar the or was ci to p with low heterogeneity i2 fig 2a among the studies eight studies reportedthe incisionrelated complication in of patients who underwent noselar and of patients who underwent aiselar the or was ci to p with no heterogeneity i2 fig 2b nine studies reported the anastomoticfistula of which ng reported zero events in bothgroups the remaining eight studies recorded anastomotic fistula in of patients sufferednoselar and of patients suffered aiselar or was ci to p withno heterogeneity i2 fig 2c a severe complication was defined based on the claviendindo classification two of the included studies recorded severecomplication claviendindo classification ¥ iii and thesevere complication in of patients withnoselar and of patients with aiselar or was ci to p withsignificant heterogeneity i2 fig 2da total of nine studies reported lymph node harvestthere was no significant difference in lymph node harvestbetween the two groups wmd ci to p no significant heterogeneity was observed i2 fig 3a the mean number of dissectedlymph nodes in the noselar group was and theaiselar group was the proximal resection marginwas reported in studies and the wmd in the upper resection edge was cm ci to cm p with no heterogeneity i2 fig 3b the distalresection margin was reported in studies and the wmdin the inferior resection edge was cm ci to cm p with no heterogeneity i2 fig3c the length of the distal resection margin in the twogroups was cm noselar group and cmaiselar groupsecondary outcomesa total of nine studies recorded operation time and intraoperative blood loss the pooled data revealed thatthe wmd of operative duration was min ci to min p heterogeneity i2 fig 4a the wmd of blood loss was ml ci to ml p heterogeneity i2 fig 4bfig forest plot comparing postoperative complications in thenoselar group and aiselar group a overall postoperativecomplication b incisionrelated complication c anastomotic fistulaand d severe complication 0che world of surgical oncology page of fig forest plot comparing pathologic outcomes in the noselar group and aiselar group a lymph nodes harvested b proximal resectionmargin and c distal resection edgesix studies provided data about the recovery of gastrointestinal function the wmd of bowel movement was day ci to day p heterogeneity i2 fig 5a the postoperative painwas recorded in studies and studies hisada and wang recorded the postoperative pain periodand the remaining reported the pain scores in postoperative day pod the wmd of pain score in pod 0che world of surgical oncology page of fig forest plot comparing intraoperative outcomes in the noselar group and aiselar group a operation time and b blood loss was ci to p heterogeneity i2 fig 5b the additional analgesicusage rate was also reported in those studies and theor of additional analgesics usage was ci to p heterogeneity i2 fig 5c theduration of hospital stay was reported in nine studiesthe wmd of hospital stay was day ci to day p heterogeneity i2 fig 5dfiveyear diseasefree survival dfs and 5year overallsurvival os were available in two studies the hazardratio hr in the 5year dfs was ci to p heterogeneity i2 fig 6a and thehr in the 5year os was ci to p heterogeneity i2 fig 6bsensitivity analysissensitivity analysis results based on the nos score ¥ and the sample size of noselar group ¥ were presented in additional table it showed a slight inconsistency in distal resection edge operation time andrecovery of gastrointestinal function and all the otheroutcomes showed a similar trend of results between thetwo groupspublication biasa funnel plot of overall postoperative complication wasperformed to detect publication bias it showed that allthe inclusive studies were within the confidenceinterval and no publication bias was found fig inaddition a harbord test confirmed there was no publication bias p discussionas a technique used for clinical treatment the safety ofnoselar should be efficiently proved morbidity isone of the most efficient indicators for assessing thesafety of an emerging technique postoperative complications may not only lead to failures of surgery but alsothreaten lives the overall postoperative complicationrate in noselar was lower than that in aiselar severe morbidity claviendindo ¥ iiiamong the two techniques was not a significant difference the operation involving digestive tract reconstruction anastomotic leakage is a potential risk once itoccurs reoperation is usually inevitable the incidence of anastomotic leakage in noselar wassimilar with that in aiselar in addition theincidence ofincisionrelated complications in noselar was significantly lower than that in theaiselar group obviously the reduction ofcomplications in noselar has largely attributed tothe decrease of incisionrelated complications althoughthe complications in noselar were reduced the riskof bacteria contamination in the peritoneal cavity shouldnot be neglected costantino had reported the peritoneal contamination in the nose group was higherthan that of the conventional group hence measures such as bowel preparation prophylactic antibioticsperitonealtransluminalwound retractor and abdominal drains are recommended to avoid the contamination of the peritonealcavity irrigationtransanallavagethe postoperative pathology results to some extentalso reflect the safety of a surgery this metaanalysisshowed lymph nodes harvested between the two groupswas comparable and it also conformed to the minimumrequirement of the guideline retrieved more than nodes in our metaanalysis the proximal marginin the noselar group was similar with the conventional group however the distal margin in the nosegroup was longer than that of the aiselar group the 0che world of surgical oncology page of potential cause of this difference was the use of transanalspecimen eversion and extraabdominal resection technique in the nose group [ ] because of thisprocedure the distal rectal resection is performed extraabdominally under direct vision moreover circumferential resection margin crm between the two groupshave no difference [ ] in addition accordingto our metaanalysis the longterm outcomes 5yeardfs and 5year os were comparable all of those indicated the nose technique was a safety procedure in thetreatment of sigmoid and rectal cancers nevertheless aconcern about tumor seeding was raised during the procedure of enterotomy and specimen extraction it is necessary to apply several measures such as the use ofprotection devices sterile specimen bags and avoidingoverpulling and compression during specimen extraction as a minimally invasive surgery noselar had moreadvantages in alleviating patients distress the reductionof pain scores in postoperative day pod was observed and this reduction could be attributed to thetrauma in noselar being further reduced owingto less pain the need for additional analgesics was alsoreduced in addition accelerating postoperative recoverywas also observed the recovery of gastrointestinal function and hospital duration in patients who sufferednoselar was much shorter besides some scholarsmay doubt if there have alterations in sexual urinary ordefecation function in the groups according to the included studies there were no differences in functionaloutcomes such as sexual urinary or defecation betweentwo groups [ ] even though a small part of patientsexperienced function alteration and the alternation wasreversible [ ] those all demonstrated thatnoselar was a safety surgery and to some extent ithad advantages in postoperative recoverynevertheless our study has several limitations firstlyintersphincteric resections were mixed with coloanalanastomoses with sigmoid cancer in our studies although there exist some differences we mixed the twotechniques and mainly considered there existing common procedures between sigmoid and rectum resectionin laparoscopic anterior resection and some studies didnot record the methods of outcome evaluation such asblood loss evaluation to some extent it reduces thecomparability of outcomes secondly the present metaanalysis relied solely on retrospective studies and someoriginal studies not presented how patients were selectedto be candidates for one technique or another the quality of all included studies was regarded as fair or good however this type of study cannot be comparedwith a randomized controlled trial and potential biascannot be ruled out thirdly this study only recruitedone multicenter research and some outcomes includedfig forest plot comparing postoperative recovery in the noselar group and aiselar group a recovery of gastrointestinalfunction b postoperative pain pod c additional analgesicsusage and d hospital stay 0che world of surgical oncology page of fig forest plot comparing longterm outcomes in the noselar group and aiselar group a 5year dfs and b 5year oslimited studies so further multicenter randomized controlled and more comprehensive studies containing adequate outcomes are needed fourthly the results ofsome pooled results among studies existed heterogeneity the sensitivity analysis could not be detected as thecause of heterogeneity although some results existedheterogeneity the major results were homogeneity andthe heterogeneity of outcomes such as operation duration blood loss and hospital stay can be explained byclinical heterogeneity such as the difference of patientssurgeons patient management and differences in surgical proficiency in nose technology in addition the results of the major parameters were robust all in all theresults of this analysis are convincedaccording to our metaanalysisthe advantages ofnose are reduced overall complications especiallyincisionrelated complications increased distal resectionedge enhanced recovery of gastrointestinal function reduced postoperative pain reduced additional analgesicsusage and shortened hospital stay and without an auxiliary patients operated by the nose technique achievebetter aesthetics however the operative time is prolonged although the nose technique has many advantagesthere are many requirements that should befollowed before the application of this technique in colorectal surgery firstly the nose should be operated byexperienced surgeons with conventionallaparoscopiccolorectal surgery secondly the indication of noseshould follow the indication of conventionallaparoscopic colorectal resection the depth of tumor invasionshould be within t3 and body mass index bmi shouldbe less than kgm2 for transanalnose and less than kgm2 for transvaginalnose transanal nose suitsfor male or female patients and the tumor diameter isfig funnel plot of the overall postoperative complications 0che world of surgical oncology page of recommended less than cm while transvaginal noseis only applied for female patients and the tumor diameter is limited within cm and the emergent conditionssuch as bowel obstruction perforation and massivebleeding are excluded all in all as surgeons follow appropriate indicationsthe noselar for sigmoid or rectal tumors is a safesurgery and the longterm outcomes between twooperations have no difference and the benefits of thenoselar in shortterm outcomes are noticeablethese findings promote enthusiasm in support ofnose surgery as an alternative approach forthetreatment of sigmoid and rectal tumorssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s1295702001982wadditional file additional file text the search strategy of pubmedadditional file additional table characteristics of the excludedstudiesadditional file additional table summary of the included studiesadditional file additional table the results of sensitivity analysisadditional file additional table quality assessment based on thenos for retrospective studiesabbreviationsnoselar laparoscopic anterior resection with natural orifice specimenextraction aiselar laparoscopic anterior resection with abdominal incisionspecimen extraction wmd weighted mean difference or odds rationos newcastleottawa scale dfs diseasefree survival os overall survivalcis confidence intervals hr hazard ratio pod postoperative day acknowledgementswe wish to thank the timely help given by junfeng hu in statistic analysisand mengdan zhou in language editingauthors contributionsguangen yang and zhong shen contributed to the conception and designof the study jun he performed the literature search and the writing of themanuscript changjian wang and jinming chen performed the data extraction haibo yao performed the data analysis qinyan yang and jianmingqiu participated in the writing of the manuscript guangen yang and haibo yao helped to revise the intellectual content the authors read and approved the final version of the manuscriptfundingthis work was funded by the zhejiang natural science foundation grantno lq19h160013 and the zhejiang medical health science and technologyproject grant no and the hangzhou health science andtechnology project grant no 2017a26availability of data and materialsall the data analyzed in this study was obtained from the included originals or related authorsethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of colorectal surgery hangzhou third hospital hangzhou zhejiang peoples republic of china 2departments ofgastroenterology pancreatic surgery zhejiang provincial peoples hospitalhangzhou zhejiang peoples republic of chinareceived february accepted july references wang cl qu g xu hw the short and longterm outcomes oflaparoscopic versus open surgery for colorectal cancer a metaanalysisint j color dis ishibe a ota m fujii s suwa y suzuki s suwa h momiyama m watanabej watanabe k taguri m midterm followup of a randomized trial ofopen surgery versus laparoscopic surgery in elderly patients withcolorectal cancer surg endosc allaix me giraudo g mistrangelo m arezzo a morino m laparoscopicversus open resection for colon cancer 10year outcomes of aprospective clinical trial surg endosc lee l aboukhalil m liberman s boutros m fried gm feldman lsincidence of incisional hernia in the specimen extraction site forlaparoscopic colorectal surgery systematic review and metaanalysissurg endosc hennessey db burke jp nidhonochu t shields c winter dc mealy k riskfactors for surgical site infection following colorectal resection a multiinstitutional study int j color dis goto s hasegawa s hata h yamaguchi t hida k nishitai r yamanokuchis nomura a yamanaka t sakai y differences in surgical site infectionbetween laparoscopic colon and rectal surgeries subanalysis of amulticenter randomized controlled trial japanmultinational trialanization prev int j color dis park js choi gs lim kh jang ys kim hj park sy jun sh clinical outcomeof laparoscopic right hemicolectomy with transvaginal resectionanastomosis and retrieval of specimen dis colon rectum ooi bs quah hm fu cw eu kw laparoscopic high anterior resection withnatural orifice specimen extraction nose for early rectal cancer techcoloproctol franklin mj kelley h kelley m brestan l portillo g torres j transvaginalextraction of the specimen after total laparoscopic right hemicolectomywith intracorporeal anastomosis surg laparosc endosc percutan tech wolthuis am de buck voa d'hoore a laparoscopic natural orificespecimen extractioncolectomy a systematic review world j gastroenterol xingmao z haitao z jianwei l huirong h junjie h zhixiang z totallylaparoscopic resection with natural orifice specimen extraction nose hasmore advantages comparing with laparoscopicassisted resection forselected patients with sigmoid colon or rectal cancer int j color dis leung al cheung hy fok bk chung cc li mk tang cn prospectiverandomized trial of hybrid notes colectomy versus conventionallaparoscopic colectomy for leftsided colonic tumors world j surg hisada m katsumata k ishizaki t enomoto m matsudo t kasuya ktsuchida a complete laparoscopic resection of the rectum using naturalorifice specimen extraction world j gastroenterol ma b huang xz gao p zhao jh song yx sun jx chen xw wang znlaparoscopic resection with natural orifice specimen extraction versusconventional laparoscopy for colorectal disease a metaanalysis int j colordis liu rj zhang cd fan yc pei jp zhang c dai dq safety and oncologicaloutcomes of laparoscopic nose surgery compared with conventionallaparoscopic surgery for colorectal diseases a metaanalysis front oncol zhou s wang x zhao c pei w zhou h liu q liang j zhou z wang xcomparison of shortterm and survival outcomes for transanal natural 0che world of surgical oncology page of orifice specimen extraction with conventional minilaparotomy afterlaparoscopic anterior resection for colorectal cancer cancer manag res xing j zhang c yang x wang h wang h yu e fu c comparison of shortterm outcomes of transrectal specimen extraction during laparoscopicsigmoid radical resection versus conventional laparoscopically assistedprocedure zhonghua wei chang wai ke za zhi wang r wei z liu q li w xiao l han hf yang s transanal versustransabdominal specimen extraction in laparoscopic rectal cancer surgery aretrospective analysis from china wideochir inne tech maloinwazyjne ng hi sun wq zhao xm jin l shen xx zhang zt wang j outcomes oftransanal natural orifice specimen extraction combined with laparoscopicanterior resection for sigmoid and rectal carcinoma an observational studymedicine baltimore 20189738e12347liu z efetov s guan x zhou h tulina i wang g tsarkov p wang x amulticenter study evaluating natural orifice specimen extraction surgery forrectal cancer j surg res hu jh li xw wang cy zhang jj ge z li bh lin xh shortterm efficacy ofnatural orifice specimen extraction surgery for low rectal cancer world jclin cases moher d liberati a tetzlaff j altman dg preferred reporting items forsystematic reviews and metaanalyses the prisma statement ann internmed w64 hawker ga mian s kendzerska t french m measures of adult pain visualanalog scale for pain vas pain numeric rating scale for pain nrs painmcgill pain questionnaire mpq shortform mcgill pain questionnaire sfmpq chronic pain grade scale cpgs short form36 bodily pain scalesf36 bps and measure of intermittent and constant osteoarthritis painicoap arthritis care res 201163suppl 11s240 higgins jp altman dg gotzsche pc juni p moher d oxman ad savovic jschulz kf weeks l sterne ja the cochrane collaborations tool forassessing risk of bias in randomised trials bmj 2011343d5928 ga wells bsdo the newcastleottawa scale nos for assessing the qualityof nonrandomised studies in metaanalyses hozo sp djulbegovic b hozo i estimating the mean and variance from themedian range and the size of a sample bmc med res methodol tierney jf stewart la ghersi d burdett s sydes mr practical methods forincorporating summary timetoevent data into metaanalysis trials denost q adam jp pontallier a celerier b laurent c rullier elaparoscopic total mesorectal excision with coloanal anastomosis for rectalcancer ann surg saurabh b chang sc ke tw huang yc kato t wang hm tzuliang cwfingerhut a natural orifice specimen extraction with single stapling colorectalanastomosis for laparoscopic anterior resection feasibility outcomes andtechnical considerations dis colon rectum dindo d demartines n clavien pa classification of surgical complicationsa new proposal with evaluation in a cohort of patients and results of asurvey ann surg almazrou am suradkar k mauro cm kiran rp characterization ofreadmission by day of rehospitalization after colorectal surgery dis colonrectum costantino fa diana m wall j leroy j mutter d marescaux j prospectiveevaluation of peritoneal fluid contamination | Colon_Cancer |
"melatonin is a sleepregulating hormone created by the pineal glandand is released at night it has been found to have biological activityin almost all living anisms including plants animals and microbes itcan quickly enter cells through the bilipid bilayer and exhibit scavenging activity towards oxygen free radicals as well as antioxidant properties due to its low molecular weight and amphiphilic nature peripheral tissues have been found to show a high afï¬nity towardsthis hormone melatonin and hence act on receptors and bindingsites studies reported by tan show that melatonin can alsobe produced in the mitochondria and hence tissue melatonin levelsare more than that of serum levels hence can be used as a moleculethat targets mitochondria the main physicochemical and biologicalproperties of melatonin are sleepinducing effects antioxidant behavior [] antiammatory activity [] antiapoptotic effects and neuroprotective effects it also regulates various corresponding authoremail address renjithsbcollegeacin r thomas101016jmolliq2020114082 elsevier bv all rights reservedphysiological functions of the brain as melatonin can diffuse quicklythrough the bloodbrain barrier it is effectively used in the treatmentof brain injuries rapid eyemovement sleepbehavior disorder patients are managed by a combination treatment of melatoninand clonazepam [] the sensing of bacteria through tolllikereceptor4 and regulation of bacteria through altered goblet cells andantimicrobial peptides are all involved in the anticolitic effects of melatonin in ammatory bowel disease melatonin is involved in theaging process growth towards puberty and modulation of blood pressure this versatile compound blocks proangiogenic andantiangiogenic effects caused by docetaxel and vinorelbine which areantitumor drugs and it enhances their tumorï¬ghting behavior this molecule can modify the redox state of the rat pancreaticstellate cellmelatonin is an endogenous hormone that is involved in circadianrhythm control it is inexpensive and safe as it has a significant effectas an antioxidant and antiammatory melatonin chemically nacetyl5methoxytryptamine is a tryptophan derivative has multiplephysiological effects and can be used to treat many diseases related tovirus infections especially respiratory diseases in covid19 patientswith digestive complications melatonin has positive effects 0cn alzaqri of molecular liquids melatonin can thus be used as an adjunctive or even as a regular therapyas no antiviral treatment is currently available electrochemical measurements of melatonin overï¬ow demonstrate that melatonin secretiondecreases with age melatonin treatments result in the enhancement of essential oil production in salvia species in the context of the recent covid pandemic melatonin can beresearched as a potential molecule to control the dangerous effect ofthis disease rising patients' tolerance and decreasing the mortality infatal virus infections would control the innate immune response and reduce ammation during this period melatonin is a molecule with respective properties as it decreases the overreaction of the innateimmune response and overshoots ammation but also facilitatesadaptive immune function even though melatonin is a critical biomolecule few works havebeen reported on the electronic structure and reactivity of this moleculeexcept for a preliminary work reported by turjanski and coworkers in using semiempirical methods in this manuscript we describe a detailed investigation into the quantum mechanical propertiesof melatonin its spectral features reactivity preferences and the resultsof docking studies with three known structural protein receptors of thenovel coronavirus2 we found that melatonin docks strongly with thethree proteins we hypothesise that this compound can be used as anadjuvant medicine for the treatment of covid19 also significant restby a person peacefully sleeping in dark surroundings will enhance theproduction of this hormone which could help in the management ofcurrent patients or as a preventive measure in the vulnerablepopulation material and methodsthe melatonin molecule was optimised using the gaussian09 software package with the dftb3lyp functional and the 6311g 2dp basis set b3lyp is a commonly used functional and 63112dpbasis set is mediumsized basis set with diffused functions over heavyatoms and polarization functions to bring accuracy we performed frequency calculations to ensure that no imaginary frequency exists suchthat the geometry determined would correspond to a global minimumfor reaching the optimised geometry we used the same geometry for calculating frontier molecular analysis natural bonding orbitals and nonlinear optical studies for uvvisible spectrum simulation we usedtimedependent density functional theory tddft with longrangecorrected camb3lyp functionals with 6311g 2dp as the basis setbecause electronic transitions are timedependent phenomena tddftcalculations are done using the optimised geometry obtained fromb3lyp6311g2dp simulations the frontier molecular orbitals wereviewed from the checkpoint ï¬le generated during the optimisation calculations a wavefunction ï¬le was generated during a single point groundstate calculation job using which the subsequent analysis performedthe melatonin molecule has more than two reaction sites for examplemethoxy carbonylamide and purine ring reaction sites of melatonincalculated using the multiwavefunction software for calculatingtotal electrostatic potential average localised ionization energies and noncovalent interactions as it is reported that melatonincan be used as an adjuvant therapeutic material to ï¬ght covid19 we decided to dock the molecule with three ncov2019 protein's rcsb site melatonin is effective in critical care patients by decreasing vessel permeability anxiety use of sedation and increasing the quality ofsleep which may also be beneï¬cial to covid19 patients for improvedclinical outcome melatonin especially has a high health proï¬le significant data indicate that melatonin reduces virusrelated diseases and willpossibly also be effective in patients with covid19 the target proteinswere downloaded cleaned removed alien atoms and molecules andthen used for docking the energy received from the swissdock software and the score values received from patchdock as well as thedocked results collected from biodiscovery studio software arepresented results and discussion geometry of melatoninthe geometry of the molecule explains its rigid structure thestructure of melatonin can be explained based on its physical parametersof bond lengths and bond angles between important atoms or groups asshown in fig the bond lengths of and arebonds of 1c14n 7c14n and 12h14n respectively and the corresponding bond angles are1090343° ° and ° for 1c14n7c 1c14n12h and 7c14n12h respectively the bond angle of° for 4c25o26c having bond lengths of and for 4c\\\\25o and 25o\\\\26c respectively the bond lengths of 18c21n21n22h 21n23c 23c\\\\24o and 23c\\\\30c are and respectively with the corresponding bondangles of ° ° ° ° ° and° for 18c21c22h 18c21c23c 22h22n23c 21n23c24o21n23c30c and 24o23c30c respectively frontier molecular orbital fmo properties of melatoninthe frontier molecular orbitals are highly reactive orbitals of othermolecules and some chemical descriptors are shown in table the higher occupied molecular orbital homo lower unoccupied molecular orbital lumo and energy gap of melatonin are and kcalmol respectively the energygap is significant indicating that the molecule is inherently stable theionization energy electron afï¬nity hardness softness chemical potential electronegativity electrophilicity index and nucleophilicity indexof melatonin are and kcalmol respectively interactionof the melatonin with the biological target can be explained by the softness value the softness value is high kcalmol indicatingthat the molecule can positively interact with biological systems andshow the desired effect electron transition study and excitedstate properties of melatonin insolutionthe electron transition study explains electrontransfer excitedstates we used the tddft formalism using camb3lyp functionalsand 6311g 2dp basis sets in an implicit solvation atmosphere ofmethanol using the iefpcm model as transmission occurs some energyis also emitted melatonin electron transitions to homo having a pyrrole ring and oxygen in methoxy homo1 over the pyrrole ring andethyl carbons and homo2 over acetamide oxygen nitrogen andethyl carbons with energies of and ev respectively melatonin electron transitions to lumo which is over the pyrrolering lumo which is over the pyrrole ring oxygen in methoxyethyl carbons and acetamide nitrogen and carbon and lumo2 having acetamide carbons acetamide nitrogen and carbons with energiesof and ev respectively the electronic spectral datausing tddft simulations indicate a significant λmax of nm in amethanol solvent the transitions are due to the movement of electronsfrom homo1 to lumo and homo to lumo with an oscillator strength of the electronic transitions are due to chargetransfer transitions from one region of the molecule to another whichindicates its inherent stability due to electronic excitations nonlinear optical behavior of melatoninscientists and technologists working in the molecular electronics ï¬eldare continuously searching for compounds with substantial nonlinearoptical nlo activity such compounds ï¬nd immense application in electronic displays surveillance equipment and consumer electronic gadgetscomputationally the ability of a molecule to act as an nlo material can be 0cn alzaqri of molecular liquids fig geometry of melatonindetermined from the polarizability and hyperpolarizability data []the nlo properties of melatonin are shown in table this is an essentialbehavior of melatonin that has a light absorption nature movement ofelectrons or protons as compared with a standard nlo material such asurea the dipole moment of melatonin is d which is times greater than urea hyperpolarizability mean polarizabilityand anisotropy of the polarizability of melatonin are and esu and which are and times greater than urea respectively the compound is not centrosymmetric hence generates secondorder spherical harmonics and betahyperpolarizability functions this compound can hence be used as an anic nonlinear optically active substance in anic electronicappliances nature of nbo study of melatonina molecule especially one with profound biological activity may havemany intramolecular electron delocalisation and hyperconjugativestabilisation regions natural bond orbital analysis which is a quantummechanical method is useful for this type of study the molecular orbitaltable frontier molecular orbital properties for melatoninchemical descriptorshomolumoionization energy i Éhomo homoelectron afï¬nity a Élumo lumoenergy gap homo lumoglobal hardness η i a global softness s ηchemical potential μ i a electronegativity Ï Î¼electrophilicity index Ï Î¼2 2ηnucleophilicity index n Ïenergy in kcalmolproperties of melatonin for the occupancy of the natural orbitals wereperformed by the nbo suite embedded in the gaussian softwarefrom donorbonding orbital Ï c1c2 with occupancy is toacceptor antibonding orbitals Ï c3c4 Ï c5c6 and Ï c7c8exhibiting the transition the energies are and kcalmol respectively from Ï c3c4 with occupancy is to Ï c5c6 and the rydberg orbital r c30 with the energies are and kcalmol respectively from Ï c5c6 having an occupancy is to Ï c3c4 r and r h33 with the energies are and kcalmol respectively from Ï n21c23 has occupancy to rydberg orbital r c30 and r h33 with the energies are and kcalmol respectively from Ï c23o24 having the occupancy is to Ï c1c2 Ï c2c3 r c23 and r h33 withthe energies are and kcalmol respectivelyfrom Ï c23c30 has occupancy is to r h17 r c18 rh19 r c23 r o24 r c30 r h33 Ï c1c2 Ï c1c6Ï c2c3 Ï c26h27 Ï c30h31 Ï c30h32 and Ï c30h33 having the energies are and kcalmol respectively from Ï o25c26 having occupancy is to r c30 andÏ c1c2 with the energies are and kcalmol respectivelyfrom Ï c26h27 to Ï c1c2 having the energy kcalmol withthe occupancy is from Ï c30h31 with the occupancy is to r h17 r c18 r h19 r c23 r o24 r c30 rh33 Ï c1c2 Ï c2c3 Ï c23o24 Ï c30h31 and Ïc30h32 having the energies are and kcalmol respectively from Ï c30h32 with occupancy is to r h17 rc18 r h19 r c23 r o24 r c30 r h33 Ï c1c2 Ïc2c3 Ï c30h32 and Ï c30h33 having the energies are and kcalmol respectively and from Ï c30h33 with the occupancy is to r h17 r c18 r c19 r c23 r c24 ro25 r c26 r h28 r c30 r h33 Ï c1c2 Ï c1c6Ï c2c3 Ï c2c4 Ï c18h19 Ï c23o24 Ï c26h27 Ï 0cn alzaqri of molecular liquids table nonlinear optics property for melatoninnonlinear propertydipole moment μhyperpolarizability βmean polarizability α0anisotropy of the polarizability δαmelatonin d esu esu esuurea d esu esu esucomparison of melatonin with urea times greater than urea times greater than urea times greater than urea times greater than ureac30c31 Ï c30h32 and Ï c30h33 having the energies are and kcalmol respectivelyfrom core bonding orbital c c23 which has the occupancy electrons move to antibonding r c23 r c30 and r h33 withthe transition energies are and kcalmol respectively from c c23 with the occupancy is to r h33 Ï c2c8 and Ï c26h28 having the energies are and kcalmol respectively from c o24 to Ï c26h29 has the energy is kcalmol with occupancy is and from c c30 having the occupancy is to r h17 r c23 r c30 r h33 Ïc2c8 Ï c26h28 Ï c30h31 and Ï c30h32 with the energies are and kcalmol respectively from lone pair orbital n n14 with the occupancy is to Ï c7c8 with the energy kcalmol from nn21 has the occupancy is to Ï c23o24 having the energy kcalmol and from n o24 having the occupancy is to rc23 r c30 r h33 Ï c1c2 and Ï c2c3 with the energiesare and kcalmol respectively fromantibonding orbital Ï c1c2 having the occupancy to rc30 and Ï c2c3 with the energies are and kcalmolrespectively from Ï c5c6 has occupancy is to r c30 withthe energy is kcalmol and from Ï c23o24 has occupancy is to r c30 with the energy is kcalmol the inherentstabilisation offrom the series ofhyperconjugative interactions presented above these interactions canalso be between the melatonin and the surrounding solvent moleculeswhich reveals its stabilisation in biological medium and also betweenthe molecule and the target proteins used in the dockingthe molecule is evident total electrostatic potentials esp and average localised ionization energy alie of melatoninthe electrostatic potential explains how reactive sites canundergo nucleophilic or electrophilic addition or substitution reactionsof melatonin shown in fig within bohr and a color changefrom blue to red indicates charges on elements from to the blue color appears in both methoxyoxygen and acetamideoxygen these are electronrich sites and electrophiles can quickly attack them the red color appears on all of the hydrogens these areelectronpoor sites and nucleophiles can quickly attack themthe alie clariï¬es the stability of any molecule based on saturatedand unsaturated bond electron movements which are localised ordelocalised the number of the resonance structure is proportionalto the stability of the molecule the alie of melatonin shown in fig iswithin the range of ± bohr color is from indigo to red and the numerical value is from to the blue color of protons in themethoxy group three protons in the sixmembered ring in the indolegroup methyl protons and both adjacent carbons in the acetamidegroup are all sites that act as electrophiles the red color ofacetamidecarbon conjugated carbon with oxygen atoms and bothacetamideamide and methoxy groups are all sites that act as nucleophiles these blue and red regions represent saturated bonds thebluishgreen regions are on indole rings to methoxy carbons via oxygenand acetamide chains this indicates that delocalised electrons and unsaturated bonds lead to several resonance structures and explains thestability of melatonin the electrophilic and nucleophilic reactive centres identiï¬ed above interact with the covid virus proteins and providevarious electrostatic and noncovalent interactions and increases drugafï¬nity noncovalent interaction nci properties of melatoninnoncovalent interactions are a valuable biological property of molecules and are nonbonded directly but are bound by some forces suchas hydrogen bonding van der waals bonding andor steric constraintsnoncovalent interactions of melatonin are shown in fig plotted as agraph with energy plotted versus a reduced density gradient hydrogen bonds appear from to au between oxygenand protons from the acetamide group the van der waals force rangesfig electrostatic potentials of melatoninfig average localised ionization energy for melatonin 0cn alzaqri of molecular liquids fig noncovalent interactions of melatoninfrom to au between acetamideoxygen and its adjacentprotons in both methyl and methoxy groups the steric force rangesfrom to au between the indole ring the methyl groupand the carbonylamide group noncovalent interactions are a groupof interactions like hydrogen bond pistacking hydrophobic interactions van der waal's forces iondipole interactions and dipoledipoleinteractions responsible for the stabilisation of the molecule and thedocking between melatonin and the covid proteins molecular dockingmolecular docking is one of the essential functions of biologically active molecules this is the theoretical evidence to design the structureand reactivity relationship of a molecule at present the covid19 pandemic caused by a new strain of the coronavirus is creating havocthroughout the world we made efforts to dock the melatonin withthe three proteins isolated from the virus represented through thepdb id 6lu7 6m03 and 6w63 were deposited in the database as mentioned in the methodology sectionwith the rapid spread of the novel coronavirus globally the designof vaccines is of great importance sarscov2 is an enveloped nonsegmented and single stranded positive sense rna virus the bestdrug target among coronaviruses is the main protease mpro also called3cl protease this is a key coronavirus enzyme and plays a vitalrole in mediating viral replication and transcription it is identiï¬ed ashaving a mechanismbased inhibitor the main targeting protease protein pdb 6lu7 is widely studied a series of frontier molecular orbital based interaction analyseswere performed on the complex between the main protease ofcovid19 and the peptidelike inhibitor whose fundamental structure was obtained from the protein pdb 6lu7 anothertargeted protease protein in an apo form pdb 6m03 shows themost stable form after binding with the selected drug threonine residue with the help of several covalent bond interactionwith a kcalmol docking afï¬nity lasinavir brecanavirtelinavir rotigaptide 13bis2ethoxycarbonylchromon5yloxy2lysyloxypropane and pimelautide can be consideredas the main protease inhibitors of covid19 by docking them tothe binding cavities of apo pdb 6m03 and holo pdb 6lu7 another protease protein pdb 6w63 is a reversible inhibitorthe ï¬avonoid narcissoside is reported to have a high afï¬nity towards the protease protein pdb 6w63 according to moleculardocking studies thus these three protease proteins pdb 6lu7pdb 6m03 and pdb 6w63 can be included in the category ofnonstructural proteins in the structure of sarscov2 from table the result from swissdock explains the biological activity of melatonin with coronavirus proteins pdb id 6lu7 6m03and 6w63 in general the total δg is more than kcalmol isright active luckily melatonin has a total δg of and kcalmol with coronavirus2 proteins pdb id 6lu7 6m03 and6w63 respectively and the total δg is directly proportional to the fullï¬tness energy values which are and kcalmol respectively it can also be seen from this table theinterfull ï¬tness intrafull ï¬tness full solvent ï¬tness full surface ï¬tnessδg complex polar solvent δg complex nonpolar solvent δg proteinpolar solvent δg protein nonpolar solvent δg ligand polar solventδg ligand nonpolar solvent δg van der waals force and δg electricforce relationships between melatonin and coronavirus2 proteins asreferredthe result from patchdock are as follows the score values are and total surface interacting area and and the minimum atomic contact energies are and kcalmol for melatonin with coronavirus2 proteins pdb id 6lu7 6m03 and 6w63 respectively figs and s1show the skeletal structure and protein residue interactions betweenmelatonin and coronavirus2 protein pdb id 6lu7 6m03 and 6w63table explains what protein residues are interacting with melatoninand details the residue names labels hydrophobic values pka valuesaverage isotropic displacements secondary structures residue solventaccessibility sidechain solvent accessibility percent solvent accessibility and percent sidechain solvent accessibility values of coronavirus2proteinstable and fig s2 show the residue structure of the favorable nonbond interactions between melatonin and coronavirus2 proteinstable lists favorable nonbond interactions of 6lu7 having conventional hydrogen bonds carbonhydrogen bonds pidonor hydrogenbonds pisulfur and pialkyl with melatonin 6m03 has pisigmapipi tshaped and pialkyl with melatonin while 6w63 has pipi tshaped pialkyl and pialkyl with melatonin along with the bond distance from chemistry fig s2 and table show the residue structure 0cn alzaqri of molecular liquids table swissdock result for melatonin with coronovirus2 proteins pdb id 6lu7 6m03 and 6w63energysimple ï¬tnessfull ï¬tnessinter full ï¬tnessintra full ï¬tnesssolvent full ï¬tnesssurface full ï¬tnessextra full ï¬tnessδg complex solvent polarδg complex solvent nonpolarδg protein solvent polarδg protein solvent nonpolarδg ligand solvent polarδg ligand solvent nonpolarδg van der waals forceδg electric forcetotal δg6lu7 kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol 6m03 kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol6w63 kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmolof the unfavorable nonbond interactions between melatonin and coronavirus2 proteins protein 6lu7 does not have any unfavorablestericinteractions protein 6m03 having three unfavorable nonbond interactions and protein 6w63 having unfavorable bumpnonbond interactions fig s2 and table show unsatisï¬ed bonds within melatonininteracting with coronavirus proteins when interacting protein 6lu7has one hydrogen donor and one oxygen acceptor protein 6m03 hastwo hydrogen donors and two oxygen acceptors and protein 6w63has one hydrogen donor and two oxygen acceptors with melatonin table shows noncovalent interactions between melatonin and coronavirus2 proteins hydrophobic groups of protein 6lu7 residues areahis41 aleu141 acys145 ahis164 amet165 and aglu166those of protein 6m03 residues are ahis41 amet49 aphe140 aleu141 acys145 amet165 aglu166 and aleu167 and those ofprotein residues 6w63 are ahis41 acys44 amet49 aleu50 amet165 aglu166 aleu167 and agln189 with melatonin as shownin fig s2 and table the hydrophilic groups of protein 6lu7 residuesare ahis41 aasn142 ahis164 aglu166 ahis172 aasp187 aarg188 and agln189 those of protein 6m03 residues are ahis41 aasn142 ahis163 ahis164 aglu166 ahis172 and agln189 andthose of protein 6w63 residues are ahis41 ahis164 aglu166 aasp187 aarg188 agln189 and agln192 with melatonin as shownin fig s3 and table neutral groups of protein 6lu7 residues are atyr54 agly143 and aser144 those of protein 6m03 residues are agly143 aser144 and apro168 and those of protein 6w63 residuesare apro52 atyr54 aarg188 and athr190 with melatonin asshown in fig s4 and table acidic groups of protein 6lu7 residuesare aglu166 and aasp187 that of protein 6m03 residue is aglu166 and protein 6w63 residues are aglu166 and aasp187 withmelatonin as shown in table and fig s5 basic group interactions ofprotein 6lu7 residues are ahis41 ahis164 ahis172 and aarg188those of protein 6m03 residues are ahis41 ahis163 ahis164 andahis172 and those of protein 6w63 residues are ahis41 ahis164and aarg188 as shown in table and fig s6 tan etal has shownthat melatonin and derivatives has excellent biological responses likeacting against oxidative stress and free radical scavenging []our studies show that melatonin molecule can interact with differentproteins present in the ncov19 virus and inhibit their proliferationthese results need further clinical follow up and could assist in the management of covid pneumonia conclusionswe conducted a detailed quantummechanical investigation of thehormone melatonin and regulation of the sleepwake cycle naturalbonding orbital studies revealed the intensity of several intramolecularinteractions the various frontier molecular orbital data explain the nature and physical parameters of melatonin and the nonlinear opticalproperties are compared with urea which is a standard materialfig skeletal structure of interactions between melatonin and 6lu7 a 6m03 b and 6w63 c coronavirus2 protein residues 0cn alzaqri of molecular liquids table interactions between melatonin and coronavirus2 protein residuesnamelabelhydrophobicitypkapdbidsavg isotropicdisplacementsecondarystructureresidue solventaccessibilitysidechain solventaccessibilitypercent solventaccessibilitypercent sidechainsolvent accessibility6m03 histidine6lu7 histidineahis41amet49methionineatyr54tyrosinealeu141leucineaasn142asparagineagly143glycineaser144serineacys145cysteineahis164histidinemethionineamet165glutamic acid aglu166ahis172histidineaasp187aspartic acidaarg188arginineglutamineagln189ahis41methionineamet49phenylalanine aphe140aleu141leucineaasn142asparagineglycineagly143aser144serineacys145cysteineahis163histidineahis164histidinemethionineamet165glutamic acid aglu166aleu167leucineapro168prolineahis172histidineagln189glutamine6w63 histidineahis41cysteineacys44methionineamet49leucinealeu50prolineapro52tyrosineatyr54histidineahis164methionineamet165glutamic acid aglu166aleu167leucineapro168prolineaasp187aspartic acidarginineaarg188agln189glutamineathr190threonineglutamineagln192helixturnhelixcoilturnturncoilturnsheetsheetsheetsheetcoilcoilcoilhelixhelixcoilcoilturnturncoilturnsheetsheetsheetsheetcoilturnsheetcoilhelixcoilcoilcoilcoilhelixsheetsheetsheetcoilturncoilcoilcoilcoilcoilwavefunction studies gave information about electrostatic potentialsaverage localised ionization and noncovalent interactions these datahelped to predict the reactivity and identify the active site of the reactivity of the molecule melatonin docks with novel coronavirus proteinsand shows a variety of interactions with an excellent docking scorewhich leads to inhibition of the virus proteins leading to its destructionhence clinicians can consider incorporating melatonin also in thecovid19 treatment regime after further studiestable favorable nonbond interactions between melatonin and coronavirus2 proteinspdb idsdistance categorytypefromfrom chemistrytoto chemistry6lu76m036w63hydrogen bondhydrogen bondhydrogen bondhydrogen bond otherhydrophobichydrophobichydrophobichydrophobichydrogen bondhydrophobichydrophobichydrophobicconventional hydrogen bondconventional hydrogen bondcarbon hydrogen bondpidonor hydrogen bond pisulfurpialkylpisigmapipi tshapedpialkylconventional hydrogen bondpipi tshapedpialkylpialkylaglu166nunk0hunk0hacys145sgunk0amet165caahis41unk0unk0hahis41unk0unk0hdonorhdonorhdonorhdonor sulfurpiorbitalschpiorbitalspiorbitalshdonorpiorbitalspiorbitalspiorbitalsunk0oahis164oaglu166ounk0acys145unk0unk0acys145aglu166ounk0amet165amet165hacceptorhacceptorhacceptorpiorbitals piorbitalsalkylpiorbitalspiorbitalsalkylhacceptorpiorbitalsalkylalkyl 0cn alzaqri of molecular liquids table unfavorable nonbond between melatonin and coronavirus2 proteinspdb idsdistance categorytypefromfrom chemistrytoto chemistry6lu76m036w63nilunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorable bumpunfavorable bumpunfavorable bump carbon hydrogen bondunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpacys145sgaglu166ounk0hagln189caagln189caagln189cbagln189cgagln189cgagln189cgagln189cgagln189cdagln189cdagln189cdagln189cdagln189oe1agln189oe1agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189haagln189haagln189haagln189hg1agln189hg2agln189hg2agln189he21agln189he21agln189he21agln189he21agln189he22agln189he22agln189he22stericstericsteric hdonorstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericunk0cunk0caglu166ounk0cunk0hunk0cunk0cunk0hunk0nunk0hunk0cunk0cunk0hunk0hunk0cunk0hunk0nunk0cunk0ounk0cunk0hunk0hunk0hunk0cunk0hunk0hunk0hunk0cunk0hunk0nunk0cunk0cunk0hunk0cunk0cunk0hstericstericsteric hacceptorstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericsterictable unsatisï¬ed bonds in melatonin with coronavirus2 proteinspdb ids6lu76m036w63nameunk0hunk0ounk0hunk0hunk0ounk0ounk0hunk0ounk0oatomunsatisï¬ed typehohhoohoodonoracceptordonordonoracceptoracceptordonoracceptoracceptorrenjith thomas conceptualization formal analysis investigation methodology project administration resources softwaresupervision validation visualization writing review editingdeclaration of competing interestthe authors declare that they have no known competing ï¬nancialinterests or personal relationships that could have appeared to uence the work reported in this paperacknowledgementsresearchers supporting project number rsp202078 king saudcredit authorship contribution statementuniversity riyadh saudi arabianabil alzaqri cenceptualization funding acquisition tpooventhiran investegation methodology original draft alialsalme investegation original draft ismail warad resourcesreview methods athira m john methodology writing draftappendix a supplementary datasupplementary data to this article can be found online at 101016jmolliq2020114082table noncovalent interactions between melatonin and coronavirus2 proteinshydrophobicityhydrophilicityneutral groupacidic groupbasic grouppdbids6lu7ahis41 aleu141 acys145 ahis164 amet165 and aglu1666m03 ahis41 amet49 aphe140 aleu141 acys145 amet165 aglu166 and aleu1676w63 ahis41 acys44 amet49 aleu50 amet165aglu166 aleu167 and agln189ahis41 aasn142 ahis164 aglu166 ahis172 aasp187 aarg188 and agln189ahis41 aasn142 ahis163 ahis164 aglu166 ahis172 and agln189ahis41 ahis164 aglu166 aasp187 aarg188 agln189 and agln192atyr54 agly143 andaser144agly143 aser144 andapro168apro52 atyr54 aarg188 and athr190aglu166 andaasp187aglu166aglu166and aasp187ahis41 ahis164 ahis172 and aarg188ahis41 ahis163 ahis164 and ahis172ahis41 ahis164 andaarg188 | Colon_Cancer |
" mucinous colon cancers mcc are characterized by abundant production of mucin muc2 proteinand are less sensitive to standard systemic chemotherapy we postulated that severepersistent endoplasmicreticulum stress ers aggravation in mcc would overwhelm compensatory cytoprotective pathways and induceapoptosisresults basal levels of ers markers were higher in mcc and dntcfls174t cells than nonmucinous tumors andthese levels were significantly increased by combinatorial treatment with ers aggravators celecoxib orlistatcombination treatment inhibited cell viability and synergistically induced apoptosis treatmentinduced cell deathwas ersdependent apoptotic pathways were not activated following knockdown of ers protein chop dual drugtreatment significantly reduced mucinous tumor growth in vivo and induced ers and apoptosis consistent within vitro experimentss novel therapies are needed since mcc are more resistant to standard systemic chemotherapy thisstudy suggests ers aggravation is a viable therapeutic strategy to reduce tumor growth in mcckeywords muc2 mucinous colon cancer xenograft colonoids endoplasmic reticulum stress mucinous colon cancers mcc account for only of colorectal cancers in the usa each year casesyear they arise from rapidly proliferating neoplastic cells with goblet celllike features thatproduce large quantities of mucin muc2 protein [] from a treatment standpoint they are less responsiveto systemic chemotherapy in the neoadjuvant and palliative setting chemoresistance of mcc has been attributed to the abundant extracellular muc2 protein thatmay act as a barrier against drug delivery or immune infiltration and forms an immunosuppressivehypoxic correspondence choudrymhupmcedu1department of surgery university of pittsburgh medical center hillmancancer center centre avenue suite pittsburgh pa usa2department of pharmacology chemical biology university of pittsburghmedical center pittsburgh pa usamicroenvironment that impairs treatment efficacy andallows cancer cells to thrive therefore our prior research has focused on targeted therapies that simultaneouslyinducemechanisms for neoplastic cell death []inhibit muc2productionandthe endoplasmic reticulum er is a major site forbiosynthesis posttranslational modification and properfolding of proteins like muc2 that are destined to be secreted from cells proteins that fail to undergo correct foldingmaturation undergo erassociated proteindegradation erad via ubiquitinproteasomeandautophagymediated pathways a variety of conditionsthat disrupt normal protein processing eg calcium imbalance hypoxia can overwhelm the ability of cells tomaintain proper protein processing in the er therebytriggering er stress ers and its associated molecularthe unfolded proteinsignaling pathways known as 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 0cdilly orphanet of rare diseases page of protective mechanismsresponse upr the upr pathways represent a coordinated effort by cells to decrease overall protein synthesisand improve protein foldingmodification or degradation however severe and persistent ers can overwhelm thesetriggermolecular pathways associated with cell death []we therefore hypothesized that mucinous colon cancerscharacterized by high muc2 protein turnover wouldexhibit elevated basal ers levels and be vulnerable totherapies that aggravate ers thereby inducing ersassociated cell death pathwaysandduring low to moderate levels of ers various prosurvival upr pathway proteins are activated these include heat shock protein grp78 glucose regulated protein also called bip immunoglobulin heavy chainbinding protein and three er transmembrane proteinsperk protein kinase activated by doublestrandedrnalike er kinase ire1 inositolrequiring enzyme and atf6 activating transcription factor duringers grp78 disassociates from these upr proteins tochaperone terminally misfolded proteins for degradationat the same time the freed upr proteins become activated and initiate signaling pathways that serve to correct or neutralize ers conversely during severe andpersistent ers proapoptotic upr signaling pathwaysare activated these include elevated expression of transcription factor chop cebp homologous protein alsocalled gadd153 inhibition of antiapoptotic proteinseg bcl2 stimulation of proapoptotic bh3only proteins eg bim and activation of caspases [ ]byinhibitionofersaggravationin this study we first characterized basal ers levels inmcc and then investigated whether aggravation of ersin these tumors would induce ersassociated cell deathwe studied a combination of two federal drug administration fda approved drugs to aggravate ers celecoxibnoncyclooxygenase [cox2] target sarcoplasmicendoplasmic reticulum calcium atpase [serca] and orlistaters activation by fatty acid synthase [fasn] inhibition[ ] serca inhibition on the er membrane activates ers by disrupting calcium homeostasis withinthe er while fasn inhibition triggers ers by a varietyof mechanisms including the disruption of protein lipidation an important process for normal protein foldingstability membrane association localization traffickingand secretion [] additional rationale for the useofthese drugs include the basal overexpression ofcox2 and fasn in mcc and prior studies demonstrating inhibition of muc2 expressionsecretion by targeting these two cancer pathways [ ] bothcelecoxib and fasn inhibitors have demonstrated effective in vitro and in vivo cellular growth suppressionin a variety of malignancies including colorectal cancerhowever clinicalthese drugs have beentrials ofdisappointing [ ] we postulate that the irrational use of such drugs against unselected cancers inclinical trials is likely responsible for lack of clinical efficacy to date and that mcc are more likely to be susceptible to such targeted therapies given their high basalers levelsresultsmcc and high muc2 producing cells exhibit elevatedbasal ers that correlates with muc2 expression levelswe compared basal ers levels in explant tissue from patients with mcc and nmcc we found significantlyhigher basal muc2 grp78 bip atf4 and chop protein expression levels in explant tissue from mcc compared to nmcc fig 1a similar results were foundwhen comparing high versus low muc2 expressingls174t cells dntcf4ls174t cells exposed to doxycycline for h high muc2 expressing cells expressedhigher levels of muc2 and upr proteins grp78 bipatf4 and chop compared to dntcf4ls174t cellslacking doxycycline exposure low muc2 expressingcells fig 1b or wildtype ls174t cells low muc2 expressing cells data not shownwe confirmed the association of muc2 expressionand upr pathway activity by demonstrating a decreasein grp78 bip levels following stable muc2 knockdown muc2 kd in ls174t cells compared to lentiviral lv control cells fig 1c these data demonstratehigher basal ers levels in mcchigh muc2 producingcells compared to nmcclow mucin producing cellsand suggest a correlation between muc2 expressionand cellular basal ers levels these results support ourhypothesis that mcc with high basal ers may be susceptible to ersaggravation as a therapeutic strategy toinduce ersmediated apoptosisers is aggravated by dual celecoxib plus orlistat drugtherapy in mcc and correlates with muc2 expressionlevelswe demonstrated a dosedependent increase in uprprotein grp78 bip levels in ls174t cells when exposed to increasing doses of celecoxib μm ororlistat μm for h consistent with theirknown role as ers aggravators fig 2a similarlymrna expression levels for grp78 bip chop andatf4 increased in ls174t cells exposed to combinationof celecoxib μm and orlistat μm for h figs2b explant tissues derived from mcc demonstratedsimilaratf4 andchop following combination therapy for h fig 2cwe demonstrated a significant increase in upr proteinsgrp78 bip atf4 peif2 [phosphorylatedeukaryotic initiation factor ] and chop in dntcf4ls174t cells exposed to doxycycline for h highincrease in upr protein levels 0cdilly orphanet of rare diseases page of fig see legend on next page 0cdilly orphanet of rare diseases page of see figure on previous pagefig mucinous colon cancers and high muc2 producing cell lines exhibit elevated basal ers that correlates with muc2 expression levels arepresentative pictures from immunofluorescence if analysis of human explant tissue comparing muc2 and ers proteins grp78 [bip] atf4chop between mucinous colon cancer mcc and nonmcc nmcc the bar graphs demonstrate mean intensity difference brepresentative pictures from if analysis of muc2 and ers proteins grp78 [bip] atf4 chop comparing highmuc2 expressing cells dntcf4ls174t cells exposed to doxycycline for h versus lowmuc2 expressing cells dntcf4ls174t cells without doxycycline exposure the bargraphs demonstrate mean intensity difference c representative pictures from if analysis of muc2 and grp78 bip comparing stable muc2knockdown kd in ls174t cells compared to lentiviral lv control cells western blot shows muc2 protein expression in muc2 kd cellscompared to lentiviral control cells error bars represents standard deviation sd from triplicate experiments p muc2 expressing cells following treatment with com μm and celecoxib μmbination of orlistatcompared to low mucin producing dntcf4ls174tcells lacking doxycycline exposure conversely combination therapyinduced ers aggravation was significantlyreduced following stable muc2 kd in ls174t cellscompared to lv control cells we found much higherlevels of grp78 atf4 peif2 and chop in lv controlcells exposed to dual drug therapy compared to levelsseen in muc2 kd cells figs 2d these results suggestthat the degree of druginduced ers aggravation correlates with muc2 expression levels and that high mucinproducing cells are more susceptible to drugmediateders aggravationtreatmenttreatment of ls174t cells with celecoxib μm ledto rapid decrease in ercytoplasmic calcium ratio at s consistent with inhibition of calcium atpase channelon the er membrane and the likely mechanism for ersaggravation fig 2e we evaluated changes in muc2sinceprotein secretion following orlistatfasn is essential for the posttranslationallipidationand secretion of muc2 protein we found that orlistat μm inhibited muc2 secretion from cos7 cellstransfected with psnmuc2mg vectorexpressingmuc2 nterminal fig 2f mechanistically we foundthat exposure to orlistat reduced fasn enzyme activitybut not enzyme expression levels fig 2g h moreover we performed abe assay in cos7 cells stably expressing muc2 nterminal our data demonstrated asignificant reduction in muc2 nterminal palmitoylation and secretion following orlistat therapy a likelymechanism for ers aggravation fig 2iers aggravation by celecoxib plus orlistat combinationtherapy induces ersmediated apoptosiswe treated ls174t cells with celecoxib μm andorlistat μm alone and in combination at varying doses for h fig 3ac the ic50 doses for celecoxib alone and orlistat alone were μm and μm respectively cell viability was significantly reduced at h following combination therapy with celecoxib μm and orlistat μm demonstrating acombination index of calculated using the computer software compusyn suggested synergy betweenin mitochondrialthe two drugs in reducing cell viability combinationtherapy induced significant apoptosis as demonstratedby tunel assay at h figs 3d ls174t cells exposedto combination therapy with celecoxib μm and orlistat μm for h demonstrated significant increasein upr proteins grp78 bip and chop proapoptoticmolecules bim noxa and puma and activatedcleaved caspase 3parp1 while single drug therapy atthese doses had minimal effect figs 3e we assessedchangestransmembrane potentialδÏm following dual drug therapy using mitochondrialmembranepermeant fluorescence dye jc1 jc1 aggregates reduced redfluorescent jaggregates were significantly reduced following cotreatment consistent withmitochondrial membrane damage and activation of intrinsic apoptosis fig 3f involvement of intrinsic mitochondrial apoptotic pathway was confirmed by cleavageof caspase but not caspase following dual drugtherapy fig 3g we also confirmed the induction ofupr and apoptotic proteins in colonoid cultures and explanttissue exposure to combination of celecoxib μm and orlistat μm for h resulted in apoptosis tunel assay in colonoid cultures fig 3h andmuch higher chop and cleaved caspase levels in explant tissue from mcc compared to nmcc suggestingtheirto ers aggravation and ersmediated apoptosis fig 3isusceptibilitysimilarly we demonstrated a significant increase inapoptotic markers puma cleaved caspase 3parp1in dntcf4ls174t cells exposure to doxycycline for h high muc2 expressing cells following combinationtreatment with orlistat μm and celecoxib μmcompared to low mucin producing dntcf4ls174tcellslacking doxycycline differentiation converselycombination therapyinduced apoptosis was significantlydampened following stable muc2 kd in ls174t cellscompared to lv control cells we found high levels ofpuma cleaved caspase 3parp1 in lv control cellsexposed to combination of orlistat and celecoxib whilethese were suppressed in muc2 kd cells figs 3j ourresults showed that combination treatmentinducedapoptosis correlated with muc2 expressionto determine whether combination therapy inducedapoptosis was ers dependent we tested this drug 0cdilly orphanet of rare diseases page of fig ers is aggravated by dual celecoxib plus orlistat drug therapyin mucinous colon cancer and correlates with muc2 expressionlevels a western blot assay of ers protein grp78 bip followingtreatment of ls174t cells with celecoxib μm or orlistat μm for and h qpcr assay of mrna expression for ersmarkers following treatment with celecoxib orlistat or combinationfor h in ls174t cells b and mcc explant tissue c representativepictures of if assay with bar graph demonstrating mean intensitydifference d western blot assay of ers markers comparing highmuc2 expressing cells dntcf4ls174t cells exposed to doxycyclinefor h versus low muc2 expressing cells dntcf4ls174t cellswithout doxycycline exposure and comparing stable muc2 kd inls174t cells compared to lv control cells following treatment withcelecoxib orlistat or combination for h e ratio of ercytoplasmiccalcium concentration at s in ls174t cells following combinationtreatment celecoxib orlistat f elisa assay of muc2 secretionfrom cos7 cells transfected with psnmuc2mg vector expressingmuc2 nterminal following treatment with celecoxib orlistat orcombination for h g fasn enzymatic activity assay in ls174tcells following combination treatment celecoxib orlistat for hand h western blot analysis of fasn expression levels i nterminalmuc2palmitoylation in cos7 cells stably expressing muc2 nterminal following single or dual drug therapy for h wasdetermined by abe assay and quantified by western blot assayhydroxylamine ham a strong reducing agent that cleavespalmitate from cysteine residues is necessary for biotinylation theomission of ham cleavage ham serves as negative control errorbars represents standard deviation sd from triplicate experiments p p p combination in chop knockdown ls174t cells chopkd we demonstrated a significant reduction in cleavedcaspase 3parp1 in chop kd cells compared to lvcontrol cells fig 3kcombination of celecoxib and orlistat reduced mucinoustumor growth in vivowe evaluated the therapeutic efficacy of this combination in vivo using ip pdx models ofluciferaselabelled ls174t cells seven days following ip tumorinoculation animals were treated ip with vehiclepbs celecoxib c alone mgkg orlistatoalone mgkg or combination of celecoxib andorlistat c o every other day for weeks animals per group drug dose selection for ip celecoxiband orlistat was based on prior publications and ourpilot studies [ ] treatmentrelated drug toxicitywas not encountered in the in vivo experiments dualdrug therapy resulted in significant reduction in mucinous tumor growth compared to either drug aloneas demonstrated by luciferase intensity measured byivis bioluminescent imaging system fig 4a tumortissue harvested from euthanized animals following weeks of therapy demonstrated a significant increasetunel positive staining with dualin apoptosistherapy fig 4b changesin upr protein levelschop and atf were consistent with those seen inin vitro studies fig 4c 0cdilly orphanet of rare diseases page of fig ers aggravation by celecoxib plus orlistat combinationtherapy induces ersmediated apoptosis mts cell proliferation assayin ls174t cells treated with increasing doses of celecoxib μm a or orlistat μm b or combination of celecoxib andorlistat for h c representative pictures of tunel assay in ls174tcells treated with single or dual drug therapy for h d westernblot analysis for ers and apoptotic markers at h e f ls174t cellswere treated with single or dual drug therapy and stained with jc1diffuse green jc1monomers indicate mitochondrial depolarizationdamage and punctate red jc1aggregates indicates intactmitochondrial membrane potential δÏm g western blot assay forcaspases in ls174t cells following single and dual drug therapy for h h representative pictures of tunel assay in colonoid culturesfrom mcc following single and dual drug therapy for h the bargraph demonstrates mean intensity difference i representativepictures from immunofluorescence if analysis of human explanttissue comparing caspase and chop between mcc and nmccfollowing single and dual drug therapy for h the bar graphdemonstrates mean intensity difference j western blot assay ofapoptotic markers comparing highmuc2 expressing cells dntcf4ls174t cells exposed to doxycycline for h versus lowmuc2expressing cells dntcf4ls174t cells without doxycycline exposureand comparing stable muc2 knockdown kd in ls174t cellscompared to lv control cells following treatment with celecoxiborlistat or combination for h k western blot assay of chop andapoptotic markers comparing stable chop knockdown kd inls174t cells compared to lv control cells following treatment withcelecoxib orlistat or combination for h asterisk represents astatistically significant difference compared with the control group p p discussionmcc are a unique histologic subtype in which greaterthan of the tumor mass is composed of extracellularmuc2 protein they demonstrate unique clinicalphenotype and molecular genotype when compared totheir nonmucinous counterparts nmcc clinicallymcc are more likely to occur in younger patients havea predilection for the proximal colon are bulkier at presentation and have a higher propensity for peritonealand distant lymph node metastases molecularly theytend to follow the serrated pathway for carcinogenesisand are more likely to have rasgnas mutationsmicrosatellite instability msi and cpg island methylator phenotype cimp positive [ ]in general many moleculartargeted therapies havedemonstrated disappointing results against advancedsolid cancers in clinical trials despite promising preclinical data this may largely be attributed to irrational useof targeted drugs against unselected cancers major considerations to improve the efficacy of targeted therapiesinclude identification of ideal drugs and drug combinations optimizing dosing schedules and enriching for patient factors eg clinical and molecular phenotypes andgenotypes that are more likely to respond to specifictargeted therapies in this study we focused on specificvulnerabilities of mucinous tumors that may make themsusceptibletherebytargeted therapiesto specific 0cdilly orphanet of rare diseases page of fig see legend on next page 0cdilly orphanet of rare diseases page of see figure on previous pagefig combination of celecoxib and orlistat reduced mucinous tumor growth in vivo murine xenograft models of intraperitoneal ip luciferaselabelled ls174t cells were treated with pbs alone control or celecoxib c alone mgkg bw or orlistat o alone mgkg bw orcombination of c o every other day starting on day following tumor implantation until they were euthanized at day gross intraabdominal tumor burden is depicted pictorially on day serial weekly changes in mean luciferase intensity during treatment for weeks areshown a error bars represent standard error of the mean among the xenografts in each treatment group harvested tumor tissue fromeuthanized mice day was subjected to tunel if assay b and if for ers markers c the bar graphs demonstrate mean intensity differenceasterisk represents a statistically significant difference compared with the control group p p applying a rational approach to treating wellselectedcancers we hypothesized that high basal ers of mccwould make them vulnerable to ers aggravation andersmediated cell death processes in this study we aggravated ers via two wellestablished pathways sercainhibition celecoxib to impair normal calcium homeostasis and fasn inhibition orlistat to exacerbate protein misfolding by inhibiting normal muc2 secretionour rationale for utilizing celecoxib was that serca is aknown noncox2 target of celecoxib and would therefore aggravate ers while at the same time celecoxib hasbeen shown to inhibit muc2 expression and decreasemucinous tumor growth in ex vivo and in vivo modelsof mucinous colonappendix cancers [ ] ourrationale for utilizing orlistat was that it inhibits fasnrequired for muc2 secretion which would induce ersthe same time reducing mucinous tumorwhile atgrowth [ ] moreoverthere is considerablecrosstalk between cox2 and fasn signaling pathwaysfasn plays a role in the conversion of excess carbohydrates to arachidonic acid a polyunsaturated fatty acidcox2 is responsible for converting arachidonic acid toprostanoids and noncox2 target pdk1 regulatesfasn activity [ ] both cox2 and fasn activitiesmodulate cancer signaling and have been shown tomodulate cell proliferation and survival [ ]in this study we first demonstrated that mcc andhigh muc2 producing cells dntcf4ls174t exposedto doxycycline had elevated basal ers levels comparedto nmcc and low muc2 producing cancer cells andthat the mucinous nature of these tumors excessivemuc2 protein production was responsible for the highbasal ers levels this laid the foundation for our targeted therapeutic approach since severe and persistenters ers aggravation can overwhelm normal protectivemechanisms upr to cope with this excessive stress andtrigger molecular pathways associated with cell deathwe then tested the therapeutic efficacy of dual drugtherapy with celecoxib and orlistat since they are bothers aggravators have demonstrated preclinical efficacyagainst a variety of cancers have mechanisms of actionthat would inhibit muc2 protein expressionsecretionand target cox2fasn cancer pathways with significant crosstalk ers aggravation as a potential mechanism to induce cell death in cancers has been tested incancer models by other investigators and we felt this approach would be especially effective in mucinous tumorsgiven the elevated basal ers levels ie rational application in wellselected tumors celecoxib is an fda approved drug used to prevent cancer progression in highrisk patients with familial adenomatous polyposis fapand continues to be investigated in clinical trials to improve the efficacy of standard chemotherapeutic drugs[ ] orlistat a reduced form of the natural productlipstatin is fda approved as an oral antiobesity drugsince it inhibits gastricpancreatic lipases and therebydecreases absorption due to its poor oral bioavailabilityintravenous formulations of orlistat are under development and have shown efficacy in preclinical modelswhile other fasn inhibitors continue to be tested asanticancer drugs in clinical trials in our studycombination treatment celecoxib orlistat aggravateders and synergistically induced markers associated withapoptotic cell death in mcc explants tissues and mccderived colonoid cultures treatmentinduced cell deathwas ersdependent since proapoptotic cell death pathways were not activated following knockdown of ersprotein chop dual drug treatment significantly reduced mucinous tumor growth in vivo and inducedmarkers of ers and apoptosis consistent with in vitroexperimentsour study has a number of limitations mechanistic studies assessing changes in ubiquitinproteosomeand autophagy markers following ers aggravationwould be importantto elucidate cellular changesthat underlie transition from cytoprotective upr tocytodestructiveapoptotic pathways such studieswould also help quantitate the magnitude of ers aggravation required to induce this transition additional studies with other ers aggravating agentswould confirm findings of this study and the proposed therapeuticthein vivo studies celecoxib and orlistat were administered ip which is not ideal for clinical trials whilecelecoxib may be given orally orlistat has poor bioavailability intravenous formulations of orlistat havebeen developed and other parenterally administeredfasn inhibitors are in clinical trials these alternate drugs and routes of administration will needto be tested in our modelsapproach for mcc for 0cdilly orphanet of rare diseases page of sin summary we found that combination of celecoxiband orlistat significantly aggravated ers and this wasmore pronounced in mcc and high muc2 producingcells dntcf4ls174t exposed to doxycycline compared to nmcc and low muc2 producing cancers andthat the mucinous nature of these tumors excessivemuc2 protein production was responsible for the highaggravated ers levels we also demonstrated that ersaggravation by dual therapy with celecoxib and orlistatinduced apoptosis and suppressed mucinoustumrowth providing a preclinical rationale for the use ofthis therapeutic strategy in the clinical settingrockfordmaterials and methodsreagentsdmem dulbeccos modified eagles medium was obtained from invitrogen carlsbad ca fetal bovineserum fbs was obtained from hyclone laboratorieslogan ut cellculture plates were purchased fromdenville scientific charlotte nc celecoxib and orlistat were obtained from cayman chemical ann arbormi cell titer aqueous one solution cell proliferation assay was obtained from promega madison withe enhanced chemiluminescence reagents ecl kitand pierce bca protein assay were obtained from thermoscientificil bd pharmigen fitcannexin v apoptosis detection kit i was obtained frombd biosciences san jose ca control muc2 andchop shrna lentiviral ps were obtained fromsanta cruz biotechnology santa cruz ca for westernblotting betaactin a1978 was obtained from sigmaaldrich st louis mo parp1 cst 46d11 grp78cst c50b12 atf4 cst d4b8 puma cstd30c10 noxa cst d8l7u chop cstl63f7and cleaved caspase 3cst asp175 were obtainedfrom cell signaling technologiesdanvers masytox orange for nucleic acid labeling was obtainedfrom life technologies grand island ny antirabbitand antimouse horseradish peroxidase hrpconjugated secondary antibodies were purchased from jacksonimmunology research west grove pacell culture and treatmentls174t cell line muc2 producing colon cancer cellswith goblet celllike characteristics was obtained fromamerican type culture collection high muc2 producing cells dntcf4 cell line under teton control system was kindly gifted by hans cleversutrechtnetherlands are able to differentiate into gobletlikecells following doxycycline induction h cellswere grown in dmem supplemented with fetal bovine serum 100iu penicillin and μgml streptomycin in co2 at °c in culture platesstable cell line generationls174t cells were incubated with muc2 or chopshorthairpin rna shrna h lentiviral ps and μgml final concentration polybrene sigmaaldrich following h of incubation the medium wasreplaced with complete dmem for h and then puromycin was added at a final concentration of μgmlcell were subcultured for weeks under puromycin selection to eliminate nontransduced cellscos7 cells were transfected with psnmuc2mgvector expressing muc2 nterminal a gift from gunnar hansson gothenburg sweden followingovernight incubation the medium was replaced withcomplete dmem for h and then g418 was added at afinal concentration of microgram per ml to eliminate nontransfected cellsconfocal imagingtumortissues were embedded in oct mediumcontaining cryomolds and immediately frozen in methylbutane then μm frozen tissue sections werecut using a cryostat and layered on super frost plusslides cells were grown on glass coverslips in wellplates for in vitro experiments the covers were incubated in paraformaldehyde for min and thenwashed and blocked for min at room temperaturethe cells were then incubated with muc2 or grp78 orchop antibody they were then washed times with1x pbs and incubated with antirabbit alexa andantimouse alexa and sytox orange for nucleicacid staining at room temperature for min repeat times washing with 1x pbs was performed glass slipswere mounted on slides using proliong gold antifadesolution from invitrogen life technologies grand island ny confocal images were taken from differentfields at random at x63 magnification using a leicaconfocal tcs sl dmre microscopecell proliferation assayscell lines were counted and plated in a 96well plateovernight the next day the cells were treated with varying concentrations of celecoxib and orlistat for or h following this treatment cell viability was determined by celltiter aqueous one solution cell proliferation mts assay according to the manufacturersprotocol promega madison wi cells were incubatedwith the combined solution of a tetrazolium compoundmts [345dimethylthiazol2yl53carboxymethoxyphenyl24sulfophenyl2htrazolium inner salt] andelectron coupling reagent pms phenazine methosulfatefor h at °c the absorbance of the product was measured at mm directly with an enzymelinked immunosorbent assay plate reader all cell treatments wereperformed in triplicate 0cdilly orphanet of rare diseases page of apoptosis analysiscells were counted and plated into a well plate for h cells were then treated with varying concentrations of celecoxib and orlistat alone and in combinationfor to h following treatment cells and mediumwere collected in ml culture tubes and apoptosis wasanalyzed using flow cytometry and bd pharmigen fitcannexin v apoptosis detection kit i bd biosciencessan jose ca per manufacturers protocol briefly cellswere washed twice with cold pbs and then resuspendedin μl of 1x binding buffer five μl of both fitcannexin v and propidium iodide pi were added twoeach tube for each cell line one tube was collected andresuspended in μl 1x binding buffer and incubatedunstained without annexin v and pi or with onlyannexin v or pi cells were incubated for min atroom temperature in the dark following this the samples were analyzed by flow cytometry using an accuric6 flow cytometer combination index was measuredusing the compusyn software combo syn inc paramus njcollected bywestern blottingcells were treated for h with varying concentrations of celecoxib and orlistat alone or in combination following treatment cells were scrapped andcollected in ml conical tubes and kept on ice acell pellet wascentrifugation andwashed twice with cold pbs cell pellets were resuspended in 1x ripa solution cell signaling technology with 1x protease inhibitorcomplete minisigma aldrich in pbs and lysis was performed usingsonication and then centrifuged for min atÃg at °c supernatant was collected proteinconcentration was determined in nonreduced samples using bca reagent thermo scientific proteinwas run on sds gels miniprotean tgxgels bio rad an | Colon_Cancer |
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 |
objectives paired box protein8 pax8 immunohistochemical expression can be used as a diagnostic marker for epithelial cells tumors this study aimed at investigating the immunohistochemical expression of pax8 among sudanese females diagnosed with cervical endometrial and ovarian cancers between december and may by studying their formalinfixed paraffin embedded blocksresults sixty patients diagnosed with female reproductive tract cancers were included who aged ± years range cervix was the most common cancer site in patients regarding cancer stage there was and of the study population had stage 3b and 2b respectively the histopathological diagnosis included and poorly moderately and well differentiated cervical squamous cell carcinoma scc as well as and endometrial adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma respectively pax8 was positively expressed in endometrial adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma poorly and moderately differentiated scc all patients diagnosed with well differentiated scc and metastatic adenocarcinoma showed no expression of pax8 a statistically significant was seen for pax8 expression and the different histopathological diagnosis p value keywords female reproductive cancer paired box protein8 immunohistochemical expressionintroductionpaired box protein8 pax8 is a member of the family paired box proteins paxs [ ] pax8 consists of amino acids with a molecular weight of approximately kilo dalton and its molecular properties are located on chromosome 2q13 [] pax8 is a transcription factor that regulates ans development during the embryonic period as well as to maintain normal cellular functions in some cells after birth [ ] during the embryonic period pax8 also plays a significant role correspondence nouh_saadoutlookcom alfarrabi college for science and technology khartoum sudanfull list of author information is available at the end of the in the development of genital ans derived from the mesonephric and the m¼llerian ducts [] in a previous experiment the deletion of the pax8 gene resulted in dysfunctional uterus absence of the endometrium and the vaginal opening also resulted in poor development of the myometrial tissue several studies have described the immunohistochemical utility of pax8 as a diagnostic marker for epithelial cells neoplasms of many glands and ans such as thyroid thymus and kidney as well as some female reproductive tract tumors [ ]in a healthy female reproductive tract pax8 shown to be overexpressed in the epithelial cells of the endocervix and the endometrium [] pax8 was found to be expressed among endometrioid carcinomas transitional the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cali a0et a0al bmc res notes page of undifferentiated cell carcinomas and the metastatic carcinomas at a range of and [ ] whereas for the ovarian carcinomas pax8 was under expressed considering that few studies have investigated the immunohistochemical expression of pax8 in carcinomas of the endometrium and uterine cervix in the different parts of the world but none from sudan yet [ ] this study aimed at investigating the immunohistochemical expression of pax8 among sudanese females diagnosed with cervical endometrial and ovarian carcinomasmain textmaterials and a0methodsstudy design and a0population characteristicsthis is a descriptive retrospective hospital based study conducted at different histopathology laboratories during the period from december till may in khartoum state sudan we retrieved archived formalin fixed paraffin embedded blocks previously collected from female patients with cervical endometrial or ovarian carcinomas the retrieved formalin fixed paraffin blocks represent all the female population admitted at the hospitals for reproductive malignancies diagnosis the participants demographic data was collected including age place of residence the clinical data including site of cancer cancer grade and the histopathological diagnosis were also collectedsections preparation for a0immunohistochemistry stainingtwo sections were cut using rotary microtome leica germany from each histopathological block then one slide was stained by hematoxylin and eosin staining technique the other slide was mounted onto 3aminopropyltriethoxysilane coated slides for immunohistochemistry to retrieve pax8 tissues antigen we treated the sections with citrate buffer at ° a0c for a0min in a waterbath then the tissue sections were rinsed first in distilled water and later with tris buffer saline tbs this was followed by treatment with peroxidase block hydrogen peroxide in methyl alcohol for a0min to quench endogenous peroxidase activity the slides were then placed in a humid chamber then the slides were drained and rinsed in two successive changes of tris buffer wash buffer for a0 min each nonspecific proteinprotein interactions were blocked by incubating and treating the tissue sections in a humid chamber with the power block casein in phosphate buffered saline for a0 min then the remaining solution was drained from the slides the sections were then incubated in the primary antibody pax8 antipax8 rabbit antihuman monoclonal antibody ab189249 abcam united kingdom at room temperature in the humid chamber according to the manufacture instructionsobserving the yellowishbrown or brown appearance of the nucleus was considered a positive result for the pax8 for the negative control we omitted the incubation with the primary antibody step instead we incubated the section in the phosphate buffer saline pbsresults interpretationsfor the interpretation of the results we depended on the intensity as well as the number of the cells that expressed the marker and the expression was graded into categories negative no staining less than of the cells were expressing the marker of the cells were expressing the marker more than of the cells were expressing the marker more than of the cells were expressing the marker the slides were interpreted and validated by two expert pathologists blindly of each other results photomicrographs were taken using olympus sp350 camera olympus imaging america inc usastatistical analysisthe statistical analysis of the results was done using ibm spss statistics vs the chisquared test was performed to compare the frequencies of categorical variables statistical significance level was defined as p value at confidence intervalresultscharacteristics of a0the a0study participantsthe study included patients diagnosed with female genital tract cancer patients aged ± a0years range a0years patients were grouped into age groups those aged a0 years constituted half of the study participants the remaining were and patients distributed across the remaining age groups of a0 years a0 years and a0 years respectively according to patients place of residence patients were originating from the four regions of sudan most of the patients were from western part of sudan followed by from the central part of sudanregarding the site of cancer the cervix was the most commonly involved patients there were and endometrial and ovarian cancer respectively based on the international federation of gynecology and obstetrics figo cancer grading the majority of the study population was diagnosed with stage 3b and 2b cancer and of the patients respectively the were and stage 4b 3a 2a 1b and 4a respectively 0cali a0et a0al bmc res notes page of no statistically significant association between figo staging and age group was found p value histologically there were squamous cell carcinoma scc all of which were cervical cancers and adenocarcinoma scc and adenocarcinoma were further classified into poorly differentiated scc moderately differentiated scc and well differentiated scc endometrium adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinomabased on age groups age group showed no statistically significant relationship with either patients place of residence cancer site cancer histological type figo staging and cancer histopathological type table a0immunohistochemical expression of a0paxthe immunohistochemical expression of pax8 was shown as a yellowishbrown or brown staining of the nucleus fig a0 based on site of cancer all endometrium carcinoma showed positive expression of pax8 with p value there were only patients who had positive expression of pax8 including adenocarcinoma and scc a statistically significant difference was noted for the pax8 staining and cancer type with p value the analysis of pax8 staining results based on the histopathological diagnosis showed that all patients who were diagnosed with well differentiated scc and metastatic adenocarcinoma had negative results for the pax8 expression while of the endometrium adenocarcinoma were found positive for the pax8 expression a statistically significan was t seen for pax8 expression and the different histopathological diagnosis p value table a0table classification of a0participants demographic and a0clinical diagnosis based on a0age groupage group no total no p value a0years a0years a0years a0yearsresidence of patient central sudan east sudan west sudan north sudansite of cancer cervix endometrium ovarycancer histological type scc adenocarcinomafigo staging stage stage 2a stage 2b stage 3a stage 3b stage 4a stage 4bhistopathological cancer grades well differentiated scc poorly differentiated scc moderately differentiated scc endometrium adenocarcinoma endocervical adenocarcinoma metastatic adenocarcinoma ovarian mucinous cyst adenocarcinomascc squamous cell carcinoma 0cali a0et a0al bmc res notes page of fig immunohistochemical expression of pax8 among the different histopathological cancer types and grades the immunohistochemical expression of pax8 is shown as a yellowishbrown or brown staining of the nucleus a well differentiated scc negative b metastatic adenocarcinoma negative c poorly differentiated scc positive d moderately differentiated scc positive e endometrium adenocarcinoma positive f ovarian mucinous cystadenocarcinoma positive g endocervical adenocarcinoma positive and h endometroid adenocarcinoma positive 0cali a0et a0al bmc res notes page of table association of a0clinical diagnosis and a0the a0immunohistochemical expression of a0pax8pax results no total no p valuepositivenegativecancer histological type scc adenocarcinomacancer site cervix endometrium ovaryfigo staging stage stage 2a stage 2b stage 3a stage 3b stage 4a stage 4bcancer histopathological grading well differentiated scc poorly differentiated scc moderately differentiated scc endometrium adenocarcinoma endocervical adenocarcinoma metastatic adenocarcinoma ovarian mucinous cyst adenocarcinomascc squamous cell carcinoma discussionprevious studies on the immunohistochemical expression of pax8 in the normal female reproductive tract showed that pax8 was expressed in the endometrial endocervical and ovarian epithelial cells as well as in nonciliated epithelial cells of the fallopian tubes [ ] this study investigated the immunohistochemical expression of pax8 in sudanese patients who were diagnosed with female reproductive tract cancers patients on the 5th decade of life were constituting half of the study participants with no statistically significant association between age group and the type of cancer however previous studies had suggested other risk factors which could contribute in the development of certain gynecological cancer [ ]regarding the place of residence the majority of patients coming from western sudan this result is in contrary with a previous study in sudan conducted by saeed et a0al in which they showed that the percentage of patients suffering from different types of cancers residing in central and northern sudan were higher compared to the other regions in sudan nevertheless these findings could suggest the involvement of environmental risk factors however the limited study samples size is insufficient to support this suggestion therefore further research with a larger samples size investigating the potential environmental risk factors is essential for strategic prevention and protection measuresthe reported number of female patients with cervical cancer was high compared to ovarian and endometrium cancer similar results were seen previously among sudanese females also the high frequency of stages 3b and 2b compared to the other stages were comparable to previous study conducted in sudan this similarity underscores a delayed response among sudanese females in seeking healthcare and urge the need for health promotion and education to encourage young sudanese females for the early signs detection and seeking healthcare as early as possible for a better treatmentregarding the classification based on the histopathological diagnosis most of the female diagnosed with scc this result was also similar to previous study investigated the prevalence of the different gynecologic cancer in sudan however the expression of pax8 among the studied samples was relatively low compared to previous studies [ ] this could be attributed 0cali a0et a0al bmc res notes page of to the site of cancer development while agrees with another study where pax8 was expressed only in patient interestingly a high frequency of pax8 expression was noted among females diagnosed with endometrium cancer compared to scc this finding is in contrary with a previous report where pax8 was expressed among only of the studied samples also the result was strongly in accordance with other studies [ ] besides that the lack of pax8 expression among those who were diagnosed with well differentiated scc and metastatic adenocarcinoma could play a significant role in either gynecologic cancer differentiation or in detection of endometrium adenocarcinoma progression to metastatic adenocarcinoma [ ]conclusionalthough pax8 showed a significant expression among adenocarcinomas lesions and negative expression was noted among those with well differentiated scc and metastatic adenocarcinoma pax8 might not be beneficial when used alone as a diagnostic marker for the tumors that occur in the female reproductive tractlimitations¢ the small sample size investigated in this study reduced the ability of using the expression of pax8 as a diagnostic marker therefore a largescale study is needed and it should include other types of malignant tumors encountered in the female reproductive systemacknowledgementsthe authors would like to acknowledge the medical staff for their interest and cooperation during the study and thanks to all who participated in completing this studyauthors contributionseta nsm and ees provided conceptual framework for the study guidance for interpretation of the data and performed data analysis eta ees irs lah and amm performed laboratory work nsm ees msm aay and aa performed the statistical analysis nsm msm ees and aa participated in the manuscript preparation revision and coordination all authors read and approved the final manuscriptfundingnot applicableavailability of data and materialsthe datasets used andor analyzed during the current study are available from the corresponding author on reasonable requestethics approval and consent to participateethical approval was obtained from the research ethics committee of the faculty of medical laboratory sciences university of khartoum sudan ethical approval no fmlsrec002042 all participant approved to participate by signing an informed consentconsent for publicationnot applicablecompeting interestsno competing interests to discloseauthor details department of histopathology and cytology faculty of medical laboratory sciences university of khartoum khartoum sudan department of histopathology and cytology faculty of medical laboratory sciences national university khartoum sudan alfarrabi college for science and technology khartoum sudan faculty of medicine sinnar university sennar sudan molecular biology department faculty of medical laboratory sciences nile university khartoum sudan faculty of dentistry ibn sina university khartoum sudan department of neurology mayo clinic jacksonville fl usa department of radiology mayo clinic jacksonville fl usa institute of endemic diseases university of khartoum khartoum sudan mycetoma research center university of khartoum khartoum sudan faculty of medicine nile university khartoum sudan received july accepted august references gruss p walther c pax in development cell mansouri a hallonet m gruss p pax genes and their roles in cell differentiation and development curr opin cell biol macchia pe lapi p krude h pirro mt missero c chiovato l souabni a baserga m tassi v pinchera a pax8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis nat genet vilain c rydlewski c duprez l heinrichs c abramowicz m malvaux p renneboog bt parma j costagliola s vassart g autosomal dominant transmission of congenital thyroid hypoplasia due to lossoffunction mutation of pax8 j clin endocrinol metab park s vk c genetics of congenital hypothyroidism j med genet dahl e koseki h balling r pax genes and anogenesis bioessays lang d powell sk plummer rs young kp ruggeri ba pax genes roles in development pathophysiology and cancer biochem pharmacol stoykova a gruss p roles of paxgenes in developing and adult brain as suggested by expression patterns j neurosci mittag j winterhager e bauer k grummer r congenital hypothyroid female pax8deficient mice are infertile despite thyroid hormone replacement therapy endocrinology bouchard m de caprona d busslinger m xu p fritzsch b pax2 and pax8 cooperate in mouse inner ear morphogenesis and innervation bmc dev biol mittag j winterhager e bauer k grummer rje congenital hypothyroid female pax8deficient mice are infertile despite thyroid hormone replacement therapy endocrinolog laury ar perets r piao h krane jf barletta ja french c chirieac lr lis r loda m hornick jl a comprehensive analysis of pax8 expression in human epithelial tumors am j surg pathol wong s hong w hui p buza n comprehensive analysis of pax8 expression in epithelial malignancies of the uterine cervix int j gynecol pathol ozcan a shen ss hamilton c anjana k coffey d krishnan b truong ld pax expression in nonneoplastic tissues primary tumors and metastatic tumors a comprehensive immunohistochemical study mod pathol bowen nj logani s dickerson eb kapa lb akhtar m benigno bb mcdonald jf emerging roles for pax8 in ovarian cancer and endosalpingeal development gynecol oncol 0cali a0et a0al bmc res notes page of ozcan a liles n coffey d shen ss truong ld pax2 and pax8 expression in primary and metastatic m¼llerian epithelial tumors a comprehensive comparison am j surg pathol distinguishing ovarian mucinous neoplasms from colonic and appendiceal mucinous neoplasm bmc res notes nesrin r kilic d risk factors for cervical cancer results from a hospital ozcan a liles n coffey d shen ss truong ldjtajosp pax2 and pax8 based casecontrol study int j hematol oncol expression in primary and metastatic m¼llerian epithelial tumors a comprehensive comparison am j surg pathol nonaka d tang y chiriboga l rivera m ghossein r diagnostic utility of thyroid transcription factors pax8 and ttf2 foxe1 in thyroid epithelial neoplasms mod pathol tacha d zhou d cheng l expression of pax8 in normal and neoplastic tissues a comprehensive immunohistochemical study appl immunohistochem mol morphol bowen nj logani s dickerson eb kapa lb akhtar m benigno bb mcdonald jfjgo emerging roles for pax8 in ovarian cancer and endosalpingeal development gynecol oncol k¶bel m kalloger se boyd n mckinney s mehl e palmer c leung s bowen nj ionescu dn rajput a ovarian carcinoma subtypes are different diseases implications for biomarker studies plos medicine 2008512e232 nonaka d chiriboga l soslow ra expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas am j surg pathol tong gx devaraj k hamelebena d yu wm turk a chen x wright jd greenebaum e pax8 a marker for carcinoma of m¼llerian origin in serous effusions diagn cytopathol laury ar perets r piao h krane jf barletta ja french c chirieac lr lis r loda m hornick jljtajosp a comprehensive analysis of pax8 expression in human epithelial tumors am j surg pathol tong gx devaraj k hamelebena d yu wm turk a chen x wright jd greenebaum ejdc pax8 a marker for carcinoma of m¼llerian origin in serous effusions diagn cytopathol chu pg chung l weiss lm lau sk determining the site of origin of mucinous adenocarcinoma an immunohistochemical study of cases am j surg pathol brunner ah riss p heinze g meltzow e brustmann h immunoexpression of pax in endometrial cancer relation to highgrade carcinoma and p53 int j gynecol pathol ozcan a shen ss hamilton c anjana k coffey d krishnan b truong ldjmp pax expression in nonneoplastic tissues primary tumors and metastatic tumors a comprehensive immunohistochemical study mod pathol aldaoud n erashdi m alkhatib s abdo n almohtaseb a graboskibauer a the utility of pax8 and satb2 immunohistochemical stains in saeed me cao j fadul b kadioglu o khalid he yassin z mustafa sm saeed e efferth t a fiveyear survey of cancer prevalence in sudan anticancer res saeed me cao j fadul b kadioglu o khalid he yassin z mustafa sm saeed e efferth tjar a fiveyear survey of cancer prevalence in sudan anticancer res mohamed keh ashmeig aaa cervical cancer our experience in sudan philadelphia aacr elhasan lme bansal d osman of enan k abd farag eab prevalence of human papillomavirus type in sudanese women diagnosed with cervical carcinoma j cancer res ther tacha d zhou d cheng ljai morphology m expression of pax8 in normal and neoplastic tissues a comprehensive immunohistochemical study appl immunohistochem mol morphol ord³±ez ng value of pax immunostaining in tumor diagnosis a review and update adv anat pathol gailey mp bellizzi am immunohistochemistry for the novel markers glypican pax8 and p40 δnp63 in squamous cell and urothelial carcinoma am j clin pathol yemelyanova a gown am holmes bj ronnett bm vang r pax8 expression in uterine adenocarcinomas and mesonephric proliferations int j gynecol pathol liang l zheng w liu j liang sx assessment of the utility of pax8 immunohistochemical stain in diagnosing endocervical glandular lesions arch pathol lab med wong s hong w hui p buza njijogp comprehensive analysis of pax8 expression in epithelial malignancies of the uterine cervix int j gynecol pathol de andrade dap da silva vd de macedo mg de lima ma de andrade vm andrade cemc schmidt rl reis rm dos reis r squamous differentiation portends poor prognosis in low and intermediaterisk endometrioid endometrial cancer plos one 20191410e0220086publishers notespringer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations ¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your ï¬eld¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold open access which fosters wider collaboration and increased citations maximum visibility for your research over 100m website views per year ¢ at bmc research is always in progresslearn more biomedcentralcomsubmissionsready to submit your research choose bmc and benefit from 0c' | Colon_Cancer |
high throughput methods in biological and biomedical fields acquire alarge number of molecular parameters or omics data by a single experimentcombining these omics data can significantly increase the capability for recoveringfinetuned structures or reducing the effects of experimental and biological noise indataresultsfhclust for identifying patient subgroups from different omics information eg geneexpression mirna expression methylation in particular hierarchical structures of patientdata are obtained in each omic or view and finally their topologies are merged byconsensus matrix one of the main aspects of this methodology is the use of ameasure of dissimilarity between sets of observations by using an appropriate metricfor each view a dendrogram is obtained by using a hierarchical clustering based on afuzzy equivalence relation with Åukasiewicz valued fuzzy similarity finally a consensusmatrix that is a representative information of all dendrograms is formed by combiningmultiple hierarchical agglomerations by an approach based on transitive consensusmatrix construction several experiments and comparisons are made on real data egglioblastoma prostate cancer to assess the proposed approachs fuzzy logic allows us to introduce more flexible data agglomerationtechniques from the analysis of scientific literature it appears to be the first time that amodel based on fuzzy logic is used for the agglomeration of multiomic data theresults suggest that fhclust provides better prognostic value and clinical significancecompared to the analysis of singleomic data alone and it is very competitive withrespect to other techniques from literaturekeywords multiomics data data integration hierarchical clustering fuzzy similarityfuzzy aggregation the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0cciaramella bmc bioinformatics 21suppl page of nowadays high throughput methods in biological and biomedical fields acquire a largenumber of molecular parameters by a single experiment in particular such measuredparameters are collected in omics datasets eg genomics transcriptomics methylomics among multiple measured parameters dna genome sequence rna expressionand dna methylation are representative instances for individually analysing suchdata several methodologies have been introduced in literature even though recentlya number of studies pointed out the best performance coming from the integration ofmultiomics data for instance analysing each omic or view in the machine learningjargon set separately fundamental patterns can be detected from data however somefinetuned structures such as cancer subtypes can be highlighted by both gene expression and dna methylation information so that multiomics analysis can reduce theeffects of experimental and biological noise in data from literature three kinds ofintegration methodologies emerge¢ early integration builds a single featurebased matrix by concatenating each omic¢ intermediate integration builds a joint representation of data given the views¢ late integration each omic is analysed separately and the solutions are integratedin general late integration methods and in particular clustering are preferred whenthe analysis combines continuous and discrete data together for a review of integrationapproaches and their comparisons the reader may refer to in this work a multiviewclustering methodology named fhclust is introduced see fig for its general schemafor identifying patient subgroups from different omics information or datasets eg geneexpression mirna expression methylation specifically for each omic dataset a fuzzybased hierarchical clustering approach is adopted and finally the results are mergedby consensus matrix the idea behind the proposed approach comes from observingthat a hierarchical clustering dendrogram can be associated with a Åukasiewicz fuzzydataset ie view and applies a singleomic analysisfig proposed approach a data preparation b data normalization and feature selection c multiomicshierarchical agglomerations d data integration e clustering and visualization 0cciaramella bmc bioinformatics 21suppl page of similaritybased equivalence relation so that a consensus matrix that is the representative information of all dendrograms is derived by combining multiple hierarchicalagglomerations following an approach based on transitive consensus matrix constructionmethodscluster analysis or clustering is an unsupervised technique that aims at agglomerating aset of patterns in homogeneous groups or clusters [ ] hierarchical clustering hc isone of several different available techniques for clustering which seeks to build a hierarchyof clusters and it can be of two types namely agglomerative where each sample starts inits own cluster and pairs of clusters are merged as one moves up the hierarchy or divisivewhere all samples start in one cluster and splits are performed recursively as one movesdown the hierarchy thus hc aims at grouping similar objects into a cluster and werethe endpoint is a set of clusters where each cluster is distinct from each other and theobjects within each cluster are broadly similar to each other hc can be performed eitheron a distance matrix or raw data agglomerative hc starts by treating each observationas a separate cluster and it repeatedly executes the following two steps identifies thetwo clusters that are closest together and merges the two most similar clusters thisprocess continues until all the clusters are merged togetherthe main output of hc is a dendrogram which shows the hierarchical relationshipbetween the clusters distances many distance metrics have been developed and thechoice should be made based on theoretical concerns from the domain of studylater on it is necessary to determine how the distance is computed eg singlelinkagecompletelinkage averagelinkage as with distance metrics the choice of linkage criteria should be based on theoretical considerations from the application domainin nonfuzzy clustering or hard clustering data is divided into distinct clusters andeach data point can only belong to exactly one cluster in fuzzy clustering data pointscan potentially belong to multiple clusters for example in hard clustering given someparameters a symptom can be in a mutually exclusive way present or absent red orblue whereas in fuzzy clustering that symptom could simultaneously be of somegrade red and some other grade blue in fig a comparison between hard and fuzzycategorisation is shown the reader can refer to for a recent comparison betweenhard and fuzzy clustering in this work we introduce a data integration methodologybased on fuzzy concepts in particular we associate a dendrogram to a fuzzy equivalencerelation ie Åukasiewicz valued fuzzy similarity so that a consensus matrix in a multiview clustering that is the representative information of all dendrograms can be obtainedfrom multiple hierarchical agglomerations [ ] the main steps of fuzzy agglomerationcan be summarised as follows characterisation of membership functions computation of a fuzzy similarity matrix or dendrogram for all models at a giventime construction of a consensus matrix for all hierarchical agglomerationsmembership functionswhen dealing with clustering tasks fuzzy logic fl permits to obtain a soft clusteringinstead of an hard clustering of data specifically data points can belong to more 0cciaramella bmc bioinformatics 21suppl page of fig hard vs fuzzy in symptom risk example a hard categorization b fuzzy categorizationthan one cluster simultaneously the fundamental concept in fl upon which all thesubsequent theory is constructed is the notion of fuzzy set a generalisation of a crisp setfrom classical set theorya fuzzy set generalises a crisp set by allowing its characteristic function ie itsmembership function assuming values in the interval [ ] rather than in the set in this way a given item belongs to the fuzzy set with a degree of truthranging from do not belong at all ie its membership function assumes value to completely belong ie the membership function assumes value in fl applications fuzzy sets make it possible to represent qualitative nonnumeric values ielinguistic variables such as high medium low for approximate reasoninginference or fuzzy control systems linguistic variables can be represented by fuzzy setsthrough a transformation step called fuzzification and it is achieved by using different types of membership functions representing the degree of truth to whicha given input sample belongs to a fuzzy set see membership functions sectionin supplementary material 0cciaramella bmc bioinformatics 21suppl page of fuzzy similarity matrixa measure of similarity or dissimilarity defines the resemblance between two samples orobjects similarity measure is a significant means for measuring uncertain informationfuzzy similarity measure is a measure that depicts the closeness among fuzzy sets and hasbeen used for dealing issues of pattern recognition and clustering analysisa binary fuzzy relation that is reflexive symmetric and transitive is known as a similarity relation fuzzy similarity relations are the generalisation of equivalence relationsin binary crisp relations to binary fuzzy relations in details a fuzzy similarity relationcan be considered to effectively group elements into crisp sets whose members are similar to each other to some specified grade and it is a generalization of classical equivalencerelation as described in fuzzy similarity section in supplementary material in orderto introduce the fuzzy similarity in the following we focus on the properties of theÅukasiewicz tnorm tl and the biresiduum in this way we obtain a fuzzy equivalencerelation that can be used for building dendrogram for more details in the derivation ofthese results see fuzzy similarity section in supplementary materialdendrogram and consensus matrixif a similarity relation is mintransitive ie t min then it implies the existence ofthe dendrogram see dendrogram and consensus matrix section in supplementarymaterial for details the mintransitive closure of a relation matrix r can be easilycomputed and the overall process is described in algorithm the last ingredient to accomplish an agglomerative clustering is a dissimilarity relationhere we considered the following result lemma letting r be a similarity relation with the elements rcid2x ycid3 [ ] and lettingd be a dissimilarity relation which is obtained from r bydx y rcid2x ycid3then d is ultrametric iif r is mintransitivein other words we have a onetoone correspondence between mintransitive similaritymatrices and dendrogram and between ultrametric dissimilarity matrices and dendrograms finally after the dendrograms have been obtained each time a consensus matrixie the representative information of all dendrograms is obtained by combining thetransitive closures ie maxmin operation the overall approach is described inalgorithm the overall workflow of the proposed approach is summarised in fig in particular for each omic data set xi a fuzzification step is adopted for obtaining thenew data set yi see supplementary material successively adopting a fuzzy similaritymeasure the similarity matrix si is computed and to guarantee the transitive closure ofthe matrix a new matrix ci is computed see algorithm finally all the ci matricesare collected for obtaining the consensus matrix a and the overall final dendrogram seealgorithm in fig we show an example that summarize a realistic agglomeration result we plotin figs 4abc three input hierarchies obtained on datasets that should be combinedin this case four sequences of patients are considered namely s1 s2 s3 and s4 respectively in fig 4d we show the final result by agglomerating dendrograms we observe that 0cciaramella bmc bioinformatics 21suppl page of fig workflow of the fuzzy based hierarchical clusteringthe output hierarchy contains clusters s1 s2 s3 and s1 s2 s3 s4 at different levels andeach of these clusters eg s1 s2 s3 are repeated at least in two out of the three inputdendrograms moreover it is worth stressing that the proposed approach based on theagglomeration of dendrograms can also be applied with commonly used metrics egeuclidean distance in fig we show a comparison between the dendrograms obtainedby using an euclidean metric and a similarity based approach ie Åukasiewicz tnormrespectively in this realistic example we simulate three omic data sets with rows ienumber of patients and columns ie features we split the single datasets in twopartitions or clusters such that the first rows are random samples from a standard normal distribution with variance and the other rows have the same distribution withfig combination algorithm abc input dendrograms d combined hierarchy 0cciaramella bmc bioinformatics 21suppl page of fig crisp hierarchical clustering vs fuzzy based hierarchical clustering a dendrogram of euclidean basedhierchical clustering b dendrogram of similarity based hierachical clusteringalgorithm mintransitive closure input relation si output transitive relation ci sti elaborate compute s if sielse ci sti si ª si ¦ sicid8 si replace si with si si and go to step i and the algorithm terminatesvariance obtaining a sort of an overlap we observe that both methods find two separated clusters but the similarity based approach in fig 5b permits to obtain a perfectseparation of the source partitionsresults and discussionin the following we describe the behaviour of the proposed methodology on multiomicsbenchmark datasetsalgorithm combination of dendrograms input ci ¤ i ¤ l l input similarity matrices dendrograms output similarity matrix dendrogram a aggregate the similarity matrices to a final similarity matrixa aggregate c1 c2 cla let a be the identity matrixb for each ci calculate e a a ª a ¦ cic if a is not changed a a and goto step else goto step 1b create the final dendrogram from aomics datasetswe consider multiomics cancer datasets available from the cancer genome atlastcga tcga is a large multiomic repository of data on thousands of cancerpatients all datasets contain three omics gene expression mirna expression and 0cciaramella bmc bioinformatics 21suppl page of table datasets description three omics are provided for each dataset respectively dna geneexpression mirna and methylationcases dnaoridatasetamlbiccoadgbmkirclihcluscskcmovsarclnrfmirnaorilnmethyorilnmultiomicsorilnrfrfrfthe number of features at each variable selection method is shown ori original variable dimension ln logarithm andnormalisation and rf random forest based on mean decrease gini indexdna methylation1 in table are summarised the main properties of the datasetsnamely acute myeloid leukemia aml breast invasive carcinoma bic colon adenocarcinoma coad glioblastoma multiforme gbm kidney renal clear cell carcinoma kirc liver hepatocellular carcinoma lihc lung squamous cell carcinomalusc skim cutaneous melanoma skcm ovarian serous cystadenocarcinoma ovsarcoma sarc the number of patients ranges from for aml to for bicmultiview clustering algorithmsfor validating the effectiveness of our model we compared it against several categories ofmultiview clustering algorithms2¢ kmeans and spectral clustering techniques ¢ lracluster it is a lowrank approximation based integrative probabilistic model¢ pins perturbation clustering for data integration and disease subtyping pinsis able to address subtype discovery as well as integration of multiple data types thealgorithm is built upon the resilience of patient connectivity and cluster ensembles toensure robustness against noise and biasto fast find the shared principal subspace across multiple data types¢ snf similarity network fusion snf allows for discovery of disease subtypesthrough integration of several types of highthroughput data on a genomic scale snfcreates a fused network of patients using a metric fusion technique and thenpartitions the data using spectral clustering snf appears to be the state of the art inthis area and has proven to be very powerful however the unstable nature ofkernelbased clustering makes the algorithm sensitive to small changes in molecularmeasurements or in its parameter settings¢ mcca multi canonical correlation analysis mcca which extends theapplication of canonical correlation analysis cca to more than two views is oneof the most widely used dimension reduction method for finding linear relationsbetween two or more multidimensional random variables1row data are available at httpacgtcstauacilmulti_omic_benchmarkdownloadhtml2httpsgithubcomshamirlabmultiomicscancerbenchmark 0cciaramella bmc bioinformatics 21suppl page of evaluation metricsin order to assess the performance of each method we adopt three evaluation metricsthat are the logrank test the enrichment of clinical labels in the clusters and the methods runtime the logrank test assumes that if clusters of patients have significantlydifferent survival they are different in a biologically meaningful way for the enrichmentof clinical labels in clusters six clinical labels are considered gender age at diagnosispathologic tumor pathologic metastases pathologic lymph nodes and pathologic stagethe four latter parameters are discrete pathological parameters measuring the progression of the tumor metastases and cancer in lymph nodes and the total progressionpathologic stage enrichment for discrete parameters was calculated using the Ï2 testfor independence and for numeric parameters using kruskalwallis test not all clinicalparameters were available for all cancer types so a total of clinical parameters wereavailable for testing to derive a pvalue for the logrank test the Ï2 test for independence the kruskalwallis test and the statistic for these three tests is assumed to have Ï2distribution preprocessingtcga datasets were preprocessed as follows patients and features with more than missing values were removed and missing values were imputed using knearest neighborimputation the sequence features were logtransformed the features with highestvariance from geneexpression and methylation omics were selected in the mirna omicfeatures with zero variance were filtered all features were then normalized to have zeromean and standard deviation for methylation we selected the features with maximal variance in each dataset and also adopted the standard pipeline proposed in whose procedure filters out the probes from the x and y chromosomes or probes that areknown to have common snps at the cpg sitea further unsupervised variable selection step has been performed by using the meandecrease gini based on random forest the mean decrease in gini is the average of a variable total decrease in node impurity weighted by the proportion of samplesfig mean performance of the algorithms on ten multiomics cancer datasets the xaxis measures thedifferential survival between clusters mean log10 of logranks test pvalue and the yaxis is the meannumber of clinical parameters enriched in the clusters 0cciaramella bmc bioinformatics 21suppl page of fig performance of the algorithms on ten multiomics cancer datasets for each plot the xaxis measuresthe differential survival between clusterslog10 of logranks test pvalue and the yaxis is the number ofclinical parameters enriched in the clusters red vertical lines indicate the threshold for significantly differentsurvival pvalue cid2 reaching that node in each individual decision tree in the forest this is effectively a measure of how important a variable is for estimating the value of the target variable acrossall of the trees that make up the forest a higher mean decrease in gini indicates highervariable importance therefore the most important variables to the model is the highestin the plot with the largest mean decrease in gini values conversely the least importantvariable is the lowest in the plot with the smallest mean decrease in gini values by following this strategy we cutoff all those variables whose importance is zero the numberof variable cutoff at each step is summarised in table experimental resultsin the experiments for all methods the number of searched clusters is selected in therange [ ] to determine the number of clusters for a method we used the elbowmethod to automatically pick out the optimal elbow rather than choose it manually weused as approximation the second derivative of a vector vv [i ] v [i ] 2v[ i] in particular we consider the index i that brings this expression to a maximum or minimum depending on whether v increases or decreases for all methods we adhered tothe guidelines for usage and parameter selection given by the developers in some casestable performance on ten multiomics number of clinical parameters enriched in the clustersfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeans 0cciaramella bmc bioinformatics 21suppl page of table performance on ten multiomics differential survival between clusters log10 of logrankstest pvaluefhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeanswhere no information was provided by the authors we devised parameter selection methods we performed the same process pipeline used in for evaluating the performanceof our method all methods were run on a multicore intelr xeonr cpu e52620v3 240ghz with gb ram in the following the obtained experimental results aredescribedfigure shows the average performance for multiomics data and for each singleomicseparately across all cancer types and fig shows the performance on the different cancer datasets all algorithms show quite similar performance in either differential survivalor enriched clinical parameters with respect to survival our fhclust method achievedthe overall best prognostic value sum of log10 pvalues while pins and mcca ranked second and third respectivelyin table the differential survival between clusters mean log10 of logranks testpvalue are reported spectral achieved the highest total number of significant clinical parameters with parameters fhclust along with lracluster and kmeansplaced themselves second with parameters snf achieved the third position with parameterswith respect to survival table fhclust outperformed its competitors achieving parameters mcca pins and snf have achieved good results with and enriched parameters respectivelywe also counted the number of datasets for which a method solution obtains significantly different survival these results are reported in table all methods that weredeveloped for multiomics data had at least four cancer types with significantly differentsurvival in this case fhclust and pins had different cancer subtypes for which itsclustering had significantly different prognosis fhclust spectral clustering and mccahad enrichment in cancer typeson average fhclust pins and mcca had better prognostic value but found lessenriched clinical labels as compared to spectral clustering methodtable for each benchmarked algorithm the number of cancer subtypes for which its clusteringhad significantly different prognosis first row and had at least one enriched clinical label secondrow are shownsignificant different survivalsignificant clinical enrichmentfhclustkmeansspectrallraclusterpinssnfmcca 0cciaramella bmc bioinformatics 21suppl page of table number of clusters chosen by the benchmarked algorithms on ten multiomics cancerdatasetsfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeansthe number of clusters found for each dataset are presented in table ranging from to because of the good methods performance in the previous analysis partitioning thedata into a relatively high number of clusters could indicate that clustering cancer patientsinto more clusters improves prognostic value and clinical significanceconcerning with methods computational burden their run times are reported intable fhclust takes on average seconds per dataset while spectral and snf gotlower timing the worst method takes roughly minutes per dataset see fig finally fig shows the benchmarked methods performance for singleomic datamoreover for each dataset and method the single omic that gave the best results forsurvival and clinical enrichment are also shown these results suggest that fhclust provides better prognostic value and clinical significance on multiomics data compared tothe analysis of singleomic data used separately nevertheless the interested reader mayrefer to the supplementary material for details on additional results concerning singleomics we also stress that the proposed method differently from other methods suchas snf does not need any hyperparameter tuning moreover clustering is embeddedin the data integration and vice versa and the use of fuzzy concepts ie tnormsfrom one hand permits to obtain a generalisation of the clustering approaches whereason the other hand gives the possibility to apply an inference system eg mamdanifor a quantitative and qualitative measure eg high medium low in cancer riskassessmentsin this work we proposed a multiview clustering methodology for identifying patientsubgroups from different omics data in biological and biomedical fields combining theseomics data can significantly increase data mining capabilities one of the main aspects oftable runtime in seconds of the algorithms on ten multiomics cancer datasetsovfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccacoadskcmbicamlgbmkirclihcluscsarcmeans 0cciaramella bmc bioinformatics 21suppl page of fig computational time comparisonsthis methodology is the use of a measure of dissimilarity between sets of observations byusing an appropriate metric and a consensus matrix that is a representative agglomerateinformation of all the dendrograms as emerged from the analysis of the scientific literature to the best of our knowledge our work concerns for the first time a model based onfuzzy logic used for the agglomeration of multiomic data the use of fuzzy logic allowsus to introduce more flexible data mining features also related to approximate reasoningseveral experiments and comparisons have been made on real data eg glioblastomaprostate cancer to assess the proposed methodology the results suggest that fhclustprovides better prognostic value and clinical significance compared to analysis of singleomic data alone fuzzy logic concepts and in particular membership functions permitsfig summarized performance of the algorithms across ten cancer datasets for each plot the xaxismeasures the total differential prognosis between clusters sum across all datasets of log10 of logranks testpvalue and the yaxis is the total number of clinical parameters enriched in the clusters across all cancertypes ac results for singleomic datasets d results when each method uses the single omic that achievesthe highest significance in survival e same with respect to enrichment of clinical labels 0cciaramella bmc bioinformatics 21suppl page of to develop a fuzzy inference model ie mamdani fuzzy cognitive maps for easilyobtaining a model for a quantitative and qualitative risk assessment of the cancer themodel based on approximate reasoning can be particularly useful for embedded devicesin future work it could be possible to improve results for multiomics analysis ina number of ways for instance more accurate feature selection algorithms couldbe adopted for improving the overall performance on one hand the integration oflabelled data could improve the feature selection step on the other hand some specific feature extraction strategies could be adopted indeed approaches based on thesignal analysis of gene expression data eg nonlinear principal component analysis compressive sensing could possibly further improve the performance [ ]in future it is possible to foresee a different weight for each omic data in order toobtain a more robust similarity and parametric similarity measures can be adoptedeg uninorm for generalizing the concept of and and or connections betweenclusterssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12859020035676additional file supplementary materialabbreviationsfhclust fuzzyhierarchical clustering dna deoxyribonucleic acid rna ribonucleic acid hierarchical clustering hccrisp hc crisp hierarchical clustering fl fuzzy logic tcga the cancer genome atlas aml acute myeloid leukemia bicbreast invasive carcinoma coad colon adenocarcinoma gbm glioblastoma multiforme kirc kidney renal clear cellcarcinoma lihc liver hepatocellular carcinoma lusc lung squamous cell carcinoma skcm skim cutaneousmelanoma ov ovarian serous cystadenocarcinoma sarc sarcoma pins perturbation clustering for data integrationand disease subtyping lracluster low rank approximation based multiomics data clustering snf similarity networkfusion mcca multi canonical correlation analysisabout this supplementthis has been published as part of bmc bioinformatics volume supplement proceedings from the 13thbioinformatics and computational biology international conference bbcc2018 the full contents of the supplement areavailable online at httpsbmcbioinformaticsbiomedcentralcomssupplementsvolume21supplement10authors contributionsac originally designed the methodology ac and dn worked on the developing of the method and the design of theexperiments ac dn and as contributed for interpreting and for analysing the results all authors contributed forwriting the manuscript read and approved the final manuscriptfundingpublication costs are funded by a grant from the dipartimento di scienze e tecnologie università degli studi di napoliparthenope tecniche di machine learning e soft computing per lelaborazione di dati multivariati softmulan piciaramellaavailability of data and materialscode and data of the proposed approach are available on multiomicscancerbenchmark github repositoryethics approval and consent to participateno ethics approval was required for the studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1dipartimento di scienze e tecnologie università degli studi di napoli parthenope centro direzionale c4 island naples italy 2hitachi rail sts via argine naples italypublished august 0cciaramella bmc bioinformatics 21suppl page of referencescamastra f di taranto md staiano a statistical and computational methods for genetic diseases an overviewcomput math meth med 20152015 id serra a fratello m fortino v raiconi g tagliaferri r greco d mvda a multiview genomic data integrationmethodology bmc bioinformatics rappoport n shamir r multiomic and multiview clustering algorithms review and cancer benchmark nucleicacids res reddy ck aggarwal cc data clustering boca raton chapman and hallcrc camastra f ciaramella a son lh riccio a staiano a fuzzy similaritybased hierarchical clustering for atmosphericpollutants prediction lncs ciaramella a staiano a on the role of clustering and visualization techniques in gene microarray data algorithmsbora dj gupta ak int j emerg trends technol comput sci napolitano f pinelli m raiconi g tagliaferri r ciaramella a staiano a miele g clustering and visualizationapproaches for human cell cycle gene expression data analysis int j approx reason ciaramella a cocozza sand clustering of genomic data neural netw iorio f miele g napolitano f pinelli m raiconi g tagliafer | Colon_Cancer |
the hypoxic tumour is a chaotic landscape of struggle and adaption against the adversity of oxygen starvationhypoxic cancer cells initiate a reprogramming of transcriptional activities allowing for survival metastasis andtreatment failure this makes hypoxia a crucial feature of aggressive tumours its importance to cancer and otherdiseases was recognised by the award of the nobel prize in physiology or medicine for research contributing toour understanding of the cellular response to oxygen deprivation for cancers with limited treatment options forexample those that rely heavily on radiotherapy the results of hypoxic adaption are particularly restrictive to treatmentsuccess a fundamental aspect of this hypoxic reprogramming with direct relevance to radioresistance is the alterationto the dna damage response a complex set of intermingling processes that guide the cell for good or for badtowards dna repair or cell death these alterations compounded by the fact that oxygen is required to inducedamage to dna during radiotherapy means that hypoxia represents a persistent obstacle in the treatment of manysolid tumours considerable research has been done to reverse correct or diminish hypoxias power over successfultreatment though many clinical trials have been performed or are ongoing particularly in the context of imagingstudies and biomarker discovery this research has yet to inform clinical practice indeed the only hypoxia interventionincorporated into standard of care is the use of the hypoxiaactivated prodrug nimorazole for head and neck cancerpatients in denmark decades of research have allowed us to build a picture of the shift in the dna repair capabilitiesof hypoxic cancer cells a literature consensus tells us that key signal transducers of this response are upregulatedwhere repair proteins are downregulated however a complete understanding of how these alterations lead toradioresistance is yet to comefacts hypoxia is present in almost every solid tumour hypoxia is a major barrier to effective radiotherapyand is associated with radioresistance the hypoxic tumour is highly heterogenous withregions of chronic and acute hypoxia altered ph andimmune ltration differences in gene expression and protein functioncan occur between acute or chronic and mild orsevere hypoxiacorrespondence mahvash tavassoli mahvashtavassolikclacuk1head and neck oncology group centre for host microbiome interactionkings college london hodgkin building london se1 1ul ukedited by i amelio all dna damageresponsehomologousddr pathwaysincludingnonhomologous end joining missmatch repair andthe fanconi anaemia pathways have been shown tosuffer alterations in hypoxiarecombination activation of ddr transducer protein atm is seenin severe hypoxia in the absence of classical atmactivatingstranddna breaksfeaturesdoublesuchas atr is also activated most likely in response tohypoxiainduced replication stress however downregulation of dna repair effectorproteins such as rad51 and brca12 is seen results of ddr reprogramming include geneticinstability aberrant cell cycle and apoptotic control 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 0cbegg and tavassoli cell death discovery page of open questions precisely how do alterations to the ddr in hypoxialead to radioresistance for example when genomicinstability and generation of radioresistant clonestakes several cell divisions to set in how does adecrease in dna repair ability lead to increasedradioresistance what aspects of the hypoxic response could betargeted to radiosensitise or more effectively treattumours particularly in the context of ddr forexampleupregulated ddrtransducers such as atm atr and dnapkcs orinduce synthetic lethality following downregulationof dna repair effectorscan wetarget do different types of cancers have different patternsof ddr alteration within hypoxic tumours thisparticularly needs further research as tissues havebeen shown to have different oxygen pressureslevels of hypoxia and hypoxic heterogeneity can we use the data on this subject to develop abiomarkerhypoxiainducedradioresistance as we have done using hypoxia as asingle parametersignatureof how can we monitor hypoxic tumours during thecourse of a patients disease to help guide treatmentand how can wereportingreliableensureinterpretation of in vitro and in vivo dataintroductionofprogramstranscriptionalhypoxia is present in almost every solid tumour aninevitability of cancers characteristic disanised andfunctionally inefï¬cient vasculature rapid growth anddemanding metabolism1 the result is a comprehensiverewritingupdownregulating certain genes and proteins allowing cells toevade apoptosis and migrate to areas with better oxygenperfusion crucially this microenvironmentally inducedintracellular shift also results in genomic instability gialterations to dna repair and resistance to cell killing bycancer therapies after decades of research it has becomeclear that the relevance of hypoxia for both oncogenesisand treatment resistance is inescapablein the context of radiotherapy rt a link betweenresistance and low intratumoral oxygen pressure has beenknown since the publication of a study modelling oxygenï¬ow in lung tumours years ago2 for some cancerssuch as head and neck squamous cell carcinomashnsccs hypoxia is a major contributing factor to localrt failure3 advancesin developing technologiesallowing for more precise delivery of rtimaging oftumours and sensitisation to treatment while protectingnormal tissues have led to improved locoregional controland quality ofsincelife for patients78 howeverofï¬cial of the cell death differentiation associationtreatment for many cancers like hnsccs which incidentally are also some of the most hypoxic depends onrt the hypoxic problem remains particularly pertinent9in recent years efforts have been made to correct orreverse hypoxia including administering hyperbaric oxygen therapy to patients1011 reducing cellular oxygenconsumption12 and increasing blood vesselfunctionality1314 to more precisely tackle resistance induced byhypoxia researchers have also soughtthethreshold of treatability of hypoxic tumours by use ofsensitizers15 as a third arm in our battle plan researchhas also gone into developing methods to detect hypoxiaincluding the use of specialised imaging techniques petct scanning combined with hypoxiadetecting radionuclides often studied in tandem with genetic signaturesseeking to genotypically deï¬ne these tumours1819to lowerhowever very few of these advancements have allowedus to overcome hypoxiainduced radioresistance rrthough research activity in this area remains strong amore complete understanding of how the hypoxic environment contributes to rr particularly by modulation ofpotentially targetable dna damage response ddrpathways is warranted this review will outline our current knowledge of the molecular processes that underpinhypoxic rr particularly in the reprogramming of the ddrradiotherapy mechanism of actiontherequirement of oxygenseminal work by gray and colleagues during the 1950sproved that the efï¬cacy of rt was dependent on theavailability of oxygen within the tissue22021 radiationinduces damage through the direct and indirect generation of double stranded breaks dsbs in dna in thepresence of oxygen damage induced is times morelikely to end in cell death22 this effect is best explainedby the oxygen fixation hypothesis where radicals produced directly or indirectly by ionizing radiation ir areoxidised to dna in the presence of oxygen23 making thedamage irreversible24 thistothe hypothesis with the notion that these lesions cannotbe restored to an undamaged state as the damage isï¬xed to dna by oxidisation25 thus without oxygendamage induced is transient and hypoxic cells experiencefar reduced radiationassociated damagelast pointis crucialthough crucial the requirement of oxygen to inducedamage is not where the story ends for rr as it does notfully explain the level of rr we observe this is evidencedby the fact that restoration of oxygen to tumours forexample through applying hyperbaric oxygen does notrestore radiosensitivity26 importantlyit also does notaccount for changes that occur with respect to dnarepair which have been shown to be crucial in impactingthe radiation response27 as these changes are retainedpast the point of radiotherapy administration 0cbegg and tavassoli cell death discovery page of the landscape of the hypoxic tumourin vivo the hypoxic region exists on a gradient ofoxygen pressures with oxygen levels throughout the tissue ranging between severe hypoxia mildhypoxia and anoxia with around consideredphysoxia see table for deï¬nitions tissue oxygenpressures are usually measured in mmhg however sincethe majority of research on hypoxia and the ddr hasbeen performed in vitro where oxygen levels are measured in percent for the purpose of this review o2 willbe referred to predominantly the difference betweenin vivo and in vitro measurements of oxygen is importantthough with tissue normoxia physoxia classiï¬ed ataround o2 or mmhg and in vitro normoxia beingaround o2 fig the hypoxic tumour is a space of restricted proliferationparticularly when oxygen levels are o2 cell cyclearrest and decreased protein synthesis juxtaposed againstaccelerated aggressivity microenvironmental interactionsand altered ph28 at the most oxygendepleted borderexists the barren land of necrosis with the highly proliferating and comparatively treatmentsensitive aerobiccells closest to the blood vessel fig tumour hypoxia does not develop in a linear fashionand is highly heterogenous and changeable the hypoxictumour is dynamic with ï¬uctuating vessel functionalityand cycling oxygen levels creating regions of acute andchronic hypoxia31 where part of the tissue may sufferacute hypoxia after temporary occlusion of a blood vesselsocalled perfusion limited in which oxygendeprivationcycles last sometimes minutes sometimes hours beforesubsequent reoxygenation chronic hypoxia is diffusionlimited where oxygen levels become a factor of distancefrom the blood vessel31 compounded with this is thediffering rates of oxygen consumption and responsivenessto oxygen availability in cells within the tissue to be ableto fully understand and ultimately treat the hypoxictumour it must be remembered that the changeability ofoxygen concentrations in the hypoxic tumour also predictably uences its behaviour and response to treatment in the context of radiotherapy cells with o2 levels is where we see most resistance so called radiobiological hypoxia24 whether this is acute and thereforefollowed by reoxygenation or chronic oxygen deprivedfor more than h can have marked differences on theensuing genomic and proteomic changes that ultimatelyallow for hypoxic survival32 thus within one tumourdifferent regions are likely to have a completely differentresponse to the same dose of radiotherapyitaside from the oxygen status the involvement of otherenvironmentalfeatures affected by hypoxia must beconsidered an additional outcome of hypoxic adaption isthe concomitant phenotypic shift of the microenvironmentis now accepted that hypoxia can induceammation33 demonstrated even by patients whodevelop mountain sickness after prolonged periods athigh altitude34 hypoxic tumours are known to havehigher ltration of protumour immune cells such asm2 macrophages35 a feature known to be involved inrr3637 the same could also be said with respect to thehypoxicinduction of highly rt resistant cancer stemcells38 though this subject needs further research thelikelihood of an interplay between intracellular geneticreprogramming as a result of hypoxic adaption and themicroenvironment in mediating radioresponse is stronggenetic reprogrammingthe hifswithin this chaotic showground of heterogeneity andat the core of cellular adaption to hypoxia are the alteredgenetic pathways that push for survival against adversitycommonly dysregulated genes include glut1 involvedin altered glucose metabolism vegf involved intable glossary of terms multiple classiï¬cations of the terms used to describe hypoxia exist throughout the literaturethis represents a general consensus and what is used in this reviewtermhypoxianormoxiaphysoxiaanoxiasevere hypoxiamild hypoxiaacute hypoxiachronic hypoxiadeï¬nitionreduced oxygen levels usually ¤ o2 mmhg in in vitro studiesnormal atmospheric oxygen used in in vitro studies o2 mghgphysiological levels of oxygen in tissues between mmhg tissue speciï¬c see fig complete absence of oxygen o2 o2 o2incubation in hypoxic conditions hincubation in hypoxic conditions hradiobiological hypoxiaoxygen levels where the efï¬cacy of radiotherapy is half maximal mmhg04 o2ofï¬cial of the cell death differentiation association 0cbegg and tavassoli cell death discovery page of fig approximate oxygen levels reported in different tissues in mmhg used in in vivo experiments and o2 used in in vitroexperiments note that normal tissue normoxia or physoxia is considerably less than the o2 used in vitro as normoxia adapted frommckeown139 liu140 and graham26fig the heterogeneity of the hypoxic tumour tumours suffer from reduced oxygen availability due to the disanised nature of thevasculature where occlusion of a blood vessel bv occurs tumours are said to be under perfusion limited hypoxia pl hypoxia where lack ofoxygen is a function of distance from the vessel cells experience diffusion limited dl hypoxia when these states are temporary h it is said tobe acute or chronic when h within hypoxia tumour cells undergo considerable genetic reprogramming contributing to therapy resistance andmetastatic behaviourofï¬cial of the cell death differentiation association 0cbegg and tavassoli cell death discovery page of neoangiogenesis and lox involved in remodelling ofthe extracellular matrix7in the nobel prize in physiology or medicine wasawarded to three scientists gregg semenza williamkaelin and sir peter ratcliffe for their contributions toour understanding of cellular oxygensensing mechanisms39 this included the discovery of a group of transcription factors regulated by hypoxia that allow forcellular adaption40 these hypoxiainducible factor hifproteins hif13 are transcription factors composed oftwo subunits the α subunits reside in the cytoplasm andto rapid degradation min41 underare subjectnormal circumstances this degradation is mediated bythe actions of prolylhydroxylasesphd14 whichhydroxylate hifα at the oxygendependentdegradationdomains oddd of note phd2 and phd3 are themselves transcriptional targets of hif alluding to possiblenegative feedback systems in place though conï¬ictingresults suggest this system doesnt always function effectively to constrain cancer growth42 subsequentlyhydroxylation by phds recruits the vonhippellandauvhl protein45 this alongside other proteins forms ane3 ubiquitin ligase complex ubiquitinating hifα forproteasomal degradationin hypoxia due to the lack of molecular oxygen neededfor hydroxylation4246 this degradation cascade does nottake place and hifα subunits translocate to the nucleusto associate with hifβ subunits47 the hif complex ininteraction with its coactivators p300 and crebbindingprotein cbp then binds to hypoxia response elementshresin dna to initiate transcription of hiftarget genesintheincludingcontrolledadditional layers of hif regulation also exist to keepthis pathway in check such as factor inhibiting hif fihwhich hydroxylates hif subunits at asparagine residuesblocking their association with p300cbp48 some evidence has shown that hifs also undergo other posttranslational modiï¬cations including phosphorylation andacetylation as a further method of regulation4247 however as with many such processes in cancer it can beaberrantlyhypoxiaindependent stabilisation of hifα by oncogenes such asegfr and mtor4950 and depletion of hifregulatoryfactors4251 the hif proteins themselves can interactwith a number of factors relevant in cancer such as p53mutants present in human papillomavirus hpvnegativehnsccs and nonsmall celllung cancers nsclcsresulting in transcriptional control of protumorigenicgenes52 since both hifs and p53 compete for binding ofp300cbp to enact transcriptional control the hifs havea unique relationship with this highly cancerrelevantprotein53 inactivation of p53s transcriptional abilities hasbeen observed54 though again conï¬icting results exist forthis55ofï¬cial of the cell death differentiation associationmost of the work investigating hifdirected transcriptional changes in hypoxia has focussed on the actions ofthe bestknown hif hif1 however both hif2 andhif3 also play a role in hypoxic transcriptional control42interestingly relative expression of the hifs has beenshown to differ between hypoxic tissues demonstratingthat each may have speciï¬c functions56 in some casesthey may indeed work in concert as hif2 has beenshown to be induced when hif1 is depleted50hifs are master regulators of the hypoxic response andconcurrent with the notion that hypoxic tumours areradioresistant depletion of hif1α in tumour modelsradiosensitises cells41 one study showed that intermittenthypoxia showed less radiationinduced cell death bothin vitro and in mice via stabilisation of hif1α57 thisinvestigation also found that intermittent hypoxia had amore signiï¬cant effect than chronic hypoxia hif1α hasalso been shown to function via the hif1αmyc pathway in which hif1α competes with the transcriptionactivator myc for sp1 binding in the target gene promoter to downregulate mismatch repair mmr genesmsh2 and msh6 in o2however how exactly hifs contribute to rr of thehypoxic tumour be itthrough their transcriptionalfunctions or interactions with other proteins is so farunresolved notably some radio and chemotherapiesthemselves upregulate or stabilise hifs41though genetic reprogramming in hypoxia can lead to anumber of alterations for the purpose of this review wewill focus on those associated with rr and ddr formore general reviews see schito60 and tsai61reprogramming of the ddrthe ddr is a complex process consisting of overlapping and interconnected pathways initiated by differentforms of dna damage arguably one ofthe mostimportant homeostatic processes it allows us to withstand constant and numerous dna damageinducinginsults the result of this protection ensures that onlyreliable genomes are passed on to the next cellular generation for cancer considering both the power ofmutagenesis in driving oncogenic potential and the factthat many cancer therapies function by inducing dnadamage the ddr has considerable relevance for therapyresistance and tumour progressionrepair of dna is a tale of three acts ï¬rstly the damagepropagates a signal that recruits sensors to the site ofdamage secondly the signal is ampliï¬ed by transducersand thirdly response pathways are initiated by effectorsfor each part of the process welldeï¬ned though notexclusive sets of proteins act as sensors transducers andeffectors respectively28 shrouding these repair processesare signals to stall the cell cycle initiated by chk1 orchk2activated cdc25 and p21to allow time for 0cbegg and tavassoli cell death discovery page of clearance of this damage and initiation of apoptoticpathways for example as initiated by atms interactionwith p53 if the repair is unsuccessful6263for the repair of radiationinduced dsbs two primarypathways are put to use homologous recombination hrand nonhomologous end joining nhej the formerconsidered less errorprone uses sister chromatids torepair dna and as such can only take place during g2sphase of the cell cycle nhej predominates in g1 but canoccur at any stage of the cell cycle and often results in thegeneration of insertiondeletion mutations which havethe potential to lead to more oncogenic alterations hr ismediated primarily by the recruitment to sites of damageof master transducer of the dsb response atm ataxiatelangiectasia mutated a phosphoinositide3kinaserelated protein kinase pikk following detection by themrn complex composed of mre11rad50nbs1which initiates activity of effectors including rad51 andbrca1 nhej occurs following sensing of damage by theku proteins ku70 and ku80 and signal transduction ofku in complex with dnapkcs dnadependent proteinkinase catalytic subunittogether forming dnapk andsubsequent activity of effectors dna ligase iv ligivand xrcc464alteration of ddr pathways has been seen across manycancers compared to normal tissue perhaps the mostwellknown are the mutations in brca12 in aggressivehereditary breast and ovarian cancers65 understandablywhere hypoxia represents an exaggerated form ofaggressive tumours the ddr pathways in hypoxia operatedifferently to those in normoxia indeed this is true forevery aspect of the ddr process dna damage in theform of dsbs is reduced in conditions of hypoxia o2and hypoxia alone does not induce dsbs2466 researchhas shown that different members of the ddr pathwayscan be either activated or downregulated in conditions oflow oxygen see tables and for reported alterationsto hr nhej and mismatch repair mmr pathwayscrucially whether the cells are in acute or chronichypoxia or h and at what level of oxygen depletion may deï¬ne the ensuing response despite this delineation there lacks within the literature proper reportingof experiments carried out in either acute or chronic mildor severe hypoxia with interchangeability in use of termssee table for a consensus of parameters used with thesedeï¬nitionssensorsthe mre11rad50nbs1 mrn complex is responsible for sensing dna damage and initiating both the hrand nhej pathways by recruitment of transducers such asatm via nbs167 while the mrn complex is consideredthe main sensor responsible for recruiting and activatingatm followingdamage atrip atrinteractingofï¬cial of the cell death differentiation associationprotein and ku7080 are sensorsresponsible forrecruitment of atr and dnapkcs respectively67 fig though there are many overlapping interactions atminatminteracting protein with roles in replication stressrs genome stability and the base excision repair berpathway has also been shown to recruit atm independent of dna damage68repression of the mrn machinery has been seen inchronic hypoxia days in a medulloblastoma modelwith transcriptomic downregulation of both mre11a andnbs1 resulting in downregulation of etoposideinducedatm and p5369 nbs1 has also been shown to stabilisehif1α particularly in response to ir70 while hif1α hasbeen shown to downregulate nbs1 the authors of onestudy where reduction of nbs1 was seen after h in o2 noted that this repression resulted in the induction ofγh2ax and 53bp1 foci in hypoxia suggesting the presence of dna breaks59 interestingly all components ofthe mrn were found to be downregulated both at themrna and protein level in nsclcs harbouring egfrmutations incubated in severe hypoxia o2 thisdownregulation in egfrmutated cells correlated withtheir increased sensitivity to egfrinhibiting drugs71sensing of damaged dna is a crucial step in theinitiation of repair and begins with changes to the chromatin72 γh2ax a phosphorylated variant of histoneh2ax is induced by mrn activation and accumulates atsites of damage in the chromatin preceding recruitmentand necessary for retention of key ddr signalling proteinsincluding mrn and atm73 studies also show thatγh2ax is crucial for retaining mediators such as 53bp1p53 binding protein mdc1 mediator of dnadamage checkpoint and brca1 at sites of damage66h2ax is primarily phosphorylated by atm but can alsobe phosphorylated by atr and dnapkcs63 indeed aswell as by radiation and chemotherapies γh2ax has alsobeen shown to be induced by hypoxia following replication fork stalling this phosphorylation has been shown inchronic severe hypoxia to occur in a hif atr or atmdependent manner74 crucially some evidence hasshown the phosphorylation of h2ax present only inproliferating cells7778 the downstream effects of thisactivation have been linked to other consequences ofhypoxic regulation including angiongenesis79 via induction of vegf80 experimentally resolution of γh2ax fociafter irradiation is often used as a marker of dsb repair astheory dictates that the phosphorylation should disappearafter damage is repaired however in hypoxia this heavilyrelied upon protocol may necessitate further ï¬netuningku70 and ku80 together forming a heterodimericcomplex tether damaged dna at breaks and are keysensors of dsbs responsible for recruitment of dnapkcs as part of the nhej pathway63 the ku complex hasbeen shown to be both upregulated and downregulated by 0cbegg and tavassoli cell death discovery page of table a nonexhaustive list showing alterations to sensors transducers and effectors of the homologoursrecombination hr pathways in hypoxiaprotein role in ddrmechanism of alterationalteration conditions and consequencesreferencenbs1sensor of dsbs in hr activated atmas part of the mrn complexcid129 pasb domain of hif1αmre11sensor of dsbs in hr activated atmas part of the mrn complexcid129 atmtransducer of hr in dsb repaircid129 autophosphorylation atser1981atrtransducer of dna repair induced byreplication stresscid129 rad51effector of dsb repair in hrrad52effector of dsb repair in hrrad54 motor protein effector of dsbrepair in hrbrca1effector of dsb repair in hrcid129 e2f4p130cid129 lsd1cid129 ezh2cid129 mir210cid129 mir373cid129 mir210cid129 cid129 e2f4p130cid129 h3k4 demethylation via lsd1cid129 downregulated in chronic mild hypoxia days o2cid129 downregulation in acute mild h o2cid129 resulted in induction of γh2ax and 53bp1 focicid129 downregulated in chronic mild hypoxia days o2cid129 activated in acute hypoxia o2cid129 increased expression and activity o2 hcid129 mediated by src and ampk signallingcid129 activated in acute o2cid129 resulted in phosphorylated p53 andaccumulation and growth arrestcid129 downregulation in chronic severe hypoxia o2 h and or hcid129 decreased radioresistancecid129 increased genomic instabilitycid129 downregulation in o2 hcid129 decreased mrna expression o2 hcid129 downregulated o2 hcid129 decreased mrna expression o2 hcid129 downregulation in chronic severe hypoxia o2 hcid129 decreased mrna expression o2 hcid129 downregulation in o2 hcid129 decreased radioresistancecid129 decreased expression o2 hcowman69to59cowman69hashimoto88bencokova28hammond75meng119bindra oliveira82meng119crosby118meng119meng119lu117bindra120oliveira82meng119brca2effector of dsb repair in hrcid129 hypoxia in different studies81 one study found downregulation of ku80 after h in mild hypoxia o282another using severe hypoxia o2 found upregulation of ku70ku80 in a431 cells alongside many othermembers of the nhej pathway and proteins generallyinvolved in metastatic progression83 a study in humanand mouse hepatoma cells found upregulation of the kuheterodimer upon incubation in hypoxia o2 or withhypoxia mimics and downregulation associated with hif1βdeï¬cient cells84 alternative subpathways of nhejalso exist possibly as insurance for when classical nhejmediators are inoperative howeverthe impact ofhypoxia on these pathways has not been extensivelystudiedofï¬cial of the cell death differentiation associationtransducersatm and atr are two of the most important proteinsinvolved in transduction of the ddr as pikk familymembers they phosphorylate a number of proteinsinvolved in propagating the signal and repairing dna aspart of both the hr and nhej pathways as well asundergoingtheresponse until dna is repairedautophosphorylationto maintainbroadly speaking atm has been shown to be activatedparticularly in acute hypoxia as shown in the study bybencokova et al the pattern of activation in this contextdoes not match rtinduced atm activation which follows mrn recruitment to dsbs85 as atm phosphorylation does not correlate with the presence of dsbs 0cbegg and tavassoli cell death discovery page of table a nonexhaustive list showing alterations to sensors transducers and effectors of the nonhomologous endjoining nhej pathways in hypoxiaproteinrole in ddrmechanism of alterationalteration conditions and consequencesreferenceku70ku80sensor in nhej pathwaysrecruits dnapkcsin complex with ku80sensor in nhej pathwaysrecruits dnapkcsin complex with ku70cid129 cid129 dnapkcstransducer of nhej pathwaycid129 autophosphorylation atser2056dna ligiv effector of nhej repairxrcc4effector of nhej repaircid129 cid129 cid129 decreased mrna expression o2 hcid129 upregulation o2 hcid129 downregulation in cervical tumour sectionscid129 upregulation o2 hcid129 upregulation o2 hcid129 downregulation o2 hcid129 downregulation in cervical tumour sectionscid129 upregulation o2 hcid129 decreased mrna expression o2 hcid129 increased expression and activity o2 hcid129 activated in mild hypoxia o2 led to positiveregulation of hif1 and upregulation of glut1cid129 decreased mrna expression o2 hcid129 decreased mrna expression o2 hmeng119ren83lara81um84oliveira82ren83lara81um84meng119hashimoto88bouquet103meng119meng119table a nonexhaustive list showing alterations to sensors transducers and effectors of the mismatch repair pathwayin hypoxiaprotein role in ddrmechanism of alterationmlh1dimerises to pms2 to form themutlα complex in mmrpms2dimerises to mlh1 to form themutlα complex in mmrmsh2dimerises with msh6 forms themutsα complex in mmrmsh6dimerises with msh6 forms themutsα complex in mmrcid129 mad1maxcid129 mntmaxcid129 dec12cid129 mir155cid129 lsd1cid129 hdaccid129 hypoacetylationhypermethylation on h3cid129 cid129 mycmaxcid129 hif1α via sp1cid129 mir155cid129 hcid129 p53cid129 hif1α via sp1cid129 mir155cid129 hdaccid129 p53cid129 hypoacetylationhypermethylation on h3ofï¬cial of the cell death differentiation associationalteration conditions andconsequencescid129 downregulation h o2cid129 downregulation in h o2cid129 increased expression h o2resulting in genomic instability instem cellscid129 downregulation at protein level h o2cid129 resulting in genomic instability instem cellscid129 downregulation h o2referencebindra121127mihaylova128nakamura129rodriguezjimenez145lu115mihaylova128rodriguezjimenez145bindra121127koshiji58cid129 downregulation h o2cid129 increased expression h o2resulting in genomic instability instem cellskoshiji58rodriguezjimenez145 0cbegg and tavassoli cell death discovery page of often reduced or absent in severe hypxoia28 the authorsof this study emphasized that atm activation was speciï¬cto the level of hypoxia only phosphorylated at o2and hifindependent since phosphorylation was maintained even in hifknockout cells activation of atmwas attributed to autophosphorylation the result of whichwas an activation of targets much like dna damageinduced atm activation but dependent on the activityof cell cycle regulator mdc128 this study did not analyserr but the results suggest that atm activation byhypoxia is likely enacted as a means of halting the cellcycle in order to allow for dna repairthe pattern of atm activation in hypoxia however isnot clearcut and may depend on cancer type atm canbe regulated by a number of factors as part of the hypoxicresponse including mrn or atmin but also by posttranslational and epigenetic factors86 one study foundthat atm was downregulated along with hif1α by amicrorna mir18 resulting in radiosensitivity87the study by hashimoto et al88 showed atm activation alongside activation of a number of other key ddr orcancerrelated proteins including dnapkcs akt andegfr and decreased expres | Colon_Cancer |
since the late a novel coronavirus officially named as severe acute respiratory syndrome coronavirus sarscov2 was identified as the pathogen to cause pneumonia as a member of the betacoronavirus genus sarscov2 has genomic nucleotides identity with human severe acute respiratory syndrome coronavirus sarscov and shares amino acid sequence identity with sarscov the world health anization who named the disease caused by sarscov2 as coronavirus disease covid19 until april the virus has swept through countries more than million cases with covid19 have been confirmed and more than cases died which has been posing significant threats to public health sarscov2 can cause respiratory diseases and may lead to acute respiratory distress syndrome ards multiple an failure and even death in severe cases in addition to typical symptoms such as cough and fever some patients developed the symptoms in multiple systems such as cardiovascular system digestive system and abbreviations ace2 angiotensin converting enzyme aki acute kidney injury ali acute liver injury alp alkaline phosphatase als artificial liver system alt alanine aminotransferase ami acute myocardial infarction ards acute respiratory distress syndrome ast aspartate aminotransferase at2 alveolar cells bun blood urea nitrogen ccle cancer cell line encyclopedia cns central nervous system covid19 coronavirus disease geo gene expression omnibus ggt gammaglutamyltransferase gi gastrointestinal injury gtex genotypetissue expression icu intensive care unit mcs mechanical circulatory support np nucleoprotein pci percutaneous coronary intervention sarscov2 severe acute respiratory syndrome coronavirus scr serum creatinine scrnaseq single cell rna sequencing stemi stelevation myocardial infarction tbil total bilirubin tem transmission electronic microscope tmprss2 transmembrane protease serine vv venousvenous who world health anization corresponding authors at department of infectious disease and institute of hepatology qingdao municipal hospital qingdao university digestive disease key laboratory of qingdao qingdao china email addresses xinyongning9812163com y xin zlk0823163com l zhuang 101016jbiopha2020110678 received june received in revised form august accepted august biomedicinepharmacotherapy1312020110678availableonline24august2020075333222020theauthorspublishedbyelseviermassonsasthisisanopenaccessundertheccbyncndlicensehttpcreativecommonslicensesbyncnd40 0cexpressed not only in the cells and tissues of lung but also in extrapulmonary ans [] fig in this section the expression levels of ace2 and tmprss2 in extrapulmonary ans including heart kidney liver digestive tract brain and other ans were reviewed heart m dong nervous system in the early stages of covid19 which brings more challenges to the timely diagnosis of patients angiotensin converting enzyme ace2 as a metalloproteinase is a carboxyterminal dipeptidyl peptidase the primary physiological role of ace2 is involved in the regulation of vasoconstriction and blood pressure [] transmembrane protease serine type2 tmprss2 belonging to the type ii transmembrane serine protease family could cleave the coronavirus spike s protein [] it was demonstrated that ace2 and tmprss2 were crucial for the entry of sarscov and sarscov2 into the host cells cell entry of sarscov2 depends on binding of the s protein to the specific cellular receptor and s protein priming by host cell proteases as shown in fig each s protein of sarscov2 consists of two subunits a globular s1 domain at the nterminal region and the membraneproximal s2 domain sarscov2 utilizes receptorbinding domain within the s1 domain to bind to the cellular receptor ace2 which could trigger the effects of tmprss2 on the cleavage of protein s at the s1 and s2 sites and priming cell membrane fusion for viral entry as receptors and mediators of virus entry are important for determining viral host and an the route of sarscov2 infection and the infected an may depend on the expression and distribution of ace2 and tmprss2 studies have shown that ace2 and tmprss2 are expressed not only in lung tissues but also in extrapulmonary ans including heart kidney liver colon esophagus brain gallbladder and testis suggesting that sarscov2 may also affect extrapulmonary ans [] in this review the distributions of ace2 and tmprss2 in extrapulmonary ans and the characteristics and clinical managements of extrapulmonary an injury caused by sarscov2 were summarized we believe that this will be important in understanding on the infection of extrapulmonary ans in patients with covid19 the mrna expressions of ace2 and tmprss2 in extrapulmonary ans the mrna expressions of ace2 in different human ans were analyzed and the results showed that ace2 was expressed in the heart furthermore chen analyzed the feature of ace2 expressions among cardiac cell types and found that ace2 was specifically expressed in pericyte moreover rna sequencing from patients with failing hearts and normal donors revealed that myocardial ace2 expressions were significantly increased in patients with heart failure which was further validated at the protein level by proteomics profiling from heart failure and normal donors another study also showed that the expression of ace2 in heart tissues of patients with underlying heart disease was higher than that in normal heart tissues these two studies suggested that the expression of ace2 in heart tissue of patients with underlying heart disease was higher than that in normal heart tissue guo et al analyzed the mrna expression of tmprss2 from the genotypetissue expression gtex database and the results showed that tmprss2 is also expressed in the heart by singlecell rna sequencing scrnaseq to profile the gene expression landscapes of cardiac cells from human embryos qi revealed that the cardiomyocytes from the heart contain ace2expressed cells and tmprss2expressed cells and the cardiovascular progenitor cells and cells tmprss2expressed cells respectively these data showed that both ace2 and tmprss2 were expressed in the heart contain ace2expressed kidney studying the viral susceptibility of extrapulmonary ans is important for a deeper understanding for the pathogenesis of sarscov infection studies have shown that ace2 and tmprss2 were expression analysis from the gtex database showed that kidney displayed the fifth high expression of ace2 to investigate the expression of ace2 in kidney lin analyzed the public singlecell transcriptome dataset of normal kidneys from healthy donors the fig entry of sarscov2 into host cells sarscov2 infected the host cells by the spike protein of the virus and the functions of ace2 and tmprss2 in host cells biomedicinepharmacotherapy13120201106782 0cm dong fig tissue distributions of ace2 and tmprss2 in human ab the schematic diagram of the expressions of ace2 a and tmprss2 b in multiple human tissues the colour strength is corresponding to the gene expression level ace2 and tmprss2 were expressed in the brain and heart ace2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while tmprss2 is expressed in the hepatocytes and cholangiocytes ace2 and tmprss2 were highly expressed in kidney and intestinal epithelial cells both ace2 and tmprss2 were also expressed in the esophagus stomach nose testis pancreas breast prostate and thyroid results showed that the ace2 was distributed across multiple cell types and was mostly enriched in proximal tubule cells fan et al confirmed the specific ace2 expression in tubular cells from the gene expression omnibus geo dataset while it was not observed in immune cells and glomerular parietal epithelial cells rna and protein expression data of ace2 in different human tissues and cancer cell lines were obtained from three online datasets including the cancer cell line encyclopedia ccle gtex database and the human protein atlas dataset and the results indicated that both mrna and protein expression levels of ace2 were relatively high in kidney cells especially in renal tubular cells meanwhile suryawanshi analyzed the data of kidney tissues in scrnaseq datasets and found that either proximal tubular cells or tubular progenitor cells in the kidney coexpressed ace2 and tmprss2 the data of the scrnaseq from geo dataset gse134355 showed that ace2 and tmprss2 expression levels were high in nephron epithelial cells epithelial cells endothelial cells and mesangial cells of the kidney recently pan also found that the tmprss2 gene was coexpressed with ace2 in kidney podocytes these data showed that both ace2 and tmprss2 were highly expressed in tissues and cells of kidney liver chai et al analyzed the scrnaseq data from geo database gse124395 to evaluate ace2 gene expression in liver the results showed that ace2 was highly expressed in cholangiocytes which level was about times higher than that in hepatocytes the gtex database also showed that both ace2 and tmprss2 were expressed in the liver zhou identified that tmprss2 is highly expressed in hepatocytes from human cell atlas database recently wen indicated that ace2 and tmprss2 are specifically coexpression in trop2 liver progenitors of human liver tissue using scrna sequencing these data indicate that ace2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while tmprss2 is expressed in hepatocytes digestive tract a previous study showed that ace2 could be found in the upper esophagus and it could be detected in stratified epithelial cells and absorptive enterocytes of the ileum and colon quantitative mrna expression profiling of ace2 across human tissues by harmer showed that ace2 was expressed at a high level in gastrointestinal tissues zhang et al analyzed datasets with singlecell transcriptomes of esophagus gastric ileum colon and lung and the data showed that ace2 was not only highly expressed in the type ii alveolar cells at2 of lung but also in the stratified epithelial cells ileum absorptive enterocytes cells and colon enterocytes similarly the immunofluorescent staining of esophagus stomach duodenum and rectum showed that ace2 was stained mainly in the cytoplasm of gastrointestinal epithelial cells besides the scrnaseq data showed that ace2 was significantly elevated in the proximal and distal enterocytes guo et al suggested that tmprss2 was highly expressed in almost all ans of the digestive tract including colon stomach small intestine and esophagus using published scrnaseq data and seven inhouse normal colon samples lee reported that the coexpressions of ace2 and tmprss2 transcripts were mainly observed in the small intestine and colon the highest expressions of tmprss2 and ace2 were found in enterocytes among the intestinal cell types these data showed that tmprss2 and aec2 are highly expressed in the digestive tract nervous system analysis using the gtex database showed that both tmprss2 and ace2 are expressed at relatively low levels in the brain cortex chen found that ace2 was relatively highly expressed in some important brain areas such as the substantia nigra and brain ventricles using seven brain transcriptome databases ace2 was expressed at high level in the piriform cortex of human brain and its expression could also be detected in many neurons including both excitatory and inhibitory neurons and some nonneuron cells including astrocytes and oligodendrocytes in human middle temporal gyrus and posterior cingulate cortex qi analyzed the scrnaseq data of substantia nigra biomedicinepharmacotherapy13120201106783 0cclinical classification of acute cardiac injury nonicucases icu cases nonicucases icucases nonsevere cases severecases1965 recoveredcases died cases nonicucases icucases survivor cases nonsurvivor cases chen hong zhou china korea china m dong and cortex of brain from geo database the results showed that both ace2 and tmprss2 were expressed in the oligodendrocyte precursor cells and the astrocytes of the substantia nigra and cortex there are limited reports on the expressions of ace2 and tmprss2 in peripheral nervous system brann analyzed the ace2 and tmprss2 expression in different cell type from human scrnaseq dataset gse139522 and found that neither olfactory sensory neurons nor olfactory bulb neurons expressed these two genes while ace2 and tmprss2 were expressed in the nonneuronal cells including the sustentacular cells and olfactory bulb pericytes these data showed that ace2 and tmprss2 could also be coexpressed in the nervous system other ans or tissues table characteristics of acute cardiac injury after sarscid0 cov2 infection study basic heart disease acute cardiac injury country subject china wang china na huang li china moreover ace2 and tmprss2 were also reported to be coexpressed in some other ans it has been revealed that both ace2 and tmprss2 are expressed in testis by scrna sequencing and expression profile analysis indicating that testicular cells might be the potential targets of sarscov2 another report revealed that multiple kinds of cells in the nose including nasal brushing epithelial cells nasal turbinate epithelial cells and nasal airway epithelial cells contained ace2expressed and tmprss2expressed cell clusters moreover ace2 and tmprss2 were also expressed in pancreas breast prostate and thyroid and these ans might also be the targets of sarscov2 infection of sarscov2 and extrapulmonary an injury of patients with covid19 sarscov2 infection and cardiac injury recently autopsy analysis by fox revealed that the histopathology of the heart was consistent with the typical pattern of viral myocarditis sarscov2 rna was detected in the cardiac tissues of the patients with covid19 these data suggested that sarscov2 may directly infect heart the epidemiology of covid19 reported that cardiac injury was one of the most severe an damages the clinical manifestations of cardiac injury in covid19 patients are complex and could present with heart failure arrhythmias or acute myocardial infarction ami [ ] inciardi reported the first case who had the symptom of heart failure at first and later the patient was positive for sarscov2 using nucleic acid test cardiac injury is a common symptom in patients with covid19 shi reported that patients with covid19 had cardiac injury moreover there were patients with acute cardiac injury in a cohort including covid19 patients and of patients with acute cardiac injury in the intensive care unit icu furthermore a study by wang showed that there were patients with acute cardiac injury and patients presented with arrhythmia of covid19 patients while acute cardiac injury was observed in of patients with civid19 in the icu these cases suggested that sarscov2 may cause serious heart damage which should be widespreadly concerned furthermore acute cardiac injury is more prevalent in severe cases with covid19 [] table and it has been reported that covid19 patients with cardiac injury had higher mortality than those without cardiac injury in this review we also summarized the possible relationship between basic heart disease and further cardiac injury [] table in a cohort of covid19 patients from renmin hospital of wuhan university china shi demonstrated that cardiac injury occurred in patients during hospitalization of which had basic heart disease including coronary heart disease and chronic heart failure and only patients with basic heart disease of covid19 patients without cardiac injury similarly liu suggested that patients with basic heart disease in covid19 patients had table comorbidity with cardiac injury in covid19 patients with basic heart disease subjects with covid19 proportion of basic heart disease patients with cardiacinjury study shi liu xu ma guo patients with basic heart disease with cardiac injury without cardiac injury cardiac injury compared with patients with basic cardiovascular diseases of covid19 patients without cardiac injury other studies also indicated that the patients with basic cardiovascular disease are more likely to present heat injury in covid19 patients [ ] in view of the points above covid19 patients with underlying cardiac conditions seem to have higher rates of cardiac injury sarscov2 infection and kidney injury recently autopsy analysis on six covid19 patients showed that varying degrees of acute tubular necrosis were observed in all the renal specimens nucleoprotein np antigens and np positive inclusion body of sarscov2 could be seen in kidney tissues from all the samples moreover viruslike ps were seen in kidney tissues by transmission electronic microscope tem su analyzed kidney abnormalities in autopsies of patients with covid19 and found that diffuse proximal tubular damage with the loss of brush border were biomedicinepharmacotherapy13120201106784 0c sarscov2 infection and liver injury m dong observed further investigation showed that diffuse necrosis can be seen under the light microscope and electron microscopic examination also showed the clusters of coronavirus ps with distinctive spikes in the tubular epithelium and podocytes it was reported that both np antigens and rna of sarscov2 were detected in urine of covid19 patients these data coincide with the finding of the sarscov2 invasion in kidney collectively sarscov2 could directly infect human renal tubules and lead to kidney damage recent studies have shown that the incidence of acute kidney injury aki in covid19 patients ranged from and higher frequency of renal function damage with elevated blood urea nitrogen bun or serum creatinine scr was observed in covid19 patients [ ] table a study of patients with covid19 indicated that levels of bun and scr were increased in and patients with covid19 respectively and routine urine tests were performed on patients among which patients were positive for urinary protein and patients were positive for hematuria another study also showed that about patients with covid19 had abnormal renal function moreover covid19 patients with more severe disease progression have higher rates of aki huang and colleagues reported that of patients with aki in the icu were observed and none of the patients who did not require care in the icu suffered aki xu found that the fatality rate was obviously higher in covid19 patients with aki than those without renal injury furthermore in another study investigating patients with covid19 at hospital admission more severe patients had higher rates of aki and the cox regression analysis also suggested that covid19 patients who developed aki had a significantly higher mortality risk therefore aki is more prevalent in severe cases with covid19 an autopsy report of a 50yearold patient with covid19 showed moderate microvesicular steatosis and mild lobular activity in liver tissues moreover zhao used human liver ductal anoids as a tool to investigate the sarscov2 infection and the tissue damage induced by sarscov2 ex vivo and the results showed that the expression of sarscov2 np was easily detected in the patchy areas of the hepatic duct indicating that liver ductal anoids were susceptible to sarscov2 infection in addition sarscov2 infection could disrupt the barrier and bile acid transporting functions of cholangiocytes which indicated that sarscov2 might directly induce cholangiocyte injury and consequently bile acid accumulation in view of the points above liver damage in the covid19 patients might be directly caused by the viral infection abnormal liver functions were frequently reported in covid19 patients epidemiologic studies showed that almost half of the patients had differing degrees of liver damage [ ] table chen reported that out of patients had elevated alanine aminotransferase alt patients had elevated aspartate aminotransferase ast and had elevated total bilirubin tbil in wuhan jinyintan hospital wuhan china similarly a nationwide study involving patients with covid19 in china showed that more than of patients had elevated alt and ast and of patients had elevated tbil it was revealed that the levels of direct bilirubin indirect bilirubin alt alkaline phosphatase alp and gammaglutamyltransferase ggt were significantly higher in males than that in females with covid19 and multivariate logistic regression analysis showed that male was an important independent risk factor for predicting acute liver injury ali in covid19 patients these data indicated that male patients with covid19 may be more susceptible to liver injury furthermore table characteristics of acute kidney injury after sarscid0 cov2 infection study chen wang huang guan xu preexisting kidney conditions na na na country china china china china china subject li chen hong cheng xiao richardson wan li qian pei china china korea china china america china china china china na na na na na na scr serum creatinine bun blood urea nitrogen aki acute kidney injury abnormal renal functional indices scr bun na scr scr scr scr bun na scr bun scr na na na scr bun scr na aki na clinical classification of aki na nonicu cases icu cases icu cases nonsevere cases severe cases mild cases severe cases critical ill cases nonsevere cases severe cases recovered cases died cases nonicu cases icu cases na nonsevere cases severe cases cured cases in hospital cases died cases mild cases severe cases nonsevere cases severe cases na moderate cases severe cases critically ill cases biomedicinepharmacotherapy13120201106785 0cm dong table characteristics of liver injury after sarscid0 cov2 infection study country subject china china china china china china china korea america china china chen wang huang guan xu li chen hong richardson et wan li al qian china na patients with preexisting liverconditions na na na patients with abnormal liver functional indices alt ast tbil na ast ast alt tbil alt ast alt ast tbil alt ast alt ast tbil ast alt ast alt ast tbil alt ast abnormal liver functional indices in the nonsevere patients ast alt na na tbil na abnormal liver functional indices in the severe patients alt na tbil na ast na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na nonsevere patients include patients without icu care and recovered patients severe patients include patients with icu care and death alt alanine aminotransferase ast aspartate aminotransferase tbil total bilirubin multiple studies found that ast alt and tbil were significantly higher in patients treated in the icu than that in nonicu patients li suggested that among the patients with abnormal liver function moderate and severe types of patients were more likely to have liver injury and respectively fu analyzed the relationship between ali and mortality risk in covid19 patients and the results showed that ali is more common in the critically ill patients and ali at the early stage increased death risk of covid19 patients together abnormal liver functions might be associated with the severity of patients with covid19 sarscov2 infection and digestive tract injury epithelial cells of the esophagus stomach duodenum and rectum in one covid19 patient tested positive for sarscov2 rna and the staining of viral np was also visualized in the cytoplasm of epithelial cells in stomach duodenum and rectum moreover minimally invasive autopsies were performed on three patients died of covid19 and the results showed that some epithelial cells of the gastrointestinal mucosa were degenerated necrotic and detached these studies strongly supported that sarscov2 may directly infect the epithelial cells of digestive tract table sarscid0 cov2 detection in gastrointestinal specimens study subject xiao zhang tan xing young holshue lescure tang wang xu gastrointestinal samples stool anal swabs rectal swab stool stool stool stool stool stool rectal swabs tested positive in gastrointestinal specimens the positive time in gastrointestinal specimens days na 6cid0 1cid0 5cid0 na 3cid0 positive time for gastrointestinal samples after respiratory samples were negative days na na 8cid0 na na na na na 2cid0 biomedicinepharmacotherapy13120201106786 0cm dong multiple studies have identified that the sarscov2 rna was detected in anal swabs rectal swabs and stool specimens [ ] of covid19 patients it has been demonstrated that sarscov2 rna could be detected in feces from more than half of covid19 patients in another study xing reported that sarscov2 rna was detected in the feces of three pediatric cases with covid19 in qingdao china and the persistence of sarscov2 in the digestive tract lasted for 6cid0 days the possibility of fecaloral transmission of sarscov2 infection needs to be taken into account furthermore as shown in table long duration of sarscov2 detection in digestive tract by rtpcr has been reported and viral rna remained detectable in the digestive tract for 2cid0 days after nucleic acid turned negative in respiratory samples [] the studies suggested that sarscov2 could be detected from respiratory tract specimens during the early period to digestive tract specimens during the late period and viral nucleic acid tests in both the respiratory and digestive tract are necessary to confirm the complete clearance of virus some covid19 patients presented gastrointestinal symptoms such as diarrhea nausea vomiting and abdominal pain holshue reported the first case of covid19 patient in the usa which had nausea and vomiting before admission multiple studies found that gastrointestinal symptoms including diarrhea nausea and vomiting and abdominal pain were common at presentation in covid19 patients [ ] table in a cohort of patients with covid19 in wuhan china gastrointestinal symptoms were described in up to moreover sun showed that critically ill patients with covid19 had gastrointestinal injury gi during hospital stay and the survival curves showed that the mortalities of patients with gi was greater than that of patients without gi jin also found that the rate of the severe type was markedly higher in covid19 patients with gi symptoms than that in those without gi symptoms these data suggested that gi is one of the common extrapulmonary an injuries in covid19 patients and may be related to the severity of the disease on the other hand many studies showed that patients with covid19 could present initially with the typical gastrointestinal symptoms and diarrhea may even occur earlier than pyrexia or respiratory symptom in some cases with covid19 [] luo reported that of covid19 cases presented initially only with gastrointestinal symptoms the covid19 patients initially only with gastrointestinal symptoms are more difficult to diagnose and might be overlooked which could lead to potentially serious consequences together digestive tract symptoms especially diarrhea are the main complications of covd19 patients which should be noticed during the outbreak of covid19 sarscov2 infection and nervous system injury transmission electron microscopy of autopsy sections showed the presence of sarscov2 virallike ps in frontal lobe brain and neural cell bodies moreover researchers confirmed the presence of sarscov2 in cerebrospinal fluid by genome sequencing the pathological mechanism may be the invasion of sarscov2 into the nervons system the virallike ps in brain capillary endothelium was also observed which suggested that hematogenous route might act as the pathway for sarscov2 to the brain in addition study using the mouse model have shown that sarscov can lead to neuroinvasion via disruption of the nasal epithelium and subsequent neuronal dissemination which suggested that coronavirus may use the olfactory nerve to enter the brain the symptoms from nervous system of covid19 patients including headache dizziness anosmia and dysgeusia have been observed in the clinic [] table disturbance of consciousness and seizures can occur as complications in the cases with severe covid19 it was convincing enough that the neurological deficits of patients with covid19 could be ongoing if it did not get noticed it was indicated that sarscov2 can cause nervous system damage the neurological deficits meningoencephalitis and acute myelitis in covid19 patients have been reported in the usa switzerland and china [] according to a recent study out of covid19 patients had central nervous system cns symptoms including dizziness headache impaired consciousness ataxia and epilepsy up to of patients with covid19 have headache and clinical manifestations of dizziness were found from of the patients with covid19 [ ] olfactory and gustatory disorders are prevalent peripheral nervous system pns symptoms in covid19 patients in patients with mild and moderate covid19 a high proportion of patients presented olfactory and gustatory dysfunctions and olfactory dysfunction appeared prior to the other symptoms in some cases [ ] these studies showed that the damage of neurological system may also act as a significant feature of covid19 table gastrointestinal symptoms after sarscid0 cov2 infection study chen wang liu xiao huang guan li chen zhou wan li [ | 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 |
" recently copy number alteration cna of 9p241 were demonstrated in of diffuse large bcelllymphoma dlbcl with gene expression and mutation profiles that were similar to those of primary mediastinallarge bcell lymphoma pmbcl however their cnabased profile and clinical impact still remain unclearmethods multiplex ligationdependent probe amplification were employed to investigate the prevalence of jak2pdl2 amplification in dlbcl and their cnabased pattern of driver genes the clinical outcome and characteristicswere also analyzedresults using unsupervised hierarchical clustering a small group of dlbcl was clustered togetherwith pmbcl as cluster_2 demonstrating amplification of jak2 and pdl2 this subgroups ofdlbcl demonstrated significant higher expression of pdl1 than those with myd88 l265p mutationp andthey exhibited dismal os and pfs as compared with dlbcl_othersp and respectively which issimilar to dlbcl with myd88 l265p mutations dlbcl with amplification of jak2pdl2 exhibits cna pattern that is similar to pmbcl anddemonstrates unfavorable clinical outcome that resembles those with myd88 l265p mutation it is essential toidentify this subgroup of dlbcl who may acquire more benefits from the jak2 and pdl1 signaling inhibitionkeywords diffuse large bcell lymphoma jak2 pdl2 amplification prognosis diffuse large bcell lymphoma dlbcl is a highly heterogeneous disease recently several distinctive geneticsubtypes were identified including schmitz r studymcd bn2 n1 and ezb subtypes and chapuy b study c0 c5 clusters [ ] godfrey j study also correspondence jmyingcicamsaccn lvningcicamsaccn xuemin xue and wenting huang are cofirst authors jianming ying andning lv are cosenior authors1department of pathology national cancer centernational clinical researchcenter for cancercancer hospital chinese academy of medical sciencesand peking union medical college beijing chinafull list of author information is available at the end of the identified an unique biological subset of dlbcl withpdl1 gene alterations having high risk features thus the genetics of dlbcl relating to potential therapeutic targets for immune checkpoint inhibitors shouldbe paid much more attention tojanus kinase jak2 programmed cell death ligand pdl1cd274pdcd1lg1 and programmed celldeath ligand pdl2cd273pdcd1lg2 are adjacent to each other on chromosome 9p241 playing keyroles in host immune surveillance amplification of9p241 were frequently seen in celllines of classical and primaryhodgkin lymphoma chl the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cxue bmc cancer page of mediastinal large bcell lymphoma pmbcl but much less in dlbcl cell lines ] correspondingly pd1 ligands pdl1 and pdl2transcripts and proteins were more abundant in chl andpmbcl cell lines than that in dlbcl cell lines recently y wang study demonstrated that ofdlbcl had copy number alteration cna of 9p241with a gene expression and mutation profile similar tothose of pmbcl however their cnabased profileand clinical impact still remain unclearin thisthereforestudy we employed multiplexligationdependent probe amplification mlpa to investigate the prevalence of jak2pdl2 amplification indlbcl and their cnabased pattern of driver genesincluding bcl2 cdkn2a and tp53 and we analyzed their longterm survival outcome after treatmentof rchoplike regimemethodscase selectionwe collected consecutive cases of dlbcl and pmbclin our clinical ffpe archives of excisional biopsy database between jan and oct and cases ofdlbcl and cases of pmbcl were found after confirmation one case of dlbcl was diagnosed as pmbclthus cases of dlbcl and cases of pmbcl wereacquired finally see additional file all patients werediagnosed at national cancer centernational clinicalresearch center for cancercancer hospital chineseacademy of medical sciences and peking union medicalcollege according to the revised 4th edition ofthewho classification of tumours of haematopoietic andlymphoid tissues the data regarding treatment andprognosis were acquired by means of medical recordconsultation and telephone conversationmultiplex ligationdependent probe amplification mlpagenomic dna were extracted from formalinfixedparaffinembedded ffpe blocks using qiaamp dnaffpe tissue kit qiagen valencia ca then dnacopy number quantification and myd88 l265p mutation were detected using mlpa kitmrchollandnetherlands the pcr products were detected on anabi genetic analyzer applied biosystems usaand the final result were analyzed using coffalyser software the relative peak ratio prr of probe largerthan was defined as amplification and less than was defined as deletion see additional file geneswhich had two or more probes covering two differentexomes were put into final analysis including jak2 pdl2 mdm2 rel pus10 bcl2 nfatc1 spib foxp1nfkbiz bcl6 prdm1 tnfaip3 cdkn2a ptening1 and tp53 the details of mlpa probes of drivergenes in dlbcl are shown in the online supportingmaterial see additional file true amplification of onegene was regarded only when all probes of this gene exhibited amplification and vice versa see additional file myd88 l265p mutation was identified when theprobe had a high peak myd88 wildtype didnt show anypeak see additional file immunohistochemistry ihc staining of pdl122c3ihc staining was performed on dako autostainer link asl48 platform each ffpe block were cut at athickness of 4μm and then deparaffinized antigen retrieval were performed using the envision¢ flex targetretrieval solution at low ph monoclonal pdl1clone 22c3 dako were used as primary antibodyfollowed by incubation with envision¢ flex mouselinker and then envision¢ flex hrp reagent finally the ihc was visualized by envision¢ flex dabeach ihc slide contained a positive controllungcarcinomaihc score of pdl1 were calculated by multiplyingthe percentage of positive cells with mean intensity no staining weak staining moderate staining strong staining which was reported in previous study the results were evaluated by an experienced hematopathologist xueminstatistical analysisthe differences of clinicopathological characteristicsamong different groups were analyzed using chisquaretest fisher exact test or kruskalwallis rank sum testpdl1 ihc score between different groups was analyzedusing wilcoxon test overall survival os and progressfree survival pfs times were defined from the date ofpathologic diagnosis to the date of the event or the lastfollowup the hazard ratio hr of each parameter wascalculated by univariate cox proportional regressionanalysis firstly in which parameters with p wereevaluated together using multivariate cox proportionalregression analysis the survival curve were made according to kaplanmeier procedure the day oflastfollowup was march 1st all statistical analysiswere two sided and p was defined as significanceunsupervised hierarchical clustering was carried outusing euclidean distance and complete method heatmap was plot using pheatmap packageall above statistical analyses were run in r statistic softwareresultsunsupervised hierarchical clustering of cnas of drivergenes and its survival analysis in dlbcl and pmbclpatientsbased on array cgh lenz g study previouslyidentified specific cnas in pmbcl which were different 0cxue bmc cancer page of from abc and gcb of dlbcl abc dlbcls oftenhave cnas in foxp1 nfkbiz cdkn2a cdkn2binf4a bcl2 nfatc1 and spib while gcb dlbclsfrequently harbor cnas in rel pten mdm2 mihg1and ing1 pmbcl often demonstrate cnas of jak2 andpdl2 using unsupervised hierarchical clustering we explored the cnabased pattern of these genes in dlbcl andpmbcl the result showed that a small group of dlbcl was clustered together with pmbcl as cluster_2 with amplification of jak2 and pdl275068fig 1a this subgroup of dlbcl occurred atthe site of cervical lymph node cases gastrointestinal tract cases nasal cavity case and spleen cases fig 1atable 1additional file the frequency of jak2 and pdl2 amplification in the whole cohort of dlbcl were and while both of them were inpmbcl fig 1a see additional file meanwhile all casesin cluster_3 harbored amplification of nfkbiz which is essential for nfκb activation in abc dlbcl but noamplification of nfkbiz was found in cluster_1as to survival dlbcl in cluster_2 demonstrated significant worse os p and pfs p as compared with dlbcl in cluster_1fig 1b howevercluster_1 and cluster_3 didnt reveal significant differencein survival fig 1b we also analyzed the os and pfs between dlbcl with and without jak2pdl2 amplification and got statistical significance see additional file of note we found that dlbcl in cluster_2 enrichedfor jak2pdl2 amplification had less frequency ofmyd88_l265p mutation fig 1a table fig heatmap and survival analysis based on unsupervised hierarchical clustering and status of jak2pdl1 amplification and myd88 mutationin tcga dataset a heatmap of cnabased profiles of driver genes in dlbcl and pmbcl by using unsupervised hierarchical clustering b survivalcurves and coxregression analysis of os and pfs among three cnabased clusters after rchoplike treatment c status of amplifications of jak2pdl1cd274 and pdl2pdcd1lg2 and mutation of myd88 in dlbcl tcga pancancer atlas from cbioportal [ ] 0ccervical lymphnodefemale high_intermediatehigh_intermediatedlbclnasal cavitymalegcbbreak_apartdlbcldlbcldlbcldlbcldlbcldlbclcervical lymphnodestomachstomachcolondlbclpmbclpmbclpmbclpmbclcervical lymphnodecervical lymphnodemediastinummediastinummediastinummalelow_intermediate non_gcbnormalfemale low_intermediate non_gcbmalemalelowhighnon_gcbgcbnon_gcbnormalnormalnormalnormalfemale low_intermediate non_gcbnormalmalelowfemale female female lowlowhigh_intermediatenanananananormalnormalnormalnormalnormaljak2_amppdl2_ampxue bmc cancer page of table the clinicopathological characteristics of dlbcl with jak2pdl2 amplification and pmbclmyd88_no diagnosis sitel265pmyc_ breakapartnormalhansalgorithmnon_gcbage ipi _riskspleenfemale lowsexpmbclna not applicablemediastinummalelowwhich was supported by the cancer genome atlas datatcga pancancer atlas from cbioportal [ ] fig 1ccategory and myc breakapart didnt show any significant differences table jak2pdl2 amplification identify a distinctive cnabasedpattern of dlbcl similar to that of pmbclsince dlbcl with jak2pdl2 amplification had less frequency of myd88 l265p mutation our study separateddlbcl patients into three subgroups dlbcl with jak2pdl2 amplification dlbcl_jak2pdl2_amp dlbclwith myd88 l265p mutation dlbcl_myd88_l265pjak2pdl2 amplification norand dlbcl withoutmyd88_l265p mutation dlbcl_others fig 2a basedon the unsupervised cluster result fig 1a one patientwho had both jak2pdl2 amplification and myd88l265p mutation was clustered into cluster_2 thereforethis patient was put into dlbcl_jak2pdl2_amp subgroup accordingly we also analyzed the data when thiscase was included in dlbcl_myd88_l265p subgroupand got the similar result see additional file unlike dlbcl_myd88_l265p and dlbcl_othersdlbcl_jak2pdl2_amp showed a distinctive pattern similar to that of pmbcl with high frequencyof rel and nfkbiz amplifications but no amplification of bcl2 and nfatc1 and no deletion ofprdm1 was found fig 2awith respectto clinicopathologicdlbcl_jak2pdl2_amp tend to be youngerdlbcl_myd88_l265p p hans modelcharacteristicsthantable whileinternational prognostic index ipi riskpdl1 expression in dlbcl with jak2pdl2 amplificationwas significantly higher than that in dlbcl with myd88l265p mutationtotally cases were performed pdl1 22c3 ihc detection including dlbcl_myd88_l265p cases dlbcl_jak2pdl2_amp cases dlbcl_others cases andpmbcl cases the result showed that pdl1 expressionin dlbcl_jak2pdl2_amp was significantly higher thanthat in dlbcl_myd88_l265p p and dlbcl_others p fig 2b and d while no significant difference was found between dlbcl_jak2pdl2_amp andpmbcl p fig 2bjak2pdl2 amplification identify a subgroup of dlbclwith unfavorable survival outcome similar to that ofmyd88 l265p mutationtrying to explore the survival indication of jak2pdl2 amplification and myd88 l265p mutation cases of dlbcls who received rchoplike regimentwith or without surgical resection were enrolled toperformed cox proportional regression analysis of osand pfs the median followup time was monthsrange monthsin the univariatecompared withdlbcl_others dlbcls with myd88 l265p mutationhad significantly worse os and pfs p andanalysisas 0cxue bmc cancer page of fig comparison of cnabased pattern pdl1 expression and survival analysis among pmbcl and three subgroups of dlbcl a comparison ofcnabased patterns of driver genes among pmbcl and three subgroups of dlbcl according to the status of jak2pdl2 amplification andmyd88 l265p mutation b comparison of pdl1 expression ihc score among pmbcl and three subgroups of dlbcl c survival curves and coxregression analysis of os and pfs among three subgroups of dlbcl after rchoplike treatment d representative images of heà and pdl1à ihc in dlbcl_jak2pdl2_amp and dlbcl_ myd88_l265p respectively and the same to dlbcls withjak2pdl2 amplification p and respectively meanwhile ipi risk category were significantly associated with os and pfs fig 2c tables and in the multivariate analysis ipi risk category andthree subgroups of dlbcl were put into analysis ascompared with dlbcl_others dlbcl with myd88l265p mutation still showed poor os and pfs p and respectively and the same todlbcl with jak2pdl2 amplification for pfs andos p and respectively meanwhile ipirisk category was still an independent risk predictorsfor os and pfs fig 2c tables and either jak2pdl2 amplification or myd88 l265pmutation are frequently seen in relapserefractory dlbclwith pfs less than yearsdlbcl with pfs less than years was defined as primaryrelapserefractory cases among these cases who treated byrchoplike regime the frequency of jak2 and pdl2amplification were and meanwhilethe frequency of myd88 l265p mutation were dlbcl with either jak2pdl2 amplification ormyd88 l265p accounted for discussiondlbcl presents with a wide spectrum of genetic aberration recently shi study exhibited pdl2 amplification in pmbcl and of dlbcl chapuy demonstrated of 9p241 amplification indlbcl meanwhile dlbcl with pdl1 gene alterations was identified as a unique biological subgrouphaving high risk features y wang study demonstrated that of dlbcl had cna of 9p241 withgene expression and mutation profiles that were similarto those of pmbcl in our study by using unsupervised hierarchical clustering cases ofdlbcl were clustered together with pmbcl as cluster_2 indicating that they shared recurrent cnas theywere enriched for jak2 amplification and pdl2 amplification fig 1a 0cxue bmc cancer page of table comparison of characteristics among pmbl and three subgroups of dlbcldlbclothersmyd88_l265pjak2pdl2_amppmbclpatientsage median range bmnegativepositiveihc hans algorithmgcbnongcbipilowrisklow_intermediatehigh_intermediatehighmyc breakapartnegativepositive p_valueÏ2test kruskalwallis rank sum testusing hans model most of dlbcl in cluster_2 werenongcb and tend to be younger than othergroups of dlbcl table which was consistent withprior study therefore coupled with y wang study we confirmed that dlbcl with jak2pdl2 amplification is a unique subgroup resembling the pmbclwith respect to cna patternwith regard to survival increasingly data exhibited thatthe suppression of immune surveillance in dlbcl was associated with poor survival godfrey j study hasdemonstrated that dlbcl with pdl1 gene alterationsshowed high risk features metaanalysis also showedthat pdl1 expression was associated with poor os andadverse clinicopathologic features in dlbcl in y wang study of dlbcl harbored cnaof 9p241 of which were gains and were amplifications and as compared with those who have nogain of 9p241 dlbcl with 9p24 amplification had atrend of better efs while patients with only gain tend tothey didnthave worse prognosis unfortunatelyshow any statistical significance in our study of dlbcl were found that had cna of jak2when jak2 cna was separated into gain mlpa valuebetween and amplification mlpa value as described cases in dlbcl_jak2pdl2_amp group were found that had jak2 gain whichwas slightly lower than that in wang j study asshown in additional file and both dlbcl withjak2 gain and with amplification demonstrated significant poor prognosis as compared with rest of dlbclas shown in additional file more interesting unlikey wang study cases of pmbcl were included in our study as control all of which demonstrating jak2 gains rather than amplifications as shown inadditional file of note we found that dlbcl in cluster_2 enrichedfor jak2pdl2 amplification had less frequency ofmyd88_l265p mutation fig 1a table which was supported by the cancer genome atlas datatcga pancancer atlas from cbioportal [ ] fig 1cmyd88 l265p is a poor indicator of survival for dlbcl which may lead to primary refractoryrelapsed diseasethis is a gainoffunction driver mutation occurring in of dlbcl but absent in pmbcl [] inour study the frequency of myd88 l265p in dlbcl andpmbcl were and which were in linewith prior studies [] of greatinterest myd88l265p mutation occurred less frequently in cluster_2 which was supported by the data tcga pancancer atlas from cbioportal [ ] thus when we divided dlbcl patients into three subgroups dlbcl_jak2pdl2_amp dlbcl_myd88_l265p and dlbcl_others both dlbcl_jak2pdl2_amp and dlbcl_myd88_l265p demonstrated dismal os and pfs with amedian followup of years as compared with dlbcl_others therefore dlbcl with jak2pdl2 amplification 0cxue bmc cancer page of table os in dlbcl treated by rchoplike regimeage ¥ bmnegativepositivesiteextranodalnodalihc hans algorithmgcbnongcbmyc fish breakapartnegativepositiveipi risk categorylowlow_intermediatehigh_intermediatehightherapyrchopresection rchopcna_based_clustercluster_1cluster_2cluster_3three subgroups of dlbcldlbcl_othersdlbcl_jak2pdl2_ampdlbcl_myd88_l265poshr_u95ci p_valueoshr_m95cip_value hr_u hazard ratio by univariate analysis hr_m hazard ratio by multivariate analysis because age was contained in the ipi thus it wasnt put into multivariate analysiswas identified as a poor survival subgroup that is similar todlbcl with myd88 l265p mutationmeanwhile we also compared the cna patterns ofdriver genes among dlbcl_jak2pdl2_amp dlbcl_myd88_l265p dlbcl_others and pmbcl dlbcl_jak2pdl2_amp showed a distinctive pattern similarto pmbcl with high frequency of rel and nfkbizamplifications but no amplification of bcl2 and nfatc1 and no deletion of prdm1 was found the profile ofdlbcl_myd88_l265p was closed to dlbcl_othersshowing relatively high frequency of cdkn2a deletionnfatc1 amplification and bcl2 amplificationin our study of dlbcl_jak2pdl2_ampharbored both jak2 and pdl2 amplifications simultaneouslyindicating that they may also have the pdl1amplification because pdl1 located in the middle ofjak2 and pdl2 at 9p241 thus we hypothesized thatpdl1 expression would be upregulated in this subgroup as what we expected using pdl1 22c3 ihcdetection pdl1 expression in dlbcl_jak2pdl2_in dlbcl_amp was significantly higher than thatmyd88_l265p p and dlbcl_othersp fig 2b and d but not in pmbcl p fig 2b meanwhile pdl1 expression could be 0cxue bmc cancer page of table pfs in dlbcl treated by rchoplike regimeage ¥ bmnegativepositivesiteextranodalnodalihc hans algorithmgcbnongcbmyc breakapartnegativepositiveipilowlow_intermediatehigh_intermediatehightherapyrchopresection rchopcna_based_clusterscluster_1cluster_2cluster_3three subgroups of dlbcldlbcl_othersdlbcl_ jak2pdl2_ampdlbcl_ myd88_l265ppfshr_u95ci p_value pfshr_m95cip_value hr_u hazard ratio by univariate analysis hr_m hazard ratio by multivariate analysis because age was contained in the ipi thus it wasnt put into multivariate analysisenhanced not only by pdl1 amplification but also byjak2 activation [ ] therefore dlbcl with jak2pdl2 amplification was confirmed as an unique subtype that is different from dlbcl with myd88 l265pand othersobjective response rates orr of pd1 blockade therapy was in unselected patients with relapsedrefractory dlbcl [ ] the wide spectrum of orrmay be due to high heterogeneity of this subgroupansell sm study demonstrated patients with9p241 alteration in relapsedrefractory dlbcl inour cohort the frequency of jak2 and pdl2 amplification in relapsedrefractory dlbcl were and which were within the range of orr in the prior studies[ ] while patients were found thathad myd88 l265p mutation who may not be suitablefor antipd1 therapy thus the genetic analysis in refractoryrelapsed dlbcl is required for future therapyselection to increase the orr of immune checkpointinhibitorsjak2 amplification could augment the expression of itself and pd1 ligands pdl1 and pdl2 enhancing the 0cxue bmc cancer page of sensitivity to jak2 kinase inhibitor chemical jak2inhibition could reduce the rna transcription and protein expression of pdl1 thus selective inhibitionof jak2 would be a valuable complementary therapy forpdl1 blockadeauthors contributionsxx contributed to pdl1 ihc staining clinical followup data analysis andmanuscript writing wh contributed to ffpe tissues collection mlpa detection and clinical followup tq and lg provided experiment guidance anddata interpretation jy and nl contributed to study design coordination discussion and manuscript editing all authors read and approved the finalmanuscriptsjak2pdl2 exhibitsdlbcl with amplification ofpmbcllike cnas pattern and demonstrates unfavorable outcome resembling those with myd88l265p mutation thusit is essential to identify thissubgroup of dlbcl who may acquire more benefitsfrom the jak2 and pdl1 signaling inhibition andjak2 amplification detection by mlpa would be feasible in routine practice meanwhile the difference ofsurvival outcome between our study and wang j study indicated that pmbcllike dlbcl suggested by 9p241 cna could be an intermixed subgroup which required further explorationsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020072933additional file mlpa results and clinical followup data the clinicopathological characteristics clinical followup data and mlpa results areshowed in this fileadditional file figure s1 representative results of mlparepresentative results of mlpa are showed in this figureadditional file table s1 the details of mlpa probes of genes indlbcl the locations and lengths of mlpa probes of genes are showedin this tableadditional file the detailed information of dlbcl with jak2pdl2amplification the detailed data about clinicopathological characteristicsmorphology immunohistochemistry and treatments of dlbcl with jak2pdl2 amplification are showed in this fileadditional file figure s2 the os and pfs of dlbcl with or withoutjak2pdl2_amp the os and pfs of dlbcl with or without jak2pdl2_ampadditional file figure s3 comparison of cnabased pattern andtheir survival outcome among pmbcl and three subgroups of dlbclone case of dlbcl with jak2pdl2 amplification and myd88 l265p mutation were included in dlbcl_myd88_l265p group a comparison ofcnabased patterns of driver genes among pmbcl and three subgroupsof dlbcl according to the status of jak2pdl2 amplification and myd88l265p mutation b survival curves and coxregression analysis of os andpfs among three subgroups of dlbcl after rchoplike treatmentadditional file figure s4 the frequencies of jak2 gain andamplification and their survival analysis a the frequencies of jak2 gainand amplification in dlbcl_jak2pdl2_amp and pmbcl b the os andpfs of dlbcl with jak2 gain or with jak2 amplificationabbreviationsdlbcl diffuse large bcell lymphoma pmbcl primary mediastinal large bcell lymphoma mlpa multiplex ligationdependent probe amplificationtcga the cancer genome atlas ipi international prognostic indexffpe formalinfixed paraffinembedded os overall survival pfs progressfree survival hr hazard ratioacknowledgementsnot applicablefundingthis study was partly supported by the beijing municipal science technology commission grant number z151100004015121 the cancerfoundation of china grant number lc2014l13 and cams innovation fundfor medical sciences grant number 2016i2m1001 to perform ffpe tissuescollection and mlpa detection and was partly supported by the cancerfoundation of china grant number lc2018b10 and pumc youth fundand the fundamental research funds for the central universities grantnumber to conduct pdl1 ihc staining and clinical followupand collect fish data of cmycavailability of data and materialsall data generated or analyzed during this study are included in thispublished and its supplementary information filesethics approval and consent to participatethis is a retrospective study that was launched in november the casesenrolled in this project were diagnosed between jan and oct whose ffpe samples were used the data regarding treatment andprognosis were acquired by means of medical record consultation andtelephone conversation thus the need for consent was waived by theindependent ethics committee of cancer hospital chinese academy ofmedical sciences national gcp center for anticancer drugs ncc2015st05consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of pathology national cancer centernational clinical researchcenter for cancercancer hospital chinese academy of medical sciencesand peking union medical college beijing china 2department ofpathology national cancer centernational clinical research center forcancercancer hospital shenzhen hospital chinese academy of medicalsciences and peking union medical college shenzhen chinareceived march accepted august referencesschmitz r wright gw huang dw johnson ca phelan jd wang jqroulland s kasbekar m young rm shaffer al genetics andpathogenesis of diffuse large bcell lymphoma n engl j med chapuy b stewart c dunford aj kim j kamburov a redd ra lawrencems roemer mgm li aj ziepert m molecular subtypes of diffuse largeb cell lymphoma are associated with distinct pathogenic mechanisms andoutcomes nat med godfrey j tumuluru s bao r leukam m venkataraman g phillip jfitzpatrick c mcelherne j macnabb bw orlowski r pdl1 genealterations identify a subset of diffuse large bcell lymphoma harboring a tcellinflamed phenotype blood green mr monti s rodig sj juszczynski p currie t o'donnell e chapuy btakeyama k neuberg d golub tr integrative analysis reveals selective9p241 amplification increased pd1 ligand expression and furtherinduction via jak2 in nodular sclerosing hodgkin lymphoma and primarymediastinal large bcell lymphoma blood wang y wenzl k manske mk asmann yw sarangi v greipp pt krull jehartert k he r feldman al amplification of 9p241 in diffuse large bcell lymphoma identifies a unique subset of cases that resemble primarymediastinal large bcell lymphoma blood cancer j 0cxue bmc cancer page of lenz g wright gw emre nc kohlhammer h dave ss davis re carty slam lt shaffer al xiao w molecular subtypes of diffuse large bcelllymphoma arise by distinct genetic pathways proc natl acad sci u s aswerdlow sh campo e harris nl jaffe es pileri sa stein h thiele j whoclassification of tumours of haematopoietic and lymphoid tissues revised4th edn lyon iarc cerami e gao j dogrusoz u gross be sumer so aksoy ba jacobsen abyrne cj heuer ml larsson e the cbio cancer genomics portal anopen platform for exploring multidimensional cancer genomics datacancer discov gao j aksoy ba dogrusoz u dresdner g gross b sumer so sun yjacobsen a sinha r larsson e integrative analysis of complex cancergenomics and clinical profiles using the cbioportal sci signal pl1 nogai h wenzel ss hailfinger s grau m kaergel e seitz v wollertwulf bpfeifer m wolf a frick m ikappabzeta controls the constitutive nfkappab target gene network and survival of abc dlbcl blood shi m roemer mg chapuy b liao x sun h pinkus gs shipp ma freemangj rodig sj expression of programmed cell death ligand pdl2 is adistinguishing feature of primary mediastinal thymic large bcelllymphoma and associated with pdcd1lg2 copy gain am j surg pathol qiu l zheng h zhao x the prognostic and clinicopathological significanceof pdl1 expression in patients with diffuse large bcell lymphoma a metaanalysis bmc cancer moelans cb monsuur hn de pinth jh radersma rd de weger ra vandiest pj esr1 amplification is rare in breast cancer and is associated withhigh grade and high proliferation a multiplex ligationdependent probeamplification study anal cell pathol amst fernandezrodriguez c bellosillo b garciagarcia m sanchezgonzalez bgimeno e vela mc serrano s besses c salar a myd88 l265p mutation isan independent prognostic factor for outcome in patients with diffuse largebcell lymphoma leukemia ngo vn young rm schmitz r jhavar s xiao w lim kh kohlhammer h xuw yang y zhao h oncogenically active myd88 mutations in humanlymphoma nature dubois s viailly pj bohers e bertrand p ruminy p marchand vmaingonnat c mareschal s picquenot jm penther d biological andclinical relevance of associated genomic alterations in myd88 l265p andnonl265pmutated diffuse large bcell lymphoma analysis of casesclin cancer res gupta s cheville jc jungbluth aa zhang y zhang l chen yb tickoo skfine sw gopalan a alahmadie ha jak2pdl1pdl2 9p241amplifications in renal cell carcinomas with sarcomatoid transformationimplications for clinical management mod pathol ansell sm minnema mc johnson p timmerman jm armand p shipp marodig sj ligon ah roemer mgm reddy n nivolumab for relapsedrefractory diffuse large bcell lymphoma in patients ineligible for or havingfailed autologous transplantation a singlearm phase ii study j clin oncollesokhin am ansell sm armand p scott ec halwani a gutierrez mmillenson mm cohen ad schuster sj lebovic d nivolumab inpatients with relapsed or refractory hematologic malignancy preliminaryresults of a phase ib study j clin oncol hao y chapuy b monti s sun hh rodig sj shipp ma selective jak2inhibition specifically decreases hodgkin lymphoma and mediastinal largebcell lymphoma growth in vitro and in vivo clin cancer res publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Colon_Cancer |
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 |
" 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 |
" levels of physical activity change throughout the year however little is known to what extentactivity levels can vary based on accelerometer determined sedentary and physicallyactive time the aim of thislongitudinal study was to examine older adults activity changes from a nonsnowfall season to a subsequentsnowfall season with consideration of the codependence of domains of time usemethods participants were older japanese adults women aged years living in a rural area ofheavy snowfall who had valid accelerometer active style pro hja750c data during nonsnowfall and snowfallseasons activity was classified as sedentary behavior sb lightintensity pa lpa and moderatetovigorous pamvpa compositional changes from the nonsnowfall to the snowfall season were analyzed using aitchisonsperturbation method the ratios of each component in the composition such as [sbsnowsbnonsnow lpasnowlpanonsnow mvpasnowmvpanonsnow] for seasonal changes were calculated and were then divided by thesum of these ratiosresults in men the percentages of time spent in each activity during the nonsnowfallsnowfall seasons were for sb for lpa and for mvpa these corresponded to mean seasonal compositionalchanges δsb δlpa δmvpa of and respectively in women the percentages of time spent ineach activity during the nonsnowfallsnowfall seasons were for sb for lpa and formvpa these corresponded to mean seasonal compositional changes δsb δlpa δmvpa of and respectively the degree of seasonal change was greatest in mens in older adults activity behaviors were changed unfavorably during snowfall season particularly so formen the degree of seasonal change was greatest for sb development of strategies to keep rural older adultsactive during the snowfall season may be needed for maintaining a consistentlyactive lifestyle for their healthkeywords accelerometry aging environment exercise sedentary lifestyle correspondence inouetokyomedacjp1department of preventive medicine and public health tokyo medicaluniversity shinjuku shinjukuku tokyo japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0camagasa bmc public health page of global physical activity guidelines state that older adultsleast min per week of moderateshould do atintensity physical activity atleast min vigorousintensity physical activity or an equivalent combinationof both in bouts lasting min or more physical inactivity increases the risk of many adverse health outcomes including mortality cardiovascular disease type diabetes several types of cancer and cognitive decline[] in recent years there has been significant growthin research investigating the detrimental health effects ofsedentary behavior sb put simply too much sitting reducing sb and increasing physical activity are keyingredients of initiatives to reduce the global burden ofnoncommunicable diseases [ ]there is a growing body of evidence identifying the importance of natural environments the attributes of whichcan vary greatly by season as well as built environments asinfluences on physical activity [ ] one systematic review of studies from eight different countries showed thatlevels of physical activity tended to be lowest during winter to date however most of these studies have relied onselfreport physical activity assessments and have not focusedon older adults objective assessment via accelerometers can now overcome problems of recall and reportingbias and allow more accurate and finergrained assessmentsof activity behavior patterns sb lightintensity physical activityactivitymvpa there is limited evidence on how season affects devicebased activity behaviors [] compositionaldata analysis coda allows consideration ofthe codependence of time spent in all behaviors arising within aday or other fixed period [ ] to date no previous studyhas investigated seasonal changes of activity behaviors takingtime spent in each activity behavior into accountlpa and moderatetovigorous physicalwe compared times spent in accelerometermeasuredactivity behaviors during a nonsnowfall and a snowfallseason in a sample of communitydwelling older japanese adults using the coda approach we also exploredwhich activity behaviors were most affected by seasonmethodsstudy sample and data collectionwe used longitudinal data from the neuron to environmental impact across generations study neige study themethods of this study are described in previous study participants were older adults without longterm care livingin tokamachi city japan tokamachi is a rural city officiallyregistered as a heavy snowfall region during winter locatedin the southernmost region of niigata prefecture participants were from two areas matsunoyama mountain areaor tokamachi area downtown the mountain area mmaximum snow depth had more snow than the downtownarea maximum snow depth during winter theaverage temperature at tokamachi city in february was °c lowest °c highest °c brieflyin a total of residents were recruited from a resident registry using stratified randomsampling in the nonsnowfall season autumn of we conducted a questionnaire survey and health examination of older adults who agreed to enrollment inthe neige study at the same time they were asked towear an accelerometer of these participants agreedto also wear an accelerometer during the snowfall seasonwinter of accelerometermeasured activity behaviorshabitual time spent in activity behaviors were evaluatedby active style pro hja750c omron healthcarekyoto japan active style pro is a validated accelerometer [] and comparable to the devices most commonly used in studies conducted in western countries[ ] its measurement algorithm has been explainedin detail elsewhere [ ] participants were instructedto wear an accelerometer over the waist on an elasticated belt for seven consecutive days except duringsleep and waterbased activities during snowfall andnonsnowfall season respectively in the survey duringsnowfall season participants were mailed an accelerometer no acceleration signal detected for longer than consecutive minutes was defined as nonwear participants with a wear time corresponding to at least hduring waking time per day collected over four ormore valid days were included in the analysis thedata were collected in 60s epochs activity behaviorswere classified into three intensity categories based onmetabolic equivalents mets sb mets lpa mets and mvpa ¥ mets [ ]sociodemographic biological and psychological factorsparticipants reported their age gender living arrangement with others alone and selfrated health verygood good fair poor in autumn we classified participants between the ages of and years as youngoldand those between ages and years as oldold bodymass index bmi was calculated from height and weightkgm2 measured by body composition analyzer mc780a tanita corporation tokyo japanstatistical analysesall analyses were performed using r version r foundation for statistical computing vienna austria we usedr package compositions for coda approach for all analyses pvalues were considered statistically significantanalyses were applied stratified by gender since activity behavior patterns were significantly different between men andwomen 0camagasa bmc public health page of the chisquare test or t test was performed to compare participant characteristics mcnemars test wasused to compare nonsnowfall and snowfall seasonsproportions of those adhering to physical activity guidelines ie ¥ minweek of mvpa in bouts of at least min a minbout mvpa was defined as ormore consecutive minutes above the moderate intensitythreshold with the allowance of min interruptionintervals we described activity behaviors during the nonsnowfalland snowfall season using coda approach as all participantsspent some time in every behavior there was no need for amethod to deal with zeros compositional changes [δsbδlpa δmvpa] were then determined by aitchisons perturbation method [] the ratios of each component inthe composition such as [sbsnowsbnonsnow lpasnowlpanonsnow mvpasnowmvpanonsnow] for seasonalchanges were calculated and were then divided by the sumof these ratios an equal composition of these three activitiesin the nonsnowfall and snowfall seasons would result in acompositional change of[ ] compositionalchanges were plotted as ternary diagrams with some significant guide values illustratedresultsparticipant enrollment and characteristicsof the older adults who agreed to wear an accelerometer during both the nonsnowfall and snowfallseasons response rate were excluded for notmeeting wearing time criteria n bone fracturen unreturned accelerometer n and malfunction n the analytic sample was who had validaccelerometer datawhen compared to participant characteristics betweenthose who had valid accelerometer data in snowfall season and those who did not a significant difference wasfound in age groups youngold oldold chisquare test participants who participated in thesurvey in the snowfall season were significantly morephysically active approx more minutes of mvpaper day than those who did not ttesttable presents the characteristics of the participantsthe mean sd age was years womenand most of the participants were living with others kgm2 bmi and good perceived health comparedto women men were more likely to be from the mountain area be living with others and to have attained¥ minweek of mvpa there were no significant seasonal differences in proportion of those adhering to global physical activity guidelines nonsnowfall seasonoverall men women snowfall season overall men women activity behaviors in snowfall and nonsnowfall seasonmean accelerometer wear time was minday innonsnowfallseason and minday in snowfalltable participants characteristics at baseline nonsnowfall seasonoverall n mean ± sd n ± men n mean ± sd n ± women n mean ± sd n ± ± ± pvalueage yrsage categoriesyoungold yrsoldold ¥ yrsarea of residencemountain areadowntownliving arrangementliving with othersliving alonebmi kgm2bmi categoriesnormal kgm2obese ¥ kgm2perceived healthgood very good good ± poor fair poorpvalue was calculated using t test or chisquare test as appropriate 0camagasa bmc public health page of season older adults spent most of their time on sb andlpa table presents the descriptive statistics of timespent in each activity behavior in men time spent ineach activity sb lpa and mvpa was and respectively during the nonsnowfall season and and respectively during the snowfall season corresponding to mean seasonal change of forsb for lpa and for mvpa fig sincelarger distance from indicates greater change in timespent in activity the largest seasonal change was observed in sb compared to lpa and mvpa in womentime spent in each activity was and respectively during nonsnowfall season while that was and respectively during snowfall seasoncorresponding to mean seasonal change of for sb for lpa and for mvpa significant unfavorable seasonal changes in activity behaviors were observed in both men and women but the degree ofchange was larger in men in every behavior if we contrast the changes the ratios between men and womensimilar findings were observed after stratification byresidential area but the degree of change in activity behaviors was larger in mountain area than in downtownregardless of genderdiscussionwe identified seasonal differences of accelerometer determined activity behaviors in communitydwelling olderadults in a rural snowfall area using the coda approacholder adults had more unfavorable activity behavior patterns during the snowfall season compared to nonsnowfall season with the magnitude of these differencesgreater in men no significant seasonal differences werefound in the proportions adhering to global physical activity guidelinesour findings are consistent with previous studies usingselfreport [ ] and pedometer accelerometer [] data where levels of adults physical activity duringwinter was less than the other seasons a study iniceland found that both older men and women weremore active during summer than winter with less timespent in accelerometer determined sb in men in women a study in the uk found that onlytime spent in lpa was significantly higher during springthan that during winter among older adults winter ± hday spring ± hday summer ± hday and autumn ± hday we found thatthe magnitude of decline during winter seems to belarge compared to these icelandic and uk studies thismay result from winter conditions including amount ofsnowfall furthermore our study participants were relatively more active and less sedentary during autumn thenonsnowfall season more highly active participantsmay experience activity decline to a greater degree compared to low activity participants in this sample therewere no significant seasonal changes of adherence toglobal physical activity guidelines this may be due tovery little time spent in mvpa in bouts of more than mingender differences of activity patterns were in linewith our previous findings [ ] that japanese olderwomen were more physically active than older men provided that all activity behaviors were assessed in thissample men experienced greater seasonal activity declines than did women in japan women are more responsible for household chores and thus engage in acertain amount of activities throughout the year theremay be needed to develop a strategy to combat this seasonal decrease in physical activity for menin the current study activity behavior patterns wereunfavorably changed with approximately increasein sb and decrease in mvpa the degree of the changein activity behaviors may be significant for health a previous study found decline of physical activity during winter unfavorably affected the physical performance levelafter one year in communitydwelling older women particularly its effect on maximum walking speed given that the rate of muscle mass decline is higher inolder adults compared to middleaged adults maintaining physical activity may be required to keep physicaltable compositional geometric means of time spent in activity behaviors during the nonsnowfall and snowfall seasonnonsnowfall season minday wear timewear timesb lpa snowfall season minday wear timewear timesb lpa mvpa mvpa overallmenmountain areadowntownwomenmountain areadowntownabbreviation sb sedentary behavior lpa lightintensity physical activity mvpa moderatetovigorous physical activity 0camagasa bmc public health page of fig changes in sedentary behavior sb light intensity physical activity lpa and in moderatetovigorous physical activity mvpa from thenonsnowfall to snowfall season in ruraldwelling older japanese adults a stratified by gender blue men red women b stratified by genderand residential area blue mountain area green downtown abbreviation sb sedentary behavior lpa lightintensity physical activity mvpamoderatetovigorous physical activityability and prevent a negative cycle of frailty another study showed shortterm decreased physical activity with increased sb caused impairment of metabolism which increases risk of cardiovascular disease therefore promoting physical activity during wintermay be one way to tackle agerelated diseases and lossof physical functioningstrengths and limitationsstrengths of this study included objective assessment ofactivity behaviors and a novel statistical approach eventhough there has been increasing research using objective methods [ ] no study has treated with consideration to the codependence of time spent in activitybehaviors moreover we provided resultsfrom aseldomstudied elderly population in a rural snowfallarea however several limitations should be consideredto interpret the findings we may underoverestimate ofsb and lpa since active style pro cannot provide posturalinformation also some activities eg snow removal and skiing may be not captured accuratelysecond we may underestimate the decline of activitylevel since most of the days that participants wore an accelerometer were relatively good weather in spite ofheavy snowfall area it has previously been observed thatlonger day length is associated with increased devicebased physical activity in the older population [ ]third as tokamachi city is a rural area where heavysnowfall is common during winter it is not necessarilyrepresentative ofjapanese rural areas with different 0camagasa bmc public health page of climates people in areas with less snowfall may experience a smaller decline of physical activity during snowfall season more research is needed in the differentclimate zones from different geographic areas finallyselection bias may have occurred in general accelerometry respondents have been more active and healthierthan nonparticipating older adults in this studythose had valid accelerometer data in snowfall seasonwere more physically active than those who did notimplications for future research policy and practiceour findings suggest several implications in terms of bothdevelopment of interventions to protect against seasonalphysical activity decline and physical activity surveillancemonitoring further research regarding how to stay activeduring winter may be required for health promotion particularly in regions that experience long winters and withsevere weather eg heavy snow given that approximately of the land in japan has snow and a cold winter and that a quarter of the population lives in thoseareas leading an active lifestyle during winter potentially has a significant public health impactsince sb is the most affected by season it may be better tofocus on developing strategies to reduce time spent in sittingolder adults might be particularly influenced by seasonaloutside conditions due to reduced physical functioning andmobility and spend much of their time indoors it thus maybe effective to provide supplementary resources for indooractivities eg gymnastic exercise programs sharing ofhousehold chores and making educational instrumentalsupports for safe snow removal further approach includes replacing mentallypassive with mentallyactive sbmay be effective for health particularly for preventing cognitive decline [ ] and depression [ ]as for physical activity surveillance monitoring investigators should be aware of the potential for under oroverestimation of levels of activity especially when theinterest is in its betweenindividual variation includingcommunitylevel and countrylevel comparisons seasonality also should be considered when interventionstudies are performedsaccelerometer determined activity behaviors were greatlyaffected by season in communitydwelling older adults ina rural snowfall area resulting in unfavorable changesparticularly in sb time during snowfall season development of strategies to keep rural older adults active duringthe snowfall season may be needed for maintaining aconsistentlyactive lifestyle for their healthabbreviationssb sedentary behavior lpa lightintensity physical activitymvpa moderatetovigorous physical activity coda compositional dataanalysis mets metabolic equivalentsacknowledgementswe thank the city workers and the participants for their time and effort inthe neige studyauthors contributionsys hm si and tf developed the neige study ys hm si tf hk nf mmand sa collected the data sa performed the analysis and prepared themanuscript sc and no gave critical feedback and revised the manuscript allauthors advised on the data analysis and interpretation and reviewed themanuscriptfundingthis study was funded by grant from the policy research institute ministryof agriculture forestry and fisheries in japan and the pfizer healthresearch foundation and by jsps kakenhi grant number 16h03249 k19794 k10829 and 19h03910 the funding bodies were not involved inany portion of the study design data collection and analysis interpretationof the results and preparation of the manuscriptshiho amagasa is supported by jsps research fellowships for youngscientists neville owen is supported by a national health and medicalresearch council of australia nhmrc centre of research excellence grant nhmrc senior principal research fellowship and thevictorian governments operational infrastructure support programavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable requestethics approval and consent to participatethe university ethics committees niigata university and tokyo medicaluniversity granted ethical approval written informed consent was obtainedfrom all participantsconsent for publicationnacompeting intereststhe authors declare no conflict of interestauthor details1department of preventive medicine and public health tokyo medicaluniversity shinjuku shinjukuku tokyo japan 2institute ofgerontology the university of tokyo hongo bunkyoku tokyo japan 3department of global health promotion tokyo medical anddental university yushima bunkyoku tokyo japan 4school ofhealth and life science institute of applied health research glasgowcaledonian university cowcaddens road glasgow uk 5department ofsport and movement science ghent university ghent belgium6behavioral epidemiology laboratory baker heart diabetes institute level commercial road melbourne vic australia 7centre for urbantransitions swinburne university of technology po box hawthornmelbourne australia 8division of international health niigata universitygraduate school of medical and dental sciences asahimachidoriniigata city japan 9department of active ageing niigatauniversity graduate school of medical and dental sciences asahimachidori niigata city japanreceived february accepted august references world health anization global recommendations on physical activity forhealth httpappswhointirisbitstream106654439919789241599979_engpdf accessed jan lee im shiroma ej lobelo f puska p blair sn katzmarzyk pt effect ofphysical inactivity on major noncommunicable diseases worldwide ananalysis of burden of disease and life expectancy lancet kyu hh bachman vf alexander lt mumford je afshin a estep k veermanjl delwiche k iannarone ml moyer ml physical activity and risk ofbreast cancer colon cancer diabetes ischemic heart disease and ischemic 0camagasa bmc public health page of stroke events systematic review and doseresponse metaanalysis for theglobal burden of disease study bmj 2016354i3857aune d norat t leitzmann m tonstad s vatten lj physical activity andthe risk of type diabetes a systematic review and doseresponse metaanalysis eur j epidemiol arem h moore sc patel a hartge p berrington de gonzalez avisvanathan k campbell pt freedman m weiderpass e adami ho leisure time physical activity and mortality a detailed pooled analysis of thedoseresponse relationship jama intern med sofi f valecchi d bacci d abbate r gensini gf casini a macchi c physicalactivity and risk of cognitive decline a metaanalysis of prospective studiesj intern med young dr hivert mf alhassan s camhi sm ferguson jf katzmarzyk ptlewis ce owen n perry ck siddique j yong cm sedentary behavior andcardiovascular morbidity and mortality a science advisory from theamerican heart association circulation biswas a oh pi faulkner ge bajaj rr silver ma mitchell ms alter dasedentary time and its association with risk for disease incidence mortalityand hospitalization in adults a systematic review and metaanalysis annintern med ding d lawson kd kolbealexander tl finkelstein ea katzmarzyk pt vanmechelen w pratt m the economic burden of physical inactivity a globalanalysis of major noncommunicable diseases lancet tucker p gilliland j the effect of season and weather on physical activity asystematic review public health barnett dw barnett a nathan a van cauwenberg j cerin e builtenvironmental correlates of older adults' total physical activity and walking asystematic review and metaanalysis int j behav nutr phys act shephard rj tudorlocke c the objective monitoring of physical activitycontributions of accelerometry to epidemiology exercise science andrehabilitation new york springer international publishing davis mg fox kr hillsdon m sharp dj coulson jc thompson jlobjectively measured physical activity in a diverse sample of older urbanuk adults med sci sports exerc arnardottir ny oskarsdottir nd brychta rj koster a van domelen drcaserotti p eiriksdottir g sverrisdottir je johannsson e launer lj comparison of summer and winter objectively measured physical activityand sedentary behavior in older adults age geneenvironmentsusceptibility reykjavik study int j environ res public health yasunaga a togo f watanabe e park h park s shephard rj aoyagi y sexage season and habitual physical activity of older japanese the nakanojostudy j aging phys act chastin sf palareaalbaladejo j dontje ml skelton da combined effectsof time spent in physical activity sedentary behaviors and sleep on obesityand cardiometabolic health markers a novel compositional data analysisapproach plos one 201510e0139984 dumuid d stanford te martinfernandez ja pedisic z maher ca lewis lkhron k katzmarzyk pt chaput jp fogelholm m compositional dataanalysis for physical activity sedentary time and sleep research statmethods med res shobugawa y murayama h fujiwara t inoue s cohort profile of the neigestudy in tokamachi city japan j epidemiol japan meteorological agency historical weather data search httpswwwjmagojpjmaindexehtml accessed jul ohkawara k oshima y hikihara y ishikawatakata k tabata i tanaka s realtime estimation of daily physical activity intensity by a triaxial accelerometerand a gravityremoval classification algorithm br j nutr oshima y kawaguchi k tanaka s ohkawara k hikihara y ishikawatakata ktabata i classifying household and locomotive activities using a triaxialaccelerometer gait posture park j ishikawatakata k tanaka s bessyo k tanaka s kimura t accuracyof estimating step counts and intensity using accelerometers in olderpeople with or without assistive devices j aging phys act nagayoshi s oshima y ando t aoyama t nakae s usui c kumagai stanaka s validity of estimating physical activity intensity using a triaxialaccelerometer in healthy adults and older adults bmj open sport exercmed 20195e000592kurita s yano s ishii k shibata a sasai h nakata y fukushima n inoue stanaka s sugiyama t comparability of activity monitors used in asianand westerncountry studies for assessing freeliving sedentary behaviourplos one 201712e0186523 murakami h kawakami r nakae s nakata y ishikawatakata k tanaka smiyachi m accuracy of wearable devices for estimating total energyexpenditure comparison with metabolic chamber and doubly labeledwater method jama intern med tudorlocke c camhi sm troiano rp a catalog of rules variables anddefinitions applied to accelerometer data in the national health andnutrition examination survey prev chronic dis 20129e113troiano rp berrigan d dodd kw masse lc tilert t mcdowell m physicalactivity in the united states measured by accelerometer med sci sportsexerc haskell wl lee im pate rr powell ke blair sn franklin ba macera ca heathgw thompson pd bauman a physical activity and public health updatedrecommendation for adults from the american college of sports medicineand the american heart association circulation pate rr o'neill jr lobelo f the evolving definition of sedentary exercsport sci rev aitchison j ng kw the role of perturbation in compositional data analysisstat model boogaart kg tolosanadelgado r analyzing compositional data with rberlin springerverlag berlin heidelberg winkler eah chastin s eakin eg owen n lamontagne ad moodie m dempsey pckingwell ba dunstan dw healy gn cardiometabolic impact of changing sittingstanding and stepping in the workplace med sci sports exerc uitenbroek dg seasonal variation in leisure time physical activity med scisports exerc matthews ce freedson ps hebert jr stanek ej 3rd merriam pa rosal mcebbeling cb ockene is seasonal variation in household occupational andleisure time physical activity longitudinal analyses from the seasonalvariation of blood cholesterol study am j epidemiol amagasa s fukushima n kikuchi h takamiya t oka k inoue s light andsporadic physical activity overlooked by current guidelines makes olderwomen more active than older men int j behav nutr phys act amagasa s inoue s ukawa s sasaki s nakamura k yoshimura a tanaka akimura t nakagawa t imae a ding d kikuchi h tamakoshi a are japanesewomen less physically active than men findings from the dosanco healthstudy j epidemiol httpsdoi102188jeaje20200185 mizumoto a ihira h makino k saitoh s ohnishi h furuna t physical activityduring winter in oldold women associated with physical performance afterone year a prospective study curr gerontol geriatr res volpi e nazemi r fujita s muscle tissue changes with aging curr opin clinnutr metab care fried lp tangen cm walston j newman ab hirsch c gottdiener jseeman t tracy r kop wj burke g mcburnie ma frailty in older adultsevidence for a phenotype j gerontol series a 200156m146 bowden davies ka sprung vs norman ja thompson a mitchell klhalford jcg harrold ja wilding jph kemp gj cuthbertson dj shorttermdecreased physical activity with increased sedentary behaviour causesmetabolic derangements and altered body composition effects inindividuals with and without a firstdegree relative with type diabetesdiabetologia ormazabal v nair s elfeky o aguayo c salomon c zuñiga fa associationbetween insulin resistance and the development of cardiovascular diseasecardiovasc diabetol wu yt luben r wareham n griffin s jones ap weather day length andphysical activity in older adults crosssectional results from the europeanprospective investigation into cancer and nutrition epic norfolk cohortplos one 201712e0177767schepps ma shiroma ej kamada m harris tb lee im day length isassociated with physical activity and sedentary behavior among olderwomen sci rep inoue s ohya y odagiri y takamiya t kamada m okada s tudorlocke cshimomitsu t characteristics of accelerometry respondents to a mailbasedsurveillance study j epidemiol ministry of land infrastructure transport and tourism snow disaster preventionhttpwwwmlitgojproadbosaifuyumichiprojecthtml accessed jan chastin sf de craemer m lien n bernaards c buck c oppert jm nazareja lakerveld j o'donoghue g holdsworth m the sosframeworksystems of sedentary behaviours an international transdisciplinaryconsensus framework for the study of determinants research priorities andpolicy on sedentary behaviour across the life course a dedipacstudy int jbehav nutr phys act 0camagasa bmc public health page of kesseguyot e charreire h andreeva va touvier m hercberg s galan poppert jm crosssectional and longitudinal associations of differentsedentary behaviors with cognitive performance in older adults plos one20127e47831 bakrania k edwardson cl khunti k bandelow s davies mj yates tassociations between sedentary behaviors and cognitive function crosssectional and prospective findings from the uk biobank am j epidemiol hallgren m owen n stubbs b zeebari z vancampfort d schuch fbellocco r dunstan d trolle lagerros y passive and mentallyactivesedentary behaviors and incident major depressive disorder a 13yearcohort study j affect disord hallgren m nguyen tt owen n stubbs b vancampfort d lundin adunstan d bellocco r lagerros yt crosssectional and | Colon_Cancer |
the ongoing pandemic coronavirus disease outbreak covid2019 stemming from the beta coronavirus class sarscov2 causing severe acute respiratory syndrome sars has created a global health emergency in countries around the globe huang nalla sarscov2 a novel coronavirus strain belonging to the sarbecovirus subgenus genus betacoronavirus family coronaviridae had first appeared in late in wuhan china infecting a large number of hosts million people with a mortality rate of ¥ neogi carter such putative etiopathogenic agents associated with the zoonotic viral transmission pathways are responsible for respiratory viral pneumonia and gastrointestinal infections leading to infected people or patients having multiple an failure in a b s t r a c t severe acute respiratory syndrome coronavirus sarscov2 a neoteric virus belonging to the beta coronavirus class has created a global health concern responsible for an outbreak of severe acute respiratory illness the covid19 pandemic infected hosts exhibit diverse clinical features ranging from asymptomatic to severe symptoms in their genital ans respiratory digestive and circulatory systems considering the high transmissibility r0 compared to middle east respiratory syndrome coronavirus merscov and sarscov the quest for the clinical development of suitable antiviral nanotherapeutics ntps is incessant we are presenting a systematic review of the literature published between and to validate the hypothesis that the pharmacokinetics collateral acutechronic side effects of nano drugs and spike proteins arrangement of coronaviruses can revolutionize the therapeutic approach to cure covid19 our aim is also to critically assess the slow release kinetics and specific target site chemical synthesis influenced competence of ntps and nanotoxicity based antiviral actions which are commonly exploited in the synthesis of modulated nanomedicines the pathogenesis of novel virulent pathogens at the cellular and molecular levels are also considered which is of utmost importance to characterize the emerging nanodrug agents as diagnostics or therapeutics or viral entry inhibitors such types of approaches trigger the scientists and policymakers in the development of a conceptual framework of nanobiotechnology by linking nanoscience and virology to present a smart molecular diagnosis treatment for pandemic viral infections comorbidities till date no therapeutic drug has been discovered for the treatment of sarscov2 infection though the importance of monoclonal antibodies protease and helicase inhibitors and interferons ifnsα treatments have been highlighted in a handful of literature farzin torchilin palmieri and papi development of drugresistant strains host cell toxicitytarget specific actions costs associated with the serological diagnosis and therapeutics limit the widespread application of synthetic drugs nucleoside analogs palmieri and papi torchilin harnessing the potential of nanobiotechnology in the biomedical science development of engineered nanostructured materialsnanomedicines may lead to better drug delivery advanced therapeutics and medical diagnostics at nanoscale in recent years with the advancement of clinical practices the use of metal gold silver zinc and copper corresponding author discipline of earth sciences room no 336a block indian institute of technology gandhinagar gujarat india email addresses manishenvgmailcom manishkumariitgnacin m kumar 101016jenvres2020110119 received may received in revised form july accepted august environmentalresearch1912020110119availableonline23august2020001393512020elsevierincallrightsreserved 0cs mukherjee nanops in magnetic immunoassay viral diagnostics and microfluidic technology has driven the researchers to explore the potential of nanotherapeutics makvandi farzin letko and ahlawat and narayan have investigated the multistrain inhibition of sars coronavirus sarscov herpes simplex virus hsv1 and human immunodeficiency virus hiv of tcells by sulfonated nanops binding they have reported that small p size tunable surface charges biomimetic properties faster encapsulation have made the engineered nanops smart and stable colloidal carriers for the delivery of genes and drugs the mode of action of functionalized nanops can be explained by their covalent linkages with biological substrates such as peptides proteins antibodies and nucleic acids some researchers have reported virusnanops electrostatic nonspecific interactions elsewhere ie influenza a h5n1 strain inhibition alizadeh and khodavandi rothan and byrareddy nanoparticulate drug carriers through the cellular and mechanistic establishment crossed the membranes and with the help of capping agents such as sulfate polysaccharidespolymers undergo multivalent bond interactions with virus glycoproteins ie hemagglutinin ha bachmaier balakrishna such surface proteins often act as a prime inducer of neutralizing antibodies as observed by boulware and chaturvedi and shrivastava where antigenic determinants enable nucleocapsid to take entry into the host cells as reported for in vitro sarscov inhibition of virus replication and the fusion between the viral and host cell endosomal membrane are often facilitated by some emerging nanobased technologies ie silver nanorod array surface enhanced raman spectroscopy sers substrate interferometric biosensor immunoassay chauhan chhikara choudhary the development of such biomolecular detectors enable the molecular binding of virus ps to target specific antigen phase antibodies coated waveguide which help in the fast and reliable detection of viral infections kirchdoerfer and ward cojocaru das considering the high transmissibility r0 and low to moderate pathogenicity of sarscov2 compared to middle east respiratory syndrome coronavirus merscov and sarscov the quest for clinical development of suitable antiviral nanotherapeutics is need of table a summary of biocompatible nanomaterials and antiviral nanopharmaceuticals commonly used for biomedical drug delivery action as virucidal agents souce of data weiss udugama letko jamshidi gao dong nanocarriers aunps immobilized aunps tio2 nps modified tio2nps agnps engineered agnps sinps mesoporous si fullerenes modified fullerenes fenps engineered fenps acidbasic functionalized nanotube metal functionalized nanotube carbon dots graphene oxide go polystyrene nps virucidal action immunizationviral detection viral detection inactivation of virus by photolysis virus inhibitioninactivation viral entry inhibition viral replication deformation virus detection viral entry inhibition virus destabilization inhibition of virus entry host pathogen interaction viral detectionremoval virus inactivation immunization viral inhibition viral entry hindrance mucosal vaccine development target specific cell bhk21 helacdltr vero cells c636 balbc mice na mdck vero cells human rhabdomyosarcoma vero cells hek293t na supt1 na na ncih292 grass carp pk15 marc145 vero cells na virus types hin1 h3n2 hiv1 h3n2 dengue virus h5n1 h3n2 h5n1 h1n1 h7n3 feline calicivirus influenza papilloma virus hsv1 bacteriophage λ hiv1 wild and resistant type zika virus bacteriophage ms2 h5n2 h3n2 reovirus rsv pseudorabies virus porcine epidemic diarrhea virus hiv1 mode of antiviral activity binding with peroxidasemimic enzymes and viral gp120 antibody mediated inhibition viral capsid protein interaction viral surface protein interaction inhibit cd4based binding viral envelop rupture cell mediated immunenucleic acid inhibition hinder viral attachment viral capsidenvelop attachment and interaction impairing viral polyproteinhinder gag processing viral envelopprotein binding phosphatidylserine inhibit viral tropism photoactivated mediated viral inhibition destabilization vp7dna mediated inhibition type i interferon production inhibited negative single layered sharp edged p interaction with virus viral gp120 antigen binding and mannoselectin specific inhibition action immune system inhibition cd8 t cells inhibition antibodydependent cellmediated phagocytosiscytotoxicity viral transcriptase inhibition gene silencing action viral cell entry inhibition peroxidase inhibition viral entry inhibition coagulation results from virus surface protein interaction magnb mediated enzymeatic signaling inhibition cell cycle inhibition at g2m phase glycosaminoglycan binding affinity and in vitro replication inhibition chitosan coated nps peptide coated nps protein coated nps amide coated nps nanoliposomes nanomicelle aunpscarbon nanotubes polymeric micelle aunpsagnps aufenpscarbon nanotubes nanolipid carriers dendrimer na balbc strain balbc mice neuro 2a cell lines na apc49 huh75 na male wistar rats cell mdck na vk2e6e7 vero cells helfs rabies virus influenza a virus influenza a virus hiv na hepatitis c virus h3n2 hiv h1n1 h3n2 norovirus h1n1 papilloma virus hsv12 immunization nanoparticulate vaccine influenza vaccine antiviral therapy drug delivery immunomodulator antiviral activity and bioavailable vaccines viral detection by colorimetric assay oral bioavailable drugs viral inhibition and drug delivery action viral dna detection nontoxic viral inhibition mrna vaccine na not applicable aunpsgold nanops agnpssilver nanops fenpsiron nanops sinpssilica nanops tio2 titanium nanops gographene oxides environmentalresearch19120201101192 0ccome under anic nanocarrier systems based on the their function as therapeutic agents encapsulated chemical attachment adsorbed or dissolved grein neogi gacem fig demonstrates the comparative size range of nanops whereas for hybrid nanobased systems the molecular composition and target specific action of individual engineered nanoparticulate systems play a vital role in drug delivery actions table the mode of action of such nanobased therapeutic agents can be categorised based on the permeability of vasculature passive targeting for malignancy or attachment of bioactive ligands to the selective nanotherapeutics kirchdoerfer and ward gao gadade and pekamwar role of metallic nanops as in vivo and in vitro drug discharging agents s mukherjee the hour rosenberg nalla dung though it is noteworthy to mention that r0 is not data specific to the covid19 disease rather it depends on the place and the behavior of the population it is associated with social isolation hygienic habits among others and the variation must occur in different ways viceconte and petrosillo li the drug development for antiviral treatment of sarscov2 depends on several factors such as pharmacokinetics drug properties collateral acutechronic side effects and spike proteins s arrangement of coronaviruses virus properties these can act as therapeutic targets to prevent the fusion of the virus to host cell via binding with host cell receptors trimeric scaffolds such as nsp10 a viral transcription factor used along with secondary antigens in vaccines for lung and lower respiratory tract infections these are essential to nucleate and stabilize pseudosubdomains of protein and peptide antigens which are also responsible for cell infection and polycistronic expression that may help in the nanobased therapeutic vaccine development dong grein etman peginterferon α2a2b pegasys ifnα2b show excellent drug delivery actions against hepatitis virus c whereas cationiccrosslinked nanops biodegradable polymers such as mpolyethylene glycol mpegpla clpm are found suitable for hepatitis b and c virus infections synthetic and natural polymeric nanops pnps are widely used since the last decade for the effective control of pathogenic viruses based on their individual biochemical properties immuno biocompatibility biodistribution factors table low to zero toxicity profile high stability against proteinase degradation improved safety efficacy profiles and good internalization properties make such nanozymes effective against chronic infectious diseases and also help in exerting their cytoprotective actions against cytopathic effect polymeric nanops in hybridized forms have found their way in biomedical sectors as therapeutics and diagnostics of human adenovirus influenza virus and hiv therefore the widespread application of hybridized nanoformulation systems have been documented by kerry and kim where functionalized dnazyme along with cellular peptides encoding viral envelopes help in the knockdown of hsv2 hvc and influenza a viruses for the first time the purpose of the present systematic review lays out the framework for a developing a critical understanding of selfassembling metal nanops targeting a variety of fusion proteins for vaccine development b the spatial geometry three fold symmetry axis and radial distributions that drive the rapid antigen processing and render virucidal activity c building up a deep insight for biomarkers research in both prophylactic and therapeutic approaches d the critical assessment of nanops as therapeutics pathways biodegradation this review also provides a guide map for the regulation of metabolic enzymes on selected nanopharmaceuticals through which the multigenerational effects can be evaluated this publication is designed to provide vital information on biocompatible nanocarriers active vs passive targeted drug delivery action of nanomedicines and critically analyze the possible hybrid nanobased therapy for sarscov2 inhibition our current review also highlights the stateofart molecular fingerprinting techniques of virus identification through advanced biosensing methods which critically explore integral surveillance and monitoring of novel viral genotypes mode of action and biomedical applications of biocompatible nanocarriers the presence of multiple surface binding sites in vivo clinical interaction with the targeted sites composition based multiple interactions shape luminescence and large surfacevolume ratio of inanic nanops render their multifarious biomedical applications ignatov dong the virucidal activity of silver nanops agnps allows its wider application in the annihilation or amelioration of several viral infections such as poliovirus type1 coxsackievirus b3 influenza a virus etc the mode of action of such nanops can be described either inhibition of cd4dependent cellular bindingpathogenesis or by covalent linking with sulfhydryl group virion surface hill neogi elsheekh the viral internalization can also be prevented by merging nanops with viral genomecore protein that hinders viral replicationattachment release of viral core into the cytoplasm and governing conformational changes in viruscoreceptor association letko hu this type of viral replication inhibitions are quite effective in curing of dsrna viruses infectious hsv2 and bursal disease viruses iravani jamshidi on the other hand gold nanops aunps may also exert their antiviral efficacy through hindering of gp120 fusion to cd4 that are surrounded by capping agents encapsulated aunps table hybridized and charged aunps render virucidal mechanism after associating with nanobased formulation and mimicking peroxidase enzymatic reaction where inhibited viral entry is facilitated by blocking of transcription within the host cell isida kumar 2020a 2020b 2020c kang on the other hand stabilization of magnetic nanops is often carried out by biocompatible polymers which helps in the translation of magneto responsive nanoparticulate systems in clinical diagnostics bio imaging bioresonanceimaging and cell separation the use of superparamagnetic iron nanops γfe2o3fe2o3fe3o4 is not directly involved in the therapeutics but their indirect application may inhibit viral multiplication even at postentry cellular level as seen for zika virus h5n2 and hcv zhou 2020ab dong photothermal nanotherapy high refractive index photocatalytic activities and high solubility properties of titanium nanops tinps enable them to find their wide antiviral application for bacteriophages and h3n2 viruses grein kar the clinical application of functionalized silica nanops sinps drives the diverse antiviral therapeutics or diagnostics application of nanocarriers in inhibition of hiv hepatitis b virus and other recombinant viruses kim nucleic acid hybridization and fluorescent based virus viral protein detection methods are getting popular based on the antiviral efficacy of sinps though their meso to nanoporous biocompatible surfaces allow hostpathogen interaction the emerging demand for novel antiviral nanotherapeutics triggers the researchers to ponder about immunomodulation and immunization of host cells these are essential to regulate premature drug release or inhibition of viral entry through in vivo biodistribution kalantarzadeh kerry the different forms of nanomaterials that are mostly used as antiviral agents depend largely on the pathway of drug delivery system which provide versatile forms of nanobased carriers starting from complex anic hybrid nanosystems to simple inanic metallic composites nanocapsules nanocages and nanospheres are categorised under inanic nanop based systems though nanocapsules can sometimes through and associated drug metabolism environmentalresearch19120201101193 0cs mukherjee fig schematic of the size range of nanops commonly applied in clinical practice as drug delivery agents for gene and drug delivery system quantum dots qd a type of nanosized crystals have excellent nanobased sensing which allow them to be used as antiviral therapeutics or in vitro diagnosis of virulent pathogens where strong chemical interactionsbonding render their biochemical conjugation with therapeutic molecules kostarelos kumar et al 2020a 2020b 2020c and zhou 2020ab have shown that different metallic composition pb cu ga zn hg based qd showed target specific actions against hiv1 human tlymphotropic virus1 after their binding with nh2 receptorbiotin acceptor some researchers have shown that nanoformulations based qds crossed the bbb in vitro model along with target dna and saquinavir antiviral agent which have been widely utilized as highly active antiretroviral therapy kumar 2020a 2020b 2020c lembo evaluation of anic and hybrid nanops as therapeutics and in drug delivery action anic nanopsnanocarriers onp play an important role in the drug delivery action if the therapeutic compounds are of large sized molecules nm encapsulation of such nanoagents through environmentalresearch19120201101194 0cs mukherjee specific designstructure render offtarget toxicity which is required for target specific action lopez gacem slow release kinetics and specific target site chemical synthesis have direct influence on onps therapeutic competence kumar 2020a 2020b 2020c li polymeric nanops pnps have the clinical potential to carry the target drugs to its core or can coordinate with target molecules on its planar surface mainardes and diedrich nasrollahzadeh biocorona formation ease of biodegradation strong mechanicalthermal properties of carbonbased nanocarriers nanorods nanodots etc render their antiviral activities against respiratory syncytial virus rsv hiv1 ebola virus etc table in spite of having broad spectrum activities against antiviral infections the ratelimiting phase of these carbon materials limits their further biomedical applications nikaeen and nguyen have suggested that drug conjugated nanops having excellent antiviral activities can help in the development of an influenza vaccine with matrix protein m2e and hemagglutinin haamine functionalized gelatin surface coating though more research is required to validate the immunity and protection of cellular genes for such impeccable protein vaccines against infectious virulent pathogens like sarscov2 with the advancement of medical sciences the emergence of multifunctional nanobased lipid nanocarriers opens a new door in the field of novel antiviral nanotherapeutics table read and risitano have demonstrated that podophyllotoxin stearic acidglycol loaded lipid nanocarriers are useful in maintaining in vitro slow drug release nontoxic viral inhibition and hemocompatibility of vk2e6e7 hela cells such solidlipid nanops and other nanolipid carriers can fig a and b schematic of the internalization of nanodrugs through the plasma membrane and targeted drug release a and transcytosis of nanodrugs through cell barriers b nanoparticulate drug carriers through the cellular and mechanistic establishment crossed the membranes bloodbrain barrier and bloodtestis barrier and with the help of capping agents such as sulfate polysaccharidespolymers undergo multivalent bond interactions with virus glycoproteins ie hemagglutinin ha environmentalresearch19120201101195 0cs mukherjee become the exceptional antiviral drug discharging agents for infectious viral pathogens ie hpv hiv and hepatitis c virus núËnezdelgado prather hyperbranched monodispersed easily biodegradable anic dendrimers have gained importance for more than a decade in the field of nanomedicine because they act as targeted carriers for biological systems the antiviral activity of dendrimers is still under scientific investigations which may be facilitated through conjugated drug delivery mechanisms patil palestino the efficacy of such nanoparticulated dendrimer system can be observed for the inhibition of hsv1influenza virus where antibody mediated responsecd8 t cell activation and regulation of gene expressions are achieved through small rnas inactivation shang sportelli niosomes the nonionic surfactant based liposome alike anic nanovesicle are getting popular in the advanced biomedical sciences considering their nonimmunogenicity and stable optical properties which make them a suitable carrier of both hydrolipophilic drug molecules excellent bioavailability and controlled release of specific drugs at the targeted sites make such nanocarriers ideal antiviral agents hsv virus where nanoniosome was loaded with suitable antiviral drug acyclovir s´anchezl´opez et al improved drug delivery mechanism and suitable drug release kinetics may allow such nanocarriers to be used in infectious virus diseases nanomicelles fig supramolecular globular micelles exhibit colloidal stability and super encapsulation potential which help such polymeric micelles to show antiviral activity in vitro as observed from curcumin loaded bioavailable nanoformulations hepatitis c virus sivasankarapillai some researchers used graphene oxides conjugated adnps against infectious sars and bursal viruses where the drug resistance event after such antiviral efficacy was mediated by selenium nanops senps andor amantadine am arrangement te velthuis kumar 2020a 2020b 2020c diagnosis or detection of such virulent pathogens have been documented by viceconte and petrosillo and vazquezmunoz and lopez through thiolstabilized gold cluster or enteroviruses labeling with cysteine molecule a brief overview about antiviral nanomedicines rsv virus tremiliosi udugama the size and zeta potential of silver nanops can exert inhibition effects on different human parainfluenza three virus strains or on their replication event the nanocolloidal system of vivagel® is immensely used for the control of zika virus infection tkm130803 is widely used in the treatment of ebola virus utilizing the concept of rnaibased therapy for the lipidbased nanosystem it is well doccumented that for human norovirus treatment the employment of goldcopper sulfide coreshell capsid protein binding results into excellent virucidal activity ziaie on the other hand nanotrap ps are quite often used in the inhibition of infection of target cells by capturing viral rnaviral proteins ie influenza virus treatment zhou 2020ab zhang intrinsic in vivo instability poor immunogenicity and toxicity multiple therapeutic and prophylactic approaches can be overcome by nanovaccinology where cellular and humoral immune response drive the faster uptake of mucosagut associated lymphoid tissue slow controlled release of antigens is facilitated by surface modifications of nanovaccines with antibodiescarbohydrates which results in the target specific immune response by different immune cells additionally their small size and prolonged shelf life help in the faster recognition of the hostreceptor immune system ie hepatitis a virus hav and influenza virus where epaxalexapal is used with immuno targeting agents yu yang and wang nonresponsive immune systems high dose administration coldchain transport of parenteral vaccines limit their widespread application in drug therapy particularly for mucosally administered vaccines the chitosannanop embedded system might be useful for therapeutic proteins or antigens having negative charges which makes its wider application in vaccination against hbv virus through gene delivery systems table the utilization of mouse model employing humoral and mucosal immune responses helped in the liposomebased vaccine development in case of hepaxen used for hepatitis a c and e which further utilized the recombinant surface antigen as a prophylactic vaccine waris wang 2020a 2020b the usages of inflexal v and influvac as standard virosomal vaccine against influenza virus are getting quite popular considering its active biocompatible and immunogenicity wu weiss these types of licensed subunit nanovaccines are found quite useful for older infants and middleaged group people in terms of nanosafety issue as they mimic natural infections as seen in table a cysteineguanine rich oligonucleotide combination with extracellular m2egold conjugates renders molecular protection for pr8h1n1 influenza which was further activated by thiolgold interactions zhang zhou 2020ab from the discussion provided here it is clear that most of the research has been done for influenza virus vaccination but some scanty literature also report several nanomedicines including nanovaccines are under clinical trial or at least in the stage of commercialization for the cure of infectious viral diseases table in general the use of drugs for antiviral therapy is usually employed to target different life cycles of virulent pathogens ie hiv ebola virus or hsv1 valdiglesias and laffon alnrsv01 is a commonly used lipid nanop drug for lower tract respiratory disease which targets the nucleocapsid n gene of table commercial nanomedicines or under clinical trial for the antiviral therapytreatment souce of data neogi letko dong kalantarzadeh kang nanomedicines influvac plus tkmhbv cervisil doravirine dermavir inflexal v epaxal pegasys geovax novavax fluquit curevac peglntron vivagel a pepreclinical evaluation uceunder clinical evaluation hav hepatitis a virus hiv human immunodeficiency virus hbv hepatitis b virus hpv human papillomavirus hcv hepatitis c virus hsv herpes simplex virus mode of action presence of neuraminidase and hemagglutinin rnai therapeutics gene silencing reverse trancriptase inhibitor nonnucleoside dna immunogen with hiv specific t cell precursor antigens specific on speherical carriers surface natural process mimics peroxidases pegylation control stability of protein ankaravirus alike drug therapy clinical stage antiviral nanobiotechnology gene silencing mrna technique pegylation control stability of protein dendrimer with sulphonic acid group interaction disease indication influenza hbv hpv hiv hiv influenza hav hbv hcv sarscov2 sarscov2 influenza sarscov2 hcv hsv hiv biomedical application virosome vaccine solidlipid nanop sirna therapeutic nanoparticulate formulation therapeutic vaccine liposome vaccine liposome vaccine pegylated interferon antiviral therapy nanoparticulate therapeutics sirna therapeutic infections virus vaccine pegylated interferon dendrimer yearstage of development uce preclinical evaluation ucea uce pe pe pea pe uce environmentalresearch19120201101196 0cs mukherjee vaccine research for rotanoroebola virus hpv rsv and others dung das metabolic pathway of nanotherapeutics and their limitations in clinical practice pulmonary and hepatotoxicity studies are required to build a safety profile of such nanodelivery therapeutic or diagnostic agents nanobased approach for sarscov2 infection inhibition nanops uptake cellular process in nanotherapeutics are governed by their physicochemical properties along with cellular membrane characteristics which may have direct influence on the rate of administered drug dosages and structure of engineered nanops it is hypothesized that nanops with optimum diameter of nm and high surface charge density are quite effective in crossing the cellular membranes for hivderived tat cell penetrating peptides weiss waris immunoliposomes and other carbon based nanotubesnanocarriers play an important role in the activation of the complement pathway of host immune systems to deregulate in vitro utilization of nps neogi kumar 2020a 2020b 2020c antibodies that are specifically targeted at polyethylene glycol pegmacrogol polymers and peglike nanostructures can show independent therapeutic efficacy based on their individual immunotoxicology and risk assessment strategies letko read experimental findings wang 2020a 2020b hill with nanobased therapeutic agents reveal the urgent requirement of more rigorous scientific investigations to prove their clinical efficiency in reversing the drug resistance event ie h1n1 virus through seam biodegradation process of nanotherapeutics has gained special attention considering uniform biodistribution kinetics and sustained drug release which are essential for improved drug design process distribution metabolism absorption excretion are important pharmacokinetic features which rate of biochemical features are directly governed by hydrophobichydrophilic profile and tacticity of the nano based formulations at in vitro level patil exocytosis process plays a very important role in the clearance of the foreign nanops out of the cell depending on administered nanocarriers it was hypothesized that ps with nm diameter can | Colon_Cancer |
" inflammatory bowel disease ibd is the collective term for chronic immunemediated diseases ofunknown multifactorial etiology arising from the interplay between genetic and environmental factors andincluding two main disease manifestations ulcerative colitis uc and crohns disease in the last few decadesnaturally occurring alkaloids have gained interest because of their substantial antiinflammatory effects in severalanimal models of disease studies on mouse models of ibd have demonstrated the antiinflammatory action of themain tobacco alkaloid nicotine in addition anatabine a minor tobacco alkaloid also present in peppers tomatoand eggplant presents antiinflammatory properties in vivo and in vitro in this study we aimed to evaluate theantiinflammatory properties of nicotine and anatabine in a dextran sulfate sodium dss mouse model of ucresults oral administration of anatabine but not nicotine reduced the clinical symptoms of dssinduced colitisthe result of gene expression analysis suggested that anatabine had a restorative effect on global dssinducedgene expression profiles while nicotine only had limited effects accordingly map findings revealed that anatabinereduced the colonic abundance of dssassociated cytokines and increased il10 abundances our results support the amelioration of inflammatory effects by anatabine in the dss mouse modelof uc and suggest that anatabine constitutes a promising therapeutic agent for ibd treatmentkeywords plantderived alkaloid mouse model nicotine colitis correspondence pedroantonioruizcastropmicomblainephillipspmicom juliahoengpmicom pedro a ruiz castro ulrike kogel giuseppe lo sasso blaine w phillips andalain sewer contributed equally to this work1philip morris international rd philip morris products sa quai jeanrenaud neuch¢tel switzerland2philip morris international research laboratories pte ltd science parkroad the kendall science park ii singapore singapore 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 0cruiz castro of inflammation page of crohns disease cd and ulcerative colitis uc the mainclinical phenotypes of inflammatory bowel diseases ibdare chronic relapsing inflammatory disorders that affectthe gastrointestinal tract ibd are thought to result froman inappropriate and continuing inflammatory responseto commensal microbes in a genetically susceptible hostenvironmental triggers such as increased hygiene druguse stress smoking and diet influence the onset of thedisease over the past decades naturally occurring alkaloids from plant or medicinal herb sources have sparkedconsiderable interest because of their significant antiinflammatory and antioxidant properties []alkaloidsa class of natural bioactive compoundsderived from amino acids that contain one or moreheterocyclic nitrogen atomsare produced by a widerange of living anisms such as bacteria fungi plantsand animals in plants alkaloids are produced assecondary metabolites in response to environmentalbiotic or abiotic interactions and they confer protectionthrough a range of insecticidal antimicrobial and pharmacological attributes the antiinflammatory activities ofplantderived alkaloids have been documented in severalanimal models of disease including respiratory distress spinal cord injury hepatic fibrosis cancer and ibd [ ] the protective effects of alkaloids havebeen attributed to amelioration of inflammatory responsesand colonic oxidative stress [ ] promotion of epithelial barrier function and positive regulation of gutmicrobiota pyridine alkaloids present in tobacco nicotiana tabacum have been the subject of intensive research nicotinethe major alkaloid in tobacco accounts for approximately of the total alkaloid content of tobacco while thestructurally related nornicotine and anatabine are themost abundant minor pyridine alkaloids accounting for to of total alkaloids other pyridine alkaloids intobacco such as anabasine anabaseine and cotinine arepresent in smaller amounts nicotine and all minortobacco alkaloids have been shown to be pharmacologically active upon binding to several nicotinic acetylcholinereceptors nachrs tobacco nachr agonists suchas nicotine anatabine anabasine anabaseine and cotininedisplay protective effects in animal models of several inflammatory conditions including sepsis parkinsonsdisease alzheimers disease and ibd several in vitro and in vivo studies have shown a clearnicotinedependent positive effect on inflammatory processes [ ] in a previous study oral nicotine administration reduced the severity of dssinduced colitis andreduced colonic tnfα synthesis while subcutaneous injection or minipump infusion had no effect highlightingthe crucial role of administration route for the protectiveeffects of nicotine in dss colitis nicotine has alsobeen shown to attenuate the severity of dss colitis andexpression of il6 in cd4t cells a recent studysuggested that nicotine ameliorates dssinduced inflammation through inhibition of signaltransducer andactivator oftranscription stat3 in gutinfiltratedlymphocytes and intestinal epithelial cells recentlynicotine was shown to cause a decrease in leukocyte recruitment disease activity index dai and histologicalscore in dss colitis and block tnfmediated expressionof mucosal vascular addresin cell adhesion molecule1 inendothelial cells these authors concluded that nicotinesuppressesinflammation through downregulation ofadhesion molecules in the gut nonetheless clinicalstudies on the efficacy and tolerability of nicotine haveshown thattherapeutic application of nicotine fortreatment of uc is limited because of frequent adverseeffects and nicotine inconsistent efficacy in maintainingremission in uc patients [ ]anatabine is found in plants of the solanacea familywhich includes tobacco peppers tomato and eggplant little is known about the biological properties of anatabinealthough several studies have suggested that this alkaloid is apotential candidate compound for antiinflammatory drugdevelopment [ ] anatabine was marketed in the us asa dietary supplement under the name anatabloc in aninternetbased survey approximately of all users ratedthe effect of anatabine supplementation as good or excellentfor joint pain stiffness and functionality anatabine hasbeen shown to inhibit lipopolysaccharide lpsinducedproinflammatory gene expression as well as nfκb andstat3 phosphorylation in human neuroblastoma shsy5y hek293 human microglia and human bloodmononuclear cells as well as in the brain and spleen ofmouse models of autoimmune encephalomyelitis andalzheimers disease in shsy5y cells anatabine alsoreduced the expression of betasecretase the rate limitingenzyme for amyloid peptide production which is a majorhallmark of alzheimers diseasethrough inhibition of nfκb activation in this study we aimed to assess the antiinflammatoryeffects of the tobacco alkaloids nicotine and anatabine inthe established dss mouse model of uc our resultsshow that oral administration of anatabine but notnicotine ameliorates the clinical manifestations of dsstreatment in mice the results of gene expression analysisindicated that anatabine had a partial restorative effect onglobal dssinduced gene expression profiles while nicotineonly had minimal effects moreover multianalyte profilingmap showed that anatabine but not nicotine suppressesthe production of il6 il1α tnfα granulocytecolonystimulating factor gcsf and keratinocyte chemoattractant kc while increasing il10 expression in the colon ofdsstreated mice for an overview of the study conceptand analytical procedures see online resource 0cruiz castro of inflammation page of resultsanatabine has a protective effect in the dss mousemodel of colitisto study the antiinflammatory properties of nicotineand anatabine c57bl6 mice were provided with nicotine or anatabine in drinking water for a total of dayscolitis was induced by oral administration of dssin drinking water ad libitum during days fig 1adsstreated mice developed colitis as evident from thebody weight loss fig 1b increased colon weightlengthratio fig 1c increased dai fig 1d increased stooloccult blood score fig 1e increased intestinal bleedingscore fig 1fincreased diarrhea score fig 1g andincreased colon inflammation score fig 1h in micefig clinical parameters of dsstreated mice administered nicotine or anatabine a mice were orally administered nicotine or anatabine or mgkg for a total of days colitis was induced by oral administration of dss in drinking water ad libitum during days b bodyweight changes in mice during the colitis induction and recovery phases body weight changes were calculated as percentage relative to thestarting day of dss treatment day c colon weightlength ratio are represented as mgcm of colon d dai was calculated according to weightloss colon weightlength ratio and intestinal bleeding e stool occult blood score f intestinal bleeding score g diarrhea score h coloninflammatory status data are shown as mean ± sem p p nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkg 0cruiz castro of inflammation page of not subjected to dss treatment nicotine and anatabine administration had no significant effect on these parametershowever mice treated concomitantly with anatabine anddss showed a decrease in colitis severity in particular inmice that received concomitant anatabine and dss treatment anatabine improved body weight recovery at mgkg p fig 1b caused a decrease in global dai relative to that in dsstreated mice at daily dose of p and mgkg p fig 1c and reduced the stooloccult blood score at mgkg p fig 1e interestingly nicotine but not anatabine improved the intestinalbleeding score relative to that in dssadministered mice at mgkg p fig 1f the diarrhea score fig 1gand the colon inflammation score fig 1h were notaffected by nicotine or anatabine no variation in waterconsumption was registered across the different experimental groups online resource anatabine reduces dssinduced gene expression changesin the distal colon on a global levelto complement these pathological findings we analyzed the colon transcriptome dsselicited lesions inmost mouse inbred strains including c57bl6 micehave been shown to be more pronounced in the distalcolon [ ] therefore we focused our study on thatportion of the large intestine principal componentanalysis clearly captured the effect of dss treatmenton the first principal component explainedvariance fig 2a while nicotine treatment did notproduce a pronounced effect on the first principalcomponent in the dsstreatment context anatabinetreatmentin combination with dss produced aresponse more similar to that observed in the watercontrols no dssindicating that anatabine had anameliorating effect on dssinduced gene expressionchanges the results of our differential gene expressionanalysis suggested substantial perturbation of the colontranscriptomefdr fig 2bc application of dss in drinking watercaused significant changes in nearly genes incolon tissues addition of nicotine to dsscontainingdrinking water slightly increased the number of differentially expressed genes in contrast addition of anatabine decreased the number of differentially expressedgenes upon dss treatment by approximately fig 2b and c this alleviating effect of anatabinetreatment on dssinduced gene expression profileswas also apparent in the global gene expression heatmap which showed a global reduction of expressionfold changes upon anatabine treatment fig 2d and eof note in the absence of dss anatabine treatmentdid not result in differential expression of genes andnicotine treatment alone had minor effects fdr pvalue online resource upon dsstreatmentanatabine reduces dssinduced inflammatory geneexpression in the distal colonto gain a more mechanistic understanding of the effectsof nicotine and anatabine treatment we further investigated gene expression changes at the level of functionalgene sets and a ucrelevant causal network model gsaofthe reactome database showed changes acrossmultiple functional categories fig 3b the effects onfunctional categories in dsstreated mice administerednicotine appeared rather similar to those in mice treatedwith dss alone whereas administration of anatabineresulted in a general repression of dssmediated effectswe also evaluated the interaction terms betweennicotineanatabine and dss treatment to more directlyfocus on the modulating effect of anatabine and nicotinetreatment on dss effects fig 3a online resource the following six biological categories showed significant interaction terms upon anatabine and dss treatmentsupporting asignificant suppression of dssinduced effects in thepresence of anatabine immune system extracellularmatrix anization protein localization metabolism hemostasis and signal transduction fig 3bof these we further explored immune system extracellular matrix anization and signal transductionas the most relevant categories in ibdana 20dss fdr allfiginteractiontermsp the hierarchical anization of the reactome database allowed us to investigate the underlying functionalchanges in more detail the immune system categoryincludes adaptive immune system cytokine signaling in immune system and innate immune systemin particular within these immune categories cytokinesignaling egincluding il6 family signaling andtolllike receptor cascades were modulated withsignificant3conline resource online resource within thereactome signal transduction category signaling byreceptor tyrosine kinasesby rhogtpases signaling by transforming growth factortgfbeta family members mapk family signalingcascades integrin signaling signaling by erythropoietin and signaling by gpcr were found to be significantly impacted online resource online resource within the extracellular matrix anization category almost all subcategories were perturbed includingdegradation of the extracellular matrix elastic fiberformation and extracellular matrix proteoglycansonline resource online resource signalingdegradation ofthe extracellular matrix includesbiological processes such as activation of matrix metalloproteinases mmps and collagen degradationto further follow up on the effects of nicotine andanatabine on inflammatory signaling we evaluatedthe perturbation of the ucrelevant tlril1rtnfr 0cruiz castro of inflammation page of fig transcriptomics results of colon biopsy samples a principal component pc analysis the plot displays all samples in the reduced pc1pc2plane covering of the total data variance it allows us to examine the relationships between the various groups and treatmentsremarkably pc1 captured the pure dss effect black arrow while pc2 captured the pure exposure effects of anatabine and nicotine blue andred arrows respectively b volcano plots for individual gene differential expressions the horizontal axis represents the log2 differential expressionfold changes and the vertical axis represents its statistical significance as log10 fdr c number of differentially expressed genes for theselected pairwise comparisons the bar plot displays the number of genes with positive or negative fold changes with corresponding statisticallysignificant fdr ¤ note that the lower fdr values observed for the ana dss comparisons do not necessarily prefigure a reduction ofbiological effects because the statistics underlying the fdrp values also depend on the gene expression variance within the experimentalgroups d highlevel heatmap of the statistically significant differentially expressed genes for the selected pairwise comparisons in order toprovide a comprehensible display of the large foldchange matrix we first normalized its rows to their maximum absolute values andthen reordered them by hierarchical clustering complete linkage method on the basis of their euclidean distances e scatter plot from thecomparisons between the pure and exposuremodified dss effects by using the differential gene expression results the horizontal axis of thescatter plots represents the fold changes of the pure dss effect water dss vs water pairwise comparison whereas the vertical axis containsthe corresponding values in case of anatabine or nicotine exposure anatabinenicotine dss vs water pairwise comparisons in an idealcase included as a reference [first plot] the bestfitted straight line coincides with the diagonal indicated in green and its slope is equal to the transcriptomics effects of anatabine or nicotine exposure were quantified by the slope of the bestfitted straight lines indicated on each plotnic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkgnetwork model by using an established networkenrichment approach we inferred the activationstates of the network nodes on the basis of the observedgene expression changes and calculated the overall network perturbation amplitude for each group comparisonfig 3c dss treatment had a strong activating effect onthis signaling network including activation of several coresignaling nodes such as il1rassociated kinase irak1irak4 and myeloid differentiation primary response myd88 online resource heatmap leadingnodes of note the network perturbation induced bydss treatment was reduced only in the presence ofanatabine with this the results of network analysisfurther supported the ameliorating effect of anatabineon dssmediated inflammation whereas no similareffect was apparent upon nicotine treatment 0cruiz castro of inflammation page of fig biological interpretation of the transcriptomics results a schematic representation of the effects of anatabinenicotine exposure as amodification of the pure dss effect the measured combined anatabinenicotine dss effect captured by the pairwise comparisons anatabinenicotine dss vs water can be viewed as the sum of the pure effects of the two factors dss treatment and anatabinenicotine exposuretogether with the adjusting twofactor interaction term anatabinenicotinedss anatabinenicotine exposure is synergistic with dss treatmentif the interaction term has the same sign as the pure dss effect eg both are positive as in the schema in contrast the relationship isantagonistic if the interaction term has an opposite sign to the pure dss effect b gsa results for the top reactome pathway categories theheatmap displays the gsa scores normalized rowwise to their maximum absolute values as well as their competitive q1 statistical significancethe fdrs were calculated only among the top reactome pathway categories which are sufficiently distinct in their gene content to assumeindependence of their enrichment results c hierarchical representation of the gsa results for all pathways contained in the top reactomeimmune system category and for the twofactor ana 20dss interaction the color map corresponds to the normalized gsa scores containedin the interval [ ] whereas their statistical significance competitive q1 p values ¤ is indicated by black circles around the nodes theactual labels of the nodes ie the reactome pathway names can be found in online resource the treelike structure of the reactomepathway collection enables topdown investigation within the relevant pathway categories by identifying increasingly specific biologicalprocesses ie involving fewer and fewer genes along the longest paths connecting the statistically significant pathways d npa results for thetlril1rtnfr network model the bar plot displays the npa values for the selected contrasts and their statistical significance is indicated bythe three colored asterisks note that by definition the positive npa values depend on the square of the input genelevel fold changes andtherefore might amplify the differences between the contrasts without preserving the additive relationships among them nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkgexpressionvaluesfrom theto validate the observations obtained by microarraytranscriptomics we quantified the expression of six leadingedge genesgenes of the gene set with the highestimmunedifferentialsystem extracellular matrix anization and signaltransduction reactome categories using realtime quantitative pcr qpcr selected genes included il33 signaltransduction and immune system categoriesil6signal transduction and immune system categoriesmmp13 extracellular matrix anization categoryserpine1 signal transduction and extracellular matrixanization categories thbs1 thrombospondin signal transduction and extracellular matrix anizationcategories and timp1 tissue inhibitor of metalloproteinase extracellular matrix anization and immunesystem categories qpcr results showed a clear decreasein dssinduced expression of il33 il6 mmp13 serpine1thbs1 and timp1 expression in the presence of anatabineat mgkg thereby confirming the findings obtainedfrom the microarray data fig 0cruiz castro of inflammation page of fig validation of microarray transcriptomic results using qpcr a boxandwhisker plots for the distribution of the qpcr expression levels δcqof the selected genes the boxes represent the quartiles while the whiskers extend to the most extreme data point which is no more than times the interquartile range from the box the horizontal brackets indicate the statistical significance of the corresponding comparisons mean pvalue smaller than respectively b scatter plots comparing the mouse colon differential expression values obtained bymicroarray horizontal axis and qpcr vertical axis the following similarity metrics were indicated beta is the slope of the best interceptfreelinear fit between microarray and qpcr values r2 is the coefficient of determination measuring the goodnessoffit and pval is the pvalueassociated to the null hypothesis beta nic nicotine mgkg ana anatabine mgkganatabine decreases dssinduced proinflammatorycytokine production and promotes il10 expressionnext we sought to evaluate the impact of nicotine andanatabine on the expression of colonic inflammatorycytokines by mapin line with the previous dataanatabine but not nicotine significantly reduced theabundance of dssassociated inflammatory cytokines including il6 kc tnfα il1α and gcsf whereas itincreased the levels of the antiinflammatory cytokineil10 at a daily dose of mgkg fig interestinglyanatabine also increased the abundance of il21 andshowed a clear tendency towards increasing colonic il 0cruiz castro of inflammation page of fig map results for colon biopsies statistical assessment of the differences observed in the abundance of selected cytokines between thestudy experimental groups and water control fold changes in the treatment groups nicotine and anatabine at and mgkg dss relative towater control are illustrated with colors ranging from blue decrease to red increase statistically significant differences on the basis of raw pvalues no adjustment has been made for multiple testing grey cells highlight missing estimates on the observed differences due to lackof cytokine quantifications nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkg1 levels fig taken together the map data confirmthe antiinflammatory effects of anatabine in dssinduced colitisdiscussionour study shows that oral administration of anatabinebut not nicotine reduces the clinical manifestations ofdssinduced colitis in a mouse model in line with thesefindings anatabine demonstrated a global downregulatory effect on dssinduced gene expression changes inthe colon whereas the effects of nicotine were morelimited in particular the results of gene expression profiling further supported the reduction of inflammatorysignaling processes upon anatabine treatment includingsuppression of il6 signaling as shown by gsa findingsand tlr signaling as shown by the results of networkperturbation analysis and gsa map also showed asignificant decrease in the abundance of il6 kc tnfαil1α and gcsf and an increase in the expression of il and the antiinflammatory cytokine il10several studies have reported on the antiinflammatoryeffects of nicotine on the dss mouse model of uc subcutaneous administration of nicotine was shown toameliorate tissue injury in dss colitis and il6 expression in cd4t cells via α7nachrs nicotine wasalso shown to decrease the activation of stat3 throughinduction of mir124 in gutinfiltrated lymphocytes andintestinal epithelial cells strikingly alsharari 0cruiz castro of inflammation page of observed that oral but not subcutaneous injection ofnicotine ameliorated intestinalinflammation and colonic tnfα expression in dsstreated mice in spiteof the reported beneficial effects our results do not support a protective effect of orally administered nicotineon dss colitis of note we found that nicotine significantly reduced dssassociated intestinal bleeding whichwas the only clinical parameter affected by this tobaccoalkaloid the vasoconstrictor effects of nicotine are wellstablished in the gut mucosa nicotine decreasesblood flow and cigarette smoking decreases rectalblood flow to normal levels in patients with uc however how changes in blood flow affect the pathophysiology of uc is still unclear the possible therapeuticuse of nicotine to induce remission in uc patients hasbeen evaluated in five clinical studies [] and twometaanalyses [ ] these studies have demonstrated avariable efficacy of nicotine therapy in induction of remission with several studies showing no effect [ ]moreover a high frequency of adverse events increasedthe withdrawal rate in the nicotine group in some studiesthus limiting the therapeutic benefit of nicotine nucleotidebindingto the best of our knowledge the present study isthe first to assess the impact of anatabine on experimental colitis gene expression analysis of distal colonbiopsy specimens highlighted the antiinflammatoryproperties of anatabine in multiple functional categories including immune responses signal transduction and extracellular matrix anization which inturn encompassed several tlr and cytokine signalingpathways genes contributing to the downregulation oftlr cascades included tlr2 tlr4 and tlr6 and anumber of downstream signaling factors shared byseveral tlrs including myd88 ripk2 irak3 irak4and nod1oligomerizationdomaincontaining protein as well as the nuclearfactors elk1 fos cyclic adenosine monophosphatecamp response elementbinding protein creb1activating transcription factor atf1 and atf2 seeonline resource of the respective gene lists for thecytokine signaling pathways the contributing genes included il6 and il6 receptor α ifnγ il4 cxcl10il22 receptor α2 il2 receptor α tnf receptor superfamily member 1a il17α and il17 receptor α jak1jak2 stat3 and stat4 members of the nfκbsignaling pathway also contributed to the overall reducincluding nfκb p65tion in inflammatory cascadesp105 and p100 subunits iκbα and the nfκb activating protein tab3 in agreement with the results oftranscriptional analysis map findings showed a significant decrease in dssassociated il6 kc tnfα il1αand gcsf protein expression and an increase in il10expression in the presence of anatabine strikinglywhile tlr downstream factors modulated by anatabineare shared by most tlrs the majority of cytokineassociated signaling molecules are specific for eachpathway the seemingly pleiotropic regulatory effects ofanatabine suggest that this alkaloid reduces inflammation by inhibiting the expression of several factors involved in different proinflammatory signaling cascadesprevious studies using in vitro and in vivo diseasemodels have demonstrated the antiinflammatory effectsof anatabine [ ] paris showed that this pyridine alkaloid reduced the plasma levels of il1 il6and il17a as well as the expression of il1 infγ andtnfα in the spleen of experimental autoimmune encephalomyelitis mice these authors also showedthat anatabine suppressed stat3 and nfκb phosphorylation in the spleen and brain of these mice anatabine also prevented lps and tnfαinduced nfκb andstat3 phosphorylation in shsy5y and hek cellshuman microglia and human blood mononuclear cells additionally anatabine prevented lpsinduced il1 expression in human whole blood as well as il1il6 and tnfα production in the plasma kidney andspleen in the lps mouse model phosphorylatedstat3 tnfα and il6 were also downregulated in thepresence of anatabine in a transgenic mouse model ofalzheimers disease our results on the effects of anatabine in the dssmouse model of uc are also in line with the findings ofa substantial number of studies demonstrating the protective effects of natural alkaloids in several animalmodels of colitis [ ] intraperitoneal administrationof the minor tobacco alkaloid and nicotinic receptoragonist anabaseine was shown to reduce tissue damagemyeloperoxidase activity and colonic tnfα expressionin a tnbs mouse model of colitis these mice alsoshowed reduced nfκb activation in lamina propriamononuclear cells while mice administered a nicotinicreceptor antagonist presented worse colitis symptomsthan those treated with tnbs alone the algaealkaloid caulerpin reduces dss colitis by suppressingnfκb activation and subsequently inhibiting the colonicproduction of tnfα ifnγ il6 ifnγ and il17 oral administration of berberine also ameliorates dssinduced colitis and downregulates the expression oftnfα ifnγ kc and il17 in colonic macrophages the plantderived alkaloid nmethylcytisine andthe tea alkaloid theophylline mitigate colitis by downregulating tnfα il1 and il6 expression in dss andacetic acid models of colitis respectively [ ] induction ofthe antiinflammatory cytokine il10 in thepresence of natural alkaloids has also been reportedthus indirubin ameliorates dssinduced colitis by suppressing the expression of colonic tnfα ifnγ and il2and upregulating il10 additionally indole alkaloids caulerpin and isatin have been shown to increase 0cruiz castro of inflammation page of the expression of il10 in dss and tnbs models of ibdrespectively [ ] of note our results show anincrease in the abundance of il21 and a tendencytowards increase in colonic il1 levels in the presenceof anatabine although il21 expression is increased inmany chronic inflammatory disorders genetic deficiencyof il21 is associated with ibd and inhibition of il21in the early phases of some inflammatory disorders exacerbates disease development suggesting the dual role ofil21 in the control of immune responses il21also promotes il22 expression in mucosal tcellsthrough a mechanism involving stat3 retinoidrelatedorphan receptor γt and aryl hydrocarbon receptorthereby helping protect immunodeficient mice from dsscolitis interestingly il21 has been recently shownto induce il1 production in dendritic cells through astat3dependent but nfκbindependent mechanismthereby suggesting a link between il21 and il1 mounting evidence suggests that alkaloidsin particular isoquinoline alkaloids present in traditional medicineherbsexertthroughregulation of nfκb and stat3 signaling pathways forexample sanguinarine and cavidine suppress the expression of nfκb p65 subunit thereby reducing colonictnfα and i | Colon_Cancer |
" recently copy number alteration cna of 9p241 were demonstrated in of diffuse large bcelllymphoma dlbcl with gene expression and mutation profiles that were similar to those of primary mediastinallarge bcell lymphoma pmbcl however their cnabased profile and clinical impact still remain unclearmethods multiplex ligationdependent probe amplification were employed to investigate the prevalence of jak2pdl2 amplification in dlbcl and their cnabased pattern of driver genes the clinical outcome and characteristicswere also analyzedresults using unsupervised hierarchical clustering a small group of dlbcl was clustered togetherwith pmbcl as cluster_2 demonstrating amplification of jak2 and pdl2 this subgroups ofdlbcl demonstrated significant higher expression of pdl1 than those with myd88 l265p mutationp andthey exhibited dismal os and pfs as compared with dlbcl_othersp and respectively which issimilar to dlbcl with myd88 l265p mutations dlbcl with amplification of jak2pdl2 exhibits cna pattern that is similar to pmbcl anddemonstrates unfavorable clinical outcome that resembles those with myd88 l265p mutation it is essential toidentify this subgroup of dlbcl who may acquire more benefits from the jak2 and pdl1 signaling inhibitionkeywords diffuse large bcell lymphoma jak2 pdl2 amplification prognosis diffuse large bcell lymphoma dlbcl is a highly heterogeneous disease recently several distinctive geneticsubtypes were identified including schmitz r studymcd bn2 n1 and ezb subtypes and chapuy b study c0 c5 clusters [ ] godfrey j study also correspondence jmyingcicamsaccn lvningcicamsaccn xuemin xue and wenting huang are cofirst authors jianming ying andning lv are cosenior authors1department of pathology national cancer centernational clinical researchcenter for cancercancer hospital chinese academy of medical sciencesand peking union medical college beijing chinafull list of author information is available at the end of the identified an unique biological subset of dlbcl withpdl1 gene alterations having high risk features thus the genetics of dlbcl relating to potential therapeutic targets for immune checkpoint inhibitors shouldbe paid much more attention tojanus kinase jak2 programmed cell death ligand pdl1cd274pdcd1lg1 and programmed celldeath ligand pdl2cd273pdcd1lg2 are adjacent to each other on chromosome 9p241 playing keyroles in host immune surveillance amplification of9p241 were frequently seen in celllines of classical and primaryhodgkin lymphoma chl the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cxue bmc cancer page of mediastinal large bcell lymphoma pmbcl but much less in dlbcl cell lines ] correspondingly pd1 ligands pdl1 and pdl2transcripts and proteins were more abundant in chl andpmbcl cell lines than that in dlbcl cell lines recently y wang study demonstrated that ofdlbcl had copy number alteration cna of 9p241with a gene expression and mutation profile similar tothose of pmbcl however their cnabased profileand clinical impact still remain unclearin thisthereforestudy we employed multiplexligationdependent probe amplification mlpa to investigate the prevalence of jak2pdl2 amplification indlbcl and their cnabased pattern of driver genesincluding bcl2 cdkn2a and tp53 and we analyzed their longterm survival outcome after treatmentof rchoplike regimemethodscase selectionwe collected consecutive cases of dlbcl and pmbclin our clinical ffpe archives of excisional biopsy database between jan and oct and cases ofdlbcl and cases of pmbcl were found after confirmation one case of dlbcl was diagnosed as pmbclthus cases of dlbcl and cases of pmbcl wereacquired finally see additional file all patients werediagnosed at national cancer centernational clinicalresearch center for cancercancer hospital chineseacademy of medical sciences and peking union medicalcollege according to the revised 4th edition ofthewho classification of tumours of haematopoietic andlymphoid tissues the data regarding treatment andprognosis were acquired by means of medical recordconsultation and telephone conversationmultiplex ligationdependent probe amplification mlpagenomic dna were extracted from formalinfixedparaffinembedded ffpe blocks using qiaamp dnaffpe tissue kit qiagen valencia ca then dnacopy number quantification and myd88 l265p mutation were detected using mlpa kitmrchollandnetherlands the pcr products were detected on anabi genetic analyzer applied biosystems usaand the final result were analyzed using coffalyser software the relative peak ratio prr of probe largerthan was defined as amplification and less than was defined as deletion see additional file geneswhich had two or more probes covering two differentexomes were put into final analysis including jak2 pdl2 mdm2 rel pus10 bcl2 nfatc1 spib foxp1nfkbiz bcl6 prdm1 tnfaip3 cdkn2a ptening1 and tp53 the details of mlpa probes of drivergenes in dlbcl are shown in the online supportingmaterial see additional file true amplification of onegene was regarded only when all probes of this gene exhibited amplification and vice versa see additional file myd88 l265p mutation was identified when theprobe had a high peak myd88 wildtype didnt show anypeak see additional file immunohistochemistry ihc staining of pdl122c3ihc staining was performed on dako autostainer link asl48 platform each ffpe block were cut at athickness of 4μm and then deparaffinized antigen retrieval were performed using the envision¢ flex targetretrieval solution at low ph monoclonal pdl1clone 22c3 dako were used as primary antibodyfollowed by incubation with envision¢ flex mouselinker and then envision¢ flex hrp reagent finally the ihc was visualized by envision¢ flex dabeach ihc slide contained a positive controllungcarcinomaihc score of pdl1 were calculated by multiplyingthe percentage of positive cells with mean intensity no staining weak staining moderate staining strong staining which was reported in previous study the results were evaluated by an experienced hematopathologist xueminstatistical analysisthe differences of clinicopathological characteristicsamong different groups were analyzed using chisquaretest fisher exact test or kruskalwallis rank sum testpdl1 ihc score between different groups was analyzedusing wilcoxon test overall survival os and progressfree survival pfs times were defined from the date ofpathologic diagnosis to the date of the event or the lastfollowup the hazard ratio hr of each parameter wascalculated by univariate cox proportional regressionanalysis firstly in which parameters with p wereevaluated together using multivariate cox proportionalregression analysis the survival curve were made according to kaplanmeier procedure the day oflastfollowup was march 1st all statistical analysiswere two sided and p was defined as significanceunsupervised hierarchical clustering was carried outusing euclidean distance and complete method heatmap was plot using pheatmap packageall above statistical analyses were run in r statistic softwareresultsunsupervised hierarchical clustering of cnas of drivergenes and its survival analysis in dlbcl and pmbclpatientsbased on array cgh lenz g study previouslyidentified specific cnas in pmbcl which were different 0cxue bmc cancer page of from abc and gcb of dlbcl abc dlbcls oftenhave cnas in foxp1 nfkbiz cdkn2a cdkn2binf4a bcl2 nfatc1 and spib while gcb dlbclsfrequently harbor cnas in rel pten mdm2 mihg1and ing1 pmbcl often demonstrate cnas of jak2 andpdl2 using unsupervised hierarchical clustering we explored the cnabased pattern of these genes in dlbcl andpmbcl the result showed that a small group of dlbcl was clustered together with pmbcl as cluster_2 with amplification of jak2 and pdl275068fig 1a this subgroup of dlbcl occurred atthe site of cervical lymph node cases gastrointestinal tract cases nasal cavity case and spleen cases fig 1atable 1additional file the frequency of jak2 and pdl2 amplification in the whole cohort of dlbcl were and while both of them were inpmbcl fig 1a see additional file meanwhile all casesin cluster_3 harbored amplification of nfkbiz which is essential for nfκb activation in abc dlbcl but noamplification of nfkbiz was found in cluster_1as to survival dlbcl in cluster_2 demonstrated significant worse os p and pfs p as compared with dlbcl in cluster_1fig 1b howevercluster_1 and cluster_3 didnt reveal significant differencein survival fig 1b we also analyzed the os and pfs between dlbcl with and without jak2pdl2 amplification and got statistical significance see additional file of note we found that dlbcl in cluster_2 enrichedfor jak2pdl2 amplification had less frequency ofmyd88_l265p mutation fig 1a table fig heatmap and survival analysis based on unsupervised hierarchical clustering and status of jak2pdl1 amplification and myd88 mutationin tcga dataset a heatmap of cnabased profiles of driver genes in dlbcl and pmbcl by using unsupervised hierarchical clustering b survivalcurves and coxregression analysis of os and pfs among three cnabased clusters after rchoplike treatment c status of amplifications of jak2pdl1cd274 and pdl2pdcd1lg2 and mutation of myd88 in dlbcl tcga pancancer atlas from cbioportal [ ] 0ccervical lymphnodefemale high_intermediatehigh_intermediatedlbclnasal cavitymalegcbbreak_apartdlbcldlbcldlbcldlbcldlbcldlbclcervical lymphnodestomachstomachcolondlbclpmbclpmbclpmbclpmbclcervical lymphnodecervical lymphnodemediastinummediastinummediastinummalelow_intermediate non_gcbnormalfemale low_intermediate non_gcbmalemalelowhighnon_gcbgcbnon_gcbnormalnormalnormalnormalfemale low_intermediate non_gcbnormalmalelowfemale female female lowlowhigh_intermediatenanananananormalnormalnormalnormalnormaljak2_amppdl2_ampxue bmc cancer page of table the clinicopathological characteristics of dlbcl with jak2pdl2 amplification and pmbclmyd88_no diagnosis sitel265pmyc_ breakapartnormalhansalgorithmnon_gcbage ipi _riskspleenfemale lowsexpmbclna not applicablemediastinummalelowwhich was supported by the cancer genome atlas datatcga pancancer atlas from cbioportal [ ] fig 1ccategory and myc breakapart didnt show any significant differences table jak2pdl2 amplification identify a distinctive cnabasedpattern of dlbcl similar to that of pmbclsince dlbcl with jak2pdl2 amplification had less frequency of myd88 l265p mutation our study separateddlbcl patients into three subgroups dlbcl with jak2pdl2 amplification dlbcl_jak2pdl2_amp dlbclwith myd88 l265p mutation dlbcl_myd88_l265pjak2pdl2 amplification norand dlbcl withoutmyd88_l265p mutation dlbcl_others fig 2a basedon the unsupervised cluster result fig 1a one patientwho had both jak2pdl2 amplification and myd88l265p mutation was clustered into cluster_2 thereforethis patient was put into dlbcl_jak2pdl2_amp subgroup accordingly we also analyzed the data when thiscase was included in dlbcl_myd88_l265p subgroupand got the similar result see additional file unlike dlbcl_myd88_l265p and dlbcl_othersdlbcl_jak2pdl2_amp showed a distinctive pattern similar to that of pmbcl with high frequencyof rel and nfkbiz amplifications but no amplification of bcl2 and nfatc1 and no deletion ofprdm1 was found fig 2awith respectto clinicopathologicdlbcl_jak2pdl2_amp tend to be youngerdlbcl_myd88_l265p p hans modelcharacteristicsthantable whileinternational prognostic index ipi riskpdl1 expression in dlbcl with jak2pdl2 amplificationwas significantly higher than that in dlbcl with myd88l265p mutationtotally cases were performed pdl1 22c3 ihc detection including dlbcl_myd88_l265p cases dlbcl_jak2pdl2_amp cases dlbcl_others cases andpmbcl cases the result showed that pdl1 expressionin dlbcl_jak2pdl2_amp was significantly higher thanthat in dlbcl_myd88_l265p p and dlbcl_others p fig 2b and d while no significant difference was found between dlbcl_jak2pdl2_amp andpmbcl p fig 2bjak2pdl2 amplification identify a subgroup of dlbclwith unfavorable survival outcome similar to that ofmyd88 l265p mutationtrying to explore the survival indication of jak2pdl2 amplification and myd88 l265p mutation cases of dlbcls who received rchoplike regimentwith or without surgical resection were enrolled toperformed cox proportional regression analysis of osand pfs the median followup time was monthsrange monthsin the univariatecompared withdlbcl_others dlbcls with myd88 l265p mutationhad significantly worse os and pfs p andanalysisas 0cxue bmc cancer page of fig comparison of cnabased pattern pdl1 expression and survival analysis among pmbcl and three subgroups of dlbcl a comparison ofcnabased patterns of driver genes among pmbcl and three subgroups of dlbcl according to the status of jak2pdl2 amplification andmyd88 l265p mutation b comparison of pdl1 expression ihc score among pmbcl and three subgroups of dlbcl c survival curves and coxregression analysis of os and pfs among three subgroups of dlbcl after rchoplike treatment d representative images of heà and pdl1à ihc in dlbcl_jak2pdl2_amp and dlbcl_ myd88_l265p respectively and the same to dlbcls withjak2pdl2 amplification p and respectively meanwhile ipi risk category were significantly associated with os and pfs fig 2c tables and in the multivariate analysis ipi risk category andthree subgroups of dlbcl were put into analysis ascompared with dlbcl_others dlbcl with myd88l265p mutation still showed poor os and pfs p and respectively and the same todlbcl with jak2pdl2 amplification for pfs andos p and respectively meanwhile ipirisk category was still an independent risk predictorsfor os and pfs fig 2c tables and either jak2pdl2 amplification or myd88 l265pmutation are frequently seen in relapserefractory dlbclwith pfs less than yearsdlbcl with pfs less than years was defined as primaryrelapserefractory cases among these cases who treated byrchoplike regime the frequency of jak2 and pdl2amplification were and meanwhilethe frequency of myd88 l265p mutation were dlbcl with either jak2pdl2 amplification ormyd88 l265p accounted for discussiondlbcl presents with a wide spectrum of genetic aberration recently shi study exhibited pdl2 amplification in pmbcl and of dlbcl chapuy demonstrated of 9p241 amplification indlbcl meanwhile dlbcl with pdl1 gene alterations was identified as a unique biological subgrouphaving high risk features y wang study demonstrated that of dlbcl had cna of 9p241 withgene expression and mutation profiles that were similarto those of pmbcl in our study by using unsupervised hierarchical clustering cases ofdlbcl were clustered together with pmbcl as cluster_2 indicating that they shared recurrent cnas theywere enriched for jak2 amplification and pdl2 amplification fig 1a 0cxue bmc cancer page of table comparison of characteristics among pmbl and three subgroups of dlbcldlbclothersmyd88_l265pjak2pdl2_amppmbclpatientsage median range bmnegativepositiveihc hans algorithmgcbnongcbipilowrisklow_intermediatehigh_intermediatehighmyc breakapartnegativepositive p_valueÏ2test kruskalwallis rank sum testusing hans model most of dlbcl in cluster_2 werenongcb and tend to be younger than othergroups of dlbcl table which was consistent withprior study therefore coupled with y wang study we confirmed that dlbcl with jak2pdl2 amplification is a unique subgroup resembling the pmbclwith respect to cna patternwith regard to survival increasingly data exhibited thatthe suppression of immune surveillance in dlbcl was associated with poor survival godfrey j study hasdemonstrated that dlbcl with pdl1 gene alterationsshowed high risk features metaanalysis also showedthat pdl1 expression was associated with poor os andadverse clinicopathologic features in dlbcl in y wang study of dlbcl harbored cnaof 9p241 of which were gains and were amplifications and as compared with those who have nogain of 9p241 dlbcl with 9p24 amplification had atrend of better efs while patients with only gain tend tothey didnthave worse prognosis unfortunatelyshow any statistical significance in our study of dlbcl were found that had cna of jak2when jak2 cna was separated into gain mlpa valuebetween and amplification mlpa value as described cases in dlbcl_jak2pdl2_amp group were found that had jak2 gain whichwas slightly lower than that in wang j study asshown in additional file and both dlbcl withjak2 gain and with amplification demonstrated significant poor prognosis as compared with rest of dlbclas shown in additional file more interesting unlikey wang study cases of pmbcl were included in our study as control all of which demonstrating jak2 gains rather than amplifications as shown inadditional file of note we found that dlbcl in cluster_2 enrichedfor jak2pdl2 amplification had less frequency ofmyd88_l265p mutation fig 1a table which was supported by the cancer genome atlas datatcga pancancer atlas from cbioportal [ ] fig 1cmyd88 l265p is a poor indicator of survival for dlbcl which may lead to primary refractoryrelapsed diseasethis is a gainoffunction driver mutation occurring in of dlbcl but absent in pmbcl [] inour study the frequency of myd88 l265p in dlbcl andpmbcl were and which were in linewith prior studies [] of greatinterest myd88l265p mutation occurred less frequently in cluster_2 which was supported by the data tcga pancancer atlas from cbioportal [ ] thus when we divided dlbcl patients into three subgroups dlbcl_jak2pdl2_amp dlbcl_myd88_l265p and dlbcl_others both dlbcl_jak2pdl2_amp and dlbcl_myd88_l265p demonstrated dismal os and pfs with amedian followup of years as compared with dlbcl_others therefore dlbcl with jak2pdl2 amplification 0cxue bmc cancer page of table os in dlbcl treated by rchoplike regimeage ¥ bmnegativepositivesiteextranodalnodalihc hans algorithmgcbnongcbmyc fish breakapartnegativepositiveipi risk categorylowlow_intermediatehigh_intermediatehightherapyrchopresection rchopcna_based_clustercluster_1cluster_2cluster_3three subgroups of dlbcldlbcl_othersdlbcl_jak2pdl2_ampdlbcl_myd88_l265poshr_u95ci p_valueoshr_m95cip_value hr_u hazard ratio by univariate analysis hr_m hazard ratio by multivariate analysis because age was contained in the ipi thus it wasnt put into multivariate analysiswas identified as a poor survival subgroup that is similar todlbcl with myd88 l265p mutationmeanwhile we also compared the cna patterns ofdriver genes among dlbcl_jak2pdl2_amp dlbcl_myd88_l265p dlbcl_others and pmbcl dlbcl_jak2pdl2_amp showed a distinctive pattern similarto pmbcl with high frequency of rel and nfkbizamplifications but no amplification of bcl2 and nfatc1 and no deletion of prdm1 was found the profile ofdlbcl_myd88_l265p was closed to dlbcl_othersshowing relatively high frequency of cdkn2a deletionnfatc1 amplification and bcl2 amplificationin our study of dlbcl_jak2pdl2_ampharbored both jak2 and pdl2 amplifications simultaneouslyindicating that they may also have the pdl1amplification because pdl1 located in the middle ofjak2 and pdl2 at 9p241 thus we hypothesized thatpdl1 expression would be upregulated in this subgroup as what we expected using pdl1 22c3 ihcdetection pdl1 expression in dlbcl_jak2pdl2_in dlbcl_amp was significantly higher than thatmyd88_l265p p and dlbcl_othersp fig 2b and d but not in pmbcl p fig 2b meanwhile pdl1 expression could be 0cxue bmc cancer page of table pfs in dlbcl treated by rchoplike regimeage ¥ bmnegativepositivesiteextranodalnodalihc hans algorithmgcbnongcbmyc breakapartnegativepositiveipilowlow_intermediatehigh_intermediatehightherapyrchopresection rchopcna_based_clusterscluster_1cluster_2cluster_3three subgroups of dlbcldlbcl_othersdlbcl_ jak2pdl2_ampdlbcl_ myd88_l265ppfshr_u95ci p_value pfshr_m95cip_value hr_u hazard ratio by univariate analysis hr_m hazard ratio by multivariate analysis because age was contained in the ipi thus it wasnt put into multivariate analysisenhanced not only by pdl1 amplification but also byjak2 activation [ ] therefore dlbcl with jak2pdl2 amplification was confirmed as an unique subtype that is different from dlbcl with myd88 l265pand othersobjective response rates orr of pd1 blockade therapy was in unselected patients with relapsedrefractory dlbcl [ ] the wide spectrum of orrmay be due to high heterogeneity of this subgroupansell sm study demonstrated patients with9p241 alteration in relapsedrefractory dlbcl inour cohort the frequency of jak2 and pdl2 amplification in relapsedrefractory dlbcl were and which were within the range of orr in the prior studies[ ] while patients were found thathad myd88 l265p mutation who may not be suitablefor antipd1 therapy thus the genetic analysis in refractoryrelapsed dlbcl is required for future therapyselection to increase the orr of immune checkpointinhibitorsjak2 amplification could augment the expression of itself and pd1 ligands pdl1 and pdl2 enhancing the 0cxue bmc cancer page of sensitivity to jak2 kinase inhibitor chemical jak2inhibition could reduce the rna transcription and protein expression of pdl1 thus selective inhibitionof jak2 would be a valuable complementary therapy forpdl1 blockadeauthors contributionsxx contributed to pdl1 ihc staining clinical followup data analysis andmanuscript writing wh contributed to ffpe tissues collection mlpa detection and clinical followup tq and lg provided experiment guidance anddata interpretation jy and nl contributed to study design coordination discussion and manuscript editing all authors read and approved the finalmanuscriptsjak2pdl2 exhibitsdlbcl with amplification ofpmbcllike cnas pattern and demonstrates unfavorable outcome resembling those with myd88l265p mutation thusit is essential to identify thissubgroup of dlbcl who may acquire more benefitsfrom the jak2 and pdl1 signaling inhibition andjak2 amplification detection by mlpa would be feasible in routine practice meanwhile the difference ofsurvival outcome between our study and wang j study indicated that pmbcllike dlbcl suggested by 9p241 cna could be an intermixed subgroup which required further explorationsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020072933additional file mlpa results and clinical followup data the clinicopathological characteristics clinical followup data and mlpa results areshowed in this fileadditional file figure s1 representative results of mlparepresentative results of mlpa are showed in this figureadditional file table s1 the details of mlpa probes of genes indlbcl the locations and lengths of mlpa probes of genes are showedin this tableadditional file the detailed information of dlbcl with jak2pdl2amplification the detailed data about clinicopathological characteristicsmorphology immunohistochemistry and treatments of dlbcl with jak2pdl2 amplification are showed in this fileadditional file figure s2 the os and pfs of dlbcl with or withoutjak2pdl2_amp the os and pfs of dlbcl with or without jak2pdl2_ampadditional file figure s3 comparison of cnabased pattern andtheir survival outcome among pmbcl and three subgroups of dlbclone case of dlbcl with jak2pdl2 amplification and myd88 l265p mutation were included in dlbcl_myd88_l265p group a comparison ofcnabased patterns of driver genes among pmbcl and three subgroupsof dlbcl according to the status of jak2pdl2 amplification and myd88l265p mutation b survival curves and coxregression analysis of os andpfs among three subgroups of dlbcl after rchoplike treatmentadditional file figure s4 the frequencies of jak2 gain andamplification and their survival analysis a the frequencies of jak2 gainand amplification in dlbcl_jak2pdl2_amp and pmbcl b the os andpfs of dlbcl with jak2 gain or with jak2 amplificationabbreviationsdlbcl diffuse large bcell lymphoma pmbcl primary mediastinal large bcell lymphoma mlpa multiplex ligationdependent probe amplificationtcga the cancer genome atlas ipi international prognostic indexffpe formalinfixed paraffinembedded os overall survival pfs progressfree survival hr hazard ratioacknowledgementsnot applicablefundingthis study was partly supported by the beijing municipal science technology commission grant number z151100004015121 the cancerfoundation of china grant number lc2014l13 and cams innovation fundfor medical sciences grant number 2016i2m1001 to perform ffpe tissuescollection and mlpa detection and was partly supported by the cancerfoundation of china grant number lc2018b10 and pumc youth fundand the fundamental research funds for the central universities grantnumber to conduct pdl1 ihc staining and clinical followupand collect fish data of cmycavailability of data and materialsall data generated or analyzed during this study are included in thispublished and its supplementary information filesethics approval and consent to participatethis is a retrospective study that was launched in november the casesenrolled in this project were diagnosed between jan and oct whose ffpe samples were used the data regarding treatment andprognosis were acquired by means of medical record consultation andtelephone conversation thus the need for consent was waived by theindependent ethics committee of cancer hospital chinese academy ofmedical sciences national gcp center for anticancer drugs ncc2015st05consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of pathology national cancer centernational clinical researchcenter for cancercancer hospital chinese academy of medical sciencesand peking union medical college beijing china 2department ofpathology national cancer centernational clinical research center forcancercancer hospital shenzhen hospital chinese academy of medicalsciences and peking union medical college shenzhen chinareceived march accepted august referencesschmitz r wright gw huang dw johnson ca phelan jd wang jqroulland s kasbekar m young rm shaffer al genetics andpathogenesis of diffuse large bcell lymphoma n engl j med chapuy b stewart c dunford aj kim j kamburov a redd ra lawrencems roemer mgm li aj ziepert m molecular subtypes of diffuse largeb cell lymphoma are associated with distinct pathogenic mechanisms andoutcomes nat med godfrey j tumuluru s bao r leukam m venkataraman g phillip jfitzpatrick c mcelherne j macnabb bw orlowski r pdl1 genealterations identify a subset of diffuse large bcell lymphoma harboring a tcellinflamed phenotype blood green mr monti s rodig sj juszczynski p currie t o'donnell e chapuy btakeyama k neuberg d golub tr integrative analysis reveals selective9p241 amplification increased pd1 ligand expression and furtherinduction via jak2 in nodular sclerosing hodgkin lymphoma and primarymediastinal large bcell lymphoma blood wang y wenzl k manske mk asmann yw sarangi v greipp pt krull jehartert k he r feldman al amplification of 9p241 in diffuse large bcell lymphoma identifies a unique subset of cases that resemble primarymediastinal large bcell lymphoma blood cancer j 0cxue bmc cancer page of lenz g wright gw emre nc kohlhammer h dave ss davis re carty slam lt shaffer al xiao w molecular subtypes of diffuse large bcelllymphoma arise by distinct genetic pathways proc natl acad sci u s aswerdlow sh campo e harris nl jaffe es pileri sa stein h thiele j whoclassification of tumours of haematopoietic and lymphoid tissues revised4th edn lyon iarc cerami e gao j dogrusoz u gross be sumer so aksoy ba jacobsen abyrne cj heuer ml larsson e the cbio cancer genomics portal anopen platform for exploring multidimensional cancer genomics datacancer discov gao j aksoy ba dogrusoz u dresdner g gross b sumer so sun yjacobsen a sinha r larsson e integrative analysis of complex cancergenomics and clinical profiles using the cbioportal sci signal pl1 nogai h wenzel ss hailfinger s grau m kaergel e seitz v wollertwulf bpfeifer m wolf a frick m ikappabzeta controls the constitutive nfkappab target gene network and survival of abc dlbcl blood shi m roemer mg chapuy b liao x sun h pinkus gs shipp ma freemangj rodig sj expression of programmed cell death ligand pdl2 is adistinguishing feature of primary mediastinal thymic large bcelllymphoma and associated with pdcd1lg2 copy gain am j surg pathol qiu l zheng h zhao x the prognostic and clinicopathological significanceof pdl1 expression in patients with diffuse large bcell lymphoma a metaanalysis bmc cancer moelans cb monsuur hn de pinth jh radersma rd de weger ra vandiest pj esr1 amplification is rare in breast cancer and is associated withhigh grade and high proliferation a multiplex ligationdependent probeamplification study anal cell pathol amst fernandezrodriguez c bellosillo b garciagarcia m sanchezgonzalez bgimeno e vela mc serrano s besses c salar a myd88 l265p mutation isan independent prognostic factor for outcome in patients with diffuse largebcell lymphoma leukemia ngo vn young rm schmitz r jhavar s xiao w lim kh kohlhammer h xuw yang y zhao h oncogenically active myd88 mutations in humanlymphoma nature dubois s viailly pj bohers e bertrand p ruminy p marchand vmaingonnat c mareschal s picquenot jm penther d biological andclinical relevance of associated genomic alterations in myd88 l265p andnonl265pmutated diffuse large bcell lymphoma analysis of casesclin cancer res gupta s cheville jc jungbluth aa zhang y zhang l chen yb tickoo skfine sw gopalan a alahmadie ha jak2pdl1pdl2 9p241amplifications in renal cell carcinomas with sarcomatoid transformationimplications for clinical management mod pathol ansell sm minnema mc johnson p timmerman jm armand p shipp marodig sj ligon ah roemer mgm reddy n nivolumab for relapsedrefractory diffuse large bcell lymphoma in patients ineligible for or havingfailed autologous transplantation a singlearm phase ii study j clin oncollesokhin am ansell sm armand p scott ec halwani a gutierrez mmillenson mm cohen ad schuster sj lebovic d nivolumab inpatients with relapsed or refractory hematologic malignancy preliminaryresults of a phase ib study j clin oncol hao y chapuy b monti s sun hh rodig sj shipp ma selective jak2inhibition specifically decreases hodgkin lymphoma and mediastinal largebcell lymphoma growth in vitro and in vivo clin cancer res publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Colon_Cancer |
"what clinicians know about the diagnosis treatment and prevention of disease originates from studies mostly done on male cells male mice and men1 historically for multiple reasons including the purported safety of women and their offspring women of childbearing age were excluded from clinical trials as a result medical research and care have been centred on male physiology the assumption was that male and female cells and animals were biologically identical and evidencebased medicine was defined by clinical trials done predominantly in men1 in the us national institutes of health nih mandated the inclusion of women in nihfunded clinical trials but many investigators did not follow this mandate and many of those who did include women did not analyse the results by sex23 minimising the effectiveness of this policy preclinical research and drug development studies have also predominantly used male animal models and cells4 it is not surprising that a us government accountability office report found that eight of the ten prescription drugs withdrawn from the market between and posed greater health risks for women than for men most funding agencies from europe and north america have implemented policies to support and mandate researchers to consider sex and gender at all levels of medical research8 still the field of sexbased biology and medicine is often viewed as a specialised area of interest rather than a central consideration in medical research essential for the success of clinical care and translational science is awareness by clinicians and researchers that the diseases they are treating and studying are characterised by differences between women and men in epidemiology pathophysiology clinical manifestations psychological effects disease progression and response to treatmentthis review explores the role of sex biological constructs and gender social constructs as modifiers of the most common causes of death and morbidity and articulates the genetic biological and environmental determinants that underlie these differences we aim to guide clinicians and researchers to better understand and harness the importance of sex and gender as genetic wwwthelancetcom vol august biological and environmental modifiers of chronic disease ultimately it is a necessary and fundamental step towards precision medicine that will benefit women and mensex as a genetic modifier of biology and diseasesex differences in disease prevalence manifestation and response to treatment are rooted in the genetic differences between men and women genetic sex differences start at conception when the ovum fuses with a sperm cell carrying an x or a y chromosome resulting in an embryo carrying either xx or xy chromosomes this fundamental difference in chromosome complement eg genes outside the testisdetermining sry gene generates ubiquitous sex differences in the molecular makeup of all male and female cells9 first the y chromosome carries genes that exhibit subtle functional differences from their xlinked homologues eg zfy vs zfx and uty vs utx and also carries genes with no homologue at all eg sry in addition in men the x chromosome carries only maternal imprints ie epigenetic modifications made by the parent in generating the sex cellswhich alter the expression of genes in the offspring as women have x chromosomes from both parents they carry maternal and paternal imprints which target a different set of genes random inactivation of one of the x chromosomes in female cells which prevents sex differences in x chromosome gene dosage causes another degree of sex difference in gene expression as some of these xlinked genes escape inactivation in women those genes are often expressed at higher levels in women than in men9 sexspecific gene expression due to genomic search strategy and selection criteriawe searched pubmed for papers published in english between jan and june using sex or gender and the name of the disease of interest as search terms although we tried to cite seminal studies when necessary because of space limitation representative reviews were often selectedlancet diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine and southeast louisiana veterans health care system medical center new orleans la usa prof f mauvaisjarvis md barbra streisand womens heart center cedarssinai smidt heart institute los angeles ca usa prof n bairey merz md national heart lung institute imperial college london london uk prof p j barnes md department of pharmacology and department of neurology college of medicine center for innovation in brain science university of arizona tucson az usa prof r d brinton phd department of medical epidemiology and biostatistics and center for gender medicine karolinska institutet stockholm sweden prof jj carrero phd channing division of network medicine and the division of pulmonary and critical care medicine department of medicine brigham and womens hospital harvard medical school boston ma usa d l demeo md neuroscience institute and department of biology geia state university atlanta ga usa prof g j devries phd department of psychiatry university of colorado school of medicine anschutz medical campus aurora co usa prof c n epperson md division of oncology department of medicine washington university school of medicine st louis mo usa prof r govindan md w harry feinstone department of molecular microbiology and immunology the johns hopkins bloomberg school of public health baltimore md usa prof s l klein phd department of biomedical metabolic and neural sciences university of modena andreview 0creggio emilia azienda ospedalierouniversitaria di modena ospedale civile di baggiovara modena italy prof a lonardo md department of psychiatry department of psychology and department of obstetrics gynecology university of illinois at chicago chicago il usa prof p m maki phd department of neurology mcgovern medical school university of texas health science center houston tx usa prof l d mccullough md berlin institute of gender medicine charituniversittsmedizin berlin berlin germany prof v regitzzagrosek md department of cardiology university hospital z¼rich university of z¼rich switzerland prof v regitzzagrosek center for womens health research divisions of general internal medicine and cardiology university of colorado school of medicine aurora co usaimprinting extends to autosomes as well and these imprinted genes exhibit sexspecific and tissuespecific expression in humans10 thus fundamental sex differences deriving directly from genetic heterogeneity between the x and y chromosome complements and parentoforigin inher itance exist at the molecular level in all human cells these sex differences persist throughout life and are independent of sex hormones figure arguably the greatest source of differences between men and women comes from the y chromosomal sry gene which directs the development of a testis in men the ensuing developmental surge of testicular testosterone permanently masculinises the reproductive tract and the anisation of brain circuits affecting male behaviour at puberty1112 in humans the first surge occurs at the end of the first trimester of pregnancy because it alters cellular gene expression and tissue structure in multiple ans of men via epigenetic mechanisms this testosterone surge is also paramount in programming sex differences in physiology and susceptibility to diseases that will manifest in adulthood after this initial testicular testosterone surge gonadal hormone concentrations remain low until puberty which triggers lasting sex differences in circulating oestrogens and testosterone concentrations after puberty cells with androgen or afemale sexbrandom x chromosomeinactivation and escapexxxxxxxxxxxxxxxxxxxy chromosome complementmale sexsryctesticular testosterone surgefetal testistestosteroneohogenetic diï¬erences of male and female cellsepigenetic programming of male cellsfigure genetic causes of sex differencesa genetic sex differences start with cells carrying either xx or xy chromosome complement eg genes outside the testisdetermining sry gene which generates ubiquitous sex differences in the molecular makeup of all male and female cells b random inactivation of one x chromosome in female cells causes another level of sex differences in gene expression some xlinked genes escape inactivation in female individuals and have a higher expression in female than male individuals c the y chromosomal sry gene directs the development of a testis in male individuals which produces a surge of testicular testosterone at the end of pregnancy the testosterone surge programmes cellular gene expression and tissue structure in multiple ans of male individuals via epigenetic remodelling the combination of these genetic and developmental events programmes sex differences in physiology and susceptibility to diseases that will manifest in adulthoodoestrogen receptors will be affected differ ently in men and women the combination of all genetic and hormonal causes of sex differences aforementioned culminates in two different biological systems in men and women that translate into differences in disease predisposition manifestation and response to treatment therefore sex is an important modifier of physiology and disease via genetic epigenetic and hormonal regulations figure gender as a determinant of patients and doctors behaviour and as a modifier of health disease and medicinegender according to the global health definition refers to the socially constructed norms that impose and determine roles relationships and positional power for all people across their lifetime13 gender interacts with sex the biological and physical characteristics that define women men and those with intersex identities13 gender is not a binary term it includes the understanding that in many people traits of masculinity or femininity coexist and are expressed to different degrees gender attributes are fluid more than two thirds of women and men report genderrelated characteristics traditionally attributed to the opposite sex1415 in transgender people gender identity differs with the sex they were assigned at birth so far transgender people have generally been underrepresented in clinical studies to date although this underrepresentation is changing gender is an equally important variable as biological sex in human health and influences the behaviour of communities clinicians and patients1415 gender roles represent the behavioural norms applied to men and women in society which influence individuals everyday actions expectations and experiences including diet perceived stress smoking and physical activity and affect health and disease susceptibility gender identity describes the fluidity of how a person perceives oneself as a woman or a man which affects feelings and behaviours gender relations refer to how we interact with or are treated by people on the basis of our ascribed gender institutionalised gender reflects the distribution of power between men and women in the political educational and social institutions in society and shapes social norms that define perpetuate and often justify different expectations and opportunities for women and men1617 as such the distribution of genderrelated charac teristics within populations of men and women can influence health differently than biological sex together these gender constructs determine access to health care helpseeking behaviours and individual use of the healthcare system being perceived as a man or a woman triggers different responses from clinicians who might diagnose and suggest interventions differently according to gender as such gender largely determines the use of preventive measures and referral for or acceptance of invasive therapeutic strategies genderrelated behaviours contribute to risk exposure and preventive behaviour in several wwwthelancetcom vol august review 0cdiseases this postulation is well exemplified in the cardiovascular field in which women often underestimate their risk compared with men and seek consultation later than men in the clinic for treatment of myocardial infarction1819 in the genesispraxy prospective study mortality year after an acute coronary event was more strongly associated with gender than with biological sex1415 similarly control of cardio vascular risk factors hypertension diabetes depres sive symptoms was better predicted by gender than by biological sex1415 therefore including a gender dimension in clinical studies and practice will contribute to the understanding of different clinical manifestations and outcomes of diseases in women and men1617 although beyond the scope of this review it is also important to consider that regarding health and disease gender intersect with race or ethnicity and age20 sex and gender are fundamentally and frequently reciprocally interrelated in biology and disease24 sex influences behaviours eg towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and pain and nutritional habits might produce epigenetic modifications that modulate gene expression and biological phenotypes figure summarises how sex and gender are interrelated in biology and diseasesex and gender differences in major chronic diseaseshaving established the importance of sex and gender in disease we will summarise their influences on the most common causes of death and debilitating diseases in the usa as an example figure note that these sex and gender disparities are relevant to other highincome countries as well as lowincome and middleincome countries where the burden of these diseases becomes increasingly like those in highincome countries26 in most diseases efforts to separate the effects of sex and gender are still incomplete so that we just refer to the differences among women and men because current knowledge about pathophysiology diagnosis and treatment of disease is primarily based on men as representative of the human species this review focuses on how women differ from men we discuss some key aspects regarding the dimensions of men in a dedicated sectionheart diseaseepidemiology pathogenesis manifestations and diagnosisheart disease is the leading cause of death in the usa in heart disease accounted for · of all deaths for men and for · of all deaths for women figure ischaemic heart disease and heart failure are major contributors to heart disease mortality and have important sex and gender differences for example heart failure disproportionately contributes to coronary heart disease mortality in women27 potentially due to undiagnosed ischaemic heart disease in women the strength of the association with cardiovascular risk factors differ by sex biological sexsex chromosomesepigeneticeï¬ectssex hormonesohohohobehaviour of patients and doctorssocietygender constructslifestylenutritional habitsexerciseperceived stresssmokingdiseasepathophysiologymanifestationresponse to treatmentdisease perceptionhelpseeking behaviouruse of health caredecision makingtherapeutic responsesex and gender diï¬erences in health disease and medicinefigure interrelation between sex and gender in health diseases and medicinebiological sex causes sex differences through genetic and hormonal influences in disease pathophysiology clinical manifestations and response to treatment sex also influences behaviours towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and nutritional habits produce epigenetic modifications that modulate the expression of biological sex gender constructs determine patients perception of disease helpseeking behaviour and individual use of health care gender constructs also influence decision making and trigger different therapeutic responses from providers biased by gendersystolic blood pressure and hypertension smoking and diabetes are associated with higher hazard ratios for myocardial infarction in women than in men28ischaemic heart disease is the most recognised example for integrating the concept of gender and sex which shape divergent or distinct disease outcomes compared with men women suffering from ischaemic heart disease are older this difference is historically believed to be due to the protection of endogenous oestrogens29 although contemporary study refutes this simplistic explanation30 and associations cannot be inferred to be causation still women suffering from ischaemic heart disease are underdiagnosed3132 and less likely to have a prehospital diagnosis of myocardial infarction33 the reasons for this disparity reflect the intersection between sex and gender first biological sex differences exist in the pathogenesis of ischaemic heart disease whereas men are more likely to be affected by obstructive coronary artery disease of large vessels than women coronary microvascular dysfunction36 leading to chronic myocardial ischaemia without obstructive coronary artery disease has a higher prevalence in women than men37 a metaanalysis reported that following acute myocardial infarction both sexes most often presented with chest pain but compared with men women were more likely to present with pain between the shoulder blades nausea or vomiting and shortness of breathprof j g regensteiner phd department of medicine department of paediatrics and department of neuroscience washington university school of medicine st louis mo usa prof j b rubin md center for the study of sex differences in health aging and disease geetown university washington dc usa prof k sandberg phd division of gastroenterology duke university medical center durham nc usa a suzuki md and durham va medical center durham nc usa a suzukicorrespondence to prof franck mauvaisjarvis diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine new orleans la usa fmauvaistulaneeduwwwthelancetcom vol august review 0cmale individualsother·heart disease·protection might disappear after menopause45 by contrast testosterone induces adverse cardiac remod elling in the male heart44chronic liver disease ·inï¬uenza and pneumonia ·suicide ·alzheimers disease ·type diabetes ·stroke ·cpd ·injuries ·female individualsother·septicaemia ·chronic kidney disease ·inï¬uenza and pneumonia ·type diabetes ·injuries ·alzheimers disease ·stroke ·cpd ·cancer·heart disease·cancer·figure percent distribution of the ten leading causes of death by sex usa adapted from heron25 cpdchronic pulmonary diseasesecond a gender bias appears to be responsible for the absence of recognition of ischaemic heart disease presentation in women38 men and women with ischaemic heart disease who score high on feminine roles and personality traits on questionnaires designed to ascertain aspects of gender are at an increased risk of recurrent ischaemic heart disease independent of female sex39heart failure affects of adults aged years and older and more women than men in absolute numbers4041 heart failure occurs at an older age and with less ischaemic causes in women than in men however hypertension and diabetes predispose older women to heart failure to a greater extent than men heart failure with preserved ejection fraction a form of heart failure with normal systolic function is twice as prevalent in women as in men by contrast heart failure with reduced ejection fraction affects more men than women women who have heart failure with preserved ejection fraction have smaller and stiffer hearts than men inflammation and the resulting fibrosis play a sexspecific role in the pathogenesis of heart failure with preserved ejection fraction under stress premenopausal womens hearts develop less inflammation resulting in less fibrosis than mens hearts4243 this difference is partially driven by sex hormones as oestrogens produce antiinflammatory actions on endothelial and immune cells and promote cardioprotective effects in premenopausal women44 this response to treatmentcompared with men women suffering from ischaemic heart disease are less likely to receive evidencebased treatment3132 and when suffering from acute myocardial infarction they are less likely to receive reperfusion33 an stelevation myocardial infarction registry revealed that compared with men women exhibit delayed reperfusion leading to higher mortality46 women suffering from acute myocardial infarction treated by male emergency physicians have a higher mortality rate than those treated by female physicians38 additionally male physicians are more effective at treating female patients with acute myocardial infarction when they work with female colleagues and when they have experience in treating female patients38 this treatment disparity between women and men can be corrected by improving emergency recognition of stelevation myocardial infarction in women and acceleration of percutaneous coronary intervention which equilibrates gender mortality47guidelines for the treatment of heart failure are similar for women and men24 however evidence suggests that optimal survival in women occurs with lower doses of β blockers angiotensin receptor blockers and angiotensin converting enzyme inhibitors than in men48 finally fewer women undergo heart transplantation than men although women are more frequently donors suggesting a referral bias could exist41cancersepidemiology pathogenesis manifestations and diagnosiscancers are the second leading cause of death dominated by lung cancer accounting for · of deaths in men and · of deaths in women figure more men develop cancer than women49 with few exceptions eg meningioma thyroid cancer lung cancer in nonsmokers nonreproductive cancers exhibit a male predominance though for some cancers oropharynx larynx oesophagus and bladder the male versus female incidence ratios can be higher than a male predominance in cancers that affect both sexes is evident around the world in all races and at all ages50 survival is also shorter for men than women across multiple cancer types the higher cancer risk in men is partially explained by gender constructs like dietary habits or risk behaviours such as smoking and alcohol consump tion51 it is unlikely to be the only cause after appropriate adjustment for these risk factors adult men still have a higher cancer risk than women52 moreover a similar male bias in incidence and survival is seen in paediatric cancers before puberty and the adoption of highrisk cancerpromoting behaviours eg smoking53the universal male predominance in cancer incidence and differential outcomes argues for a fundamental role wwwthelancetcom vol august review 0cin inutero tumour suppressors of sex in addition to gender in cancer biology sexspecific biology includes genetic differences xx vs xy chromosomes the incomplete xinactivation in female individuals which results in biallelic expression of xencoded female cells54 y chromosomeencoded oncogenes such as the rnabinding motif on y chromosome in male cells55 and the chromatin remodelling effects of testicular testosterone in male cells56 these mechanisms have an influence on several of the hallmarks of cancer57 including metabolism growth regulation58 angiogenesis and immunity which all contribute to cancer predisposition59 a crucial example is the male predisposition to glioblastoma which is the most common form of brain cancer in glioblastoma there is a cellintrinsic predisposition of male astrocytes a subtype of glial cell to malignant transformation60 after puberty sex hormones produce additional epigenetic and acute effects on cells that further influence sex disparities in cancer for example the increased frequency and aggressive phenotype of hepatocellular carcinoma in male individuals has been linked to the stimulatory effects of androgens in male individuals and the protective effects of oestrogens in female individuals61 importantly the biology of cancer is not the same across histological and genetic diagnostic groups or even within single histol ogical subtypes thus the interaction between sex gender and cancer mechanisms cannot be expected to be constant take colon cancer the second leading cause of cancerrelated death for example although women have a lower overall incidence of colon cancer than men they have a higher incidence of rightsided colon cancers which have the worst outcomes62 tumours from women with rightsided colon cancers exhibit a distinct molecular signature of energy metabolism compared with those of women with leftsided colon cancers63 this molecular signature is not observed when comparing tumours from men with rightsided colon cancers to men with leftsided colon cancers thus overall the male predisposition to cancer is probably the consequence of genetic program ming of male cells and the effect of sex hormones after puberty interacting with genderspecific behaviours to establish cancer risksresponse to treatmentin the future cancer prevention and treatment will be improved by sexspecific and genderspecific approaches for example immune checkpoint inhibitors can improve survival for men with advanced melanomas and nonsmallcell lung cancers more than for women64 the molecular subtyping of glioblastomas based on sexspecific transcriptomes has the potential to enhance chemotherapy in a sexspecific manner65 in colon cancer sex differences in xenobiotic metabolism regulatory networks might underlie greater treatment response in women than men and require modification of approaches for men with colon cancer66 other elements of cancer metabolism are also ripe for novel sexspecific targeting in treatment cellular nutrient partitioning is sexually dimorphic so approaches such as ketogenic diets or glutaminase inhibition might be associated with substantial sexspecific responses67 furthermore data from models of development ageing and cancer all indicate that molecular pathways such as the enzyme phosphatidylinositol 3kinase and tumour suppressors such as p53 and retinoblastoma protein are sexually dimorphic and require sexspecific targeting545968chronic pulmonary diseaseepidemiology pathogenesis manifestations and diagnosischronic pulmonary disease is the third leading cause of death for women · of deaths and the fourth for men · figure it is mostly accounted for by chronic obstructive pulmonary disease and to a lesser extent by asthma chronic obstructive pulmonary disease is characterised by irreversible airflow limitation and is associated with previous exposure to smoking or air pollutants women are overrepresented among individuals with chronic obstructive pulmonary disease especially among those with earlyonset disease or those who have never smoked69 women are also twice as likely to have chronic obstructive pulmonary disease with chronic bronchitis and women with severe chronic obstructive pulmonary disease have as much emphysema as men countering the misconception of emphysema as a male form of chronic obstructive pulmonary disease70 the female lung is more susceptible to chronic obstructive pulmonary disease than the male lung and women develop symptoms of the disease at a younger age with less tobacco exposure than men71 the genetic epidemiology of chronic obstructive pulmonary disease copdgene study72 suggests that earlyonset chronic obstructive pulmonary disease might originate in utero in susceptible women from alterations in lung development potentiated by maternal asthma and smoking genetic factors or hormonal influences future studies should focus on the contribution of maternally inherited factors such as mitochondrial and x chromosome genes to understand disease pathogenesis it is important to consider gender constructs as well smoking advertising campaigns targeting women rose in the 1960s and the resulting higher smoking rates influenced womens risk for developing chronic obstructive pulmonary disease73 from a disease severity vantage point chronic obstructive pulmonary disease exacerbation rates are also higher in women than men especially at a younger age74 additionally despite the burden of symptomatology and increased rates of hospitalisations and deaths women with chronic obstructive pulmonary disease are often misdiagnosed and disproportionally suffer from comorbid conditions including anxiety and depression therefore physicians should consider chronic obstructive pulmonary disease in the differential diagnosis of women with pulmonary symptoms regardless of tobacco or pollutant exposure historieswwwthelancetcom vol august review 0casthma characterised by variable airflow obstruction and chronic airway inflammation also affects men and women differently asthma is more prevalent in prepubertal boys than girls regarding asthma both male biological sex lung development and atopy and male gender constructs related to outdoor play and indoor pet exposure are factors contributing to the devel opment of asthma and sex versus gender contributions could be difficult to separate75 from puberty onwards more female than male individuals have asthma with an increased severity in middleaged women and a higher mortality rate76 this phe nomenon could be secondary to gender differences eg symptoms perception or healthseeking behaviours however biological sex plays a crucial role in asthma and sex hormones have a major impact on female asthma symptoms and severity after puberty75 worsening of asthma occurs in women before menstruation and is known as premenstrual asthma premenstrual asthma is more common in women with severe rather than mild asthma obesity rather than normal weight and a long rather than a short duration of asthma77 premenstrual asthma is hypoth esised to be due to a fall in progesterone and patients with a severe disease respond to progestogens78 during pregnancy approximately a third of asthmatic women exhibit a worsening of asthma a third show an improvement and the remainder are unaffected79response to treatmentthe menopausal transition represents a pivotal time of accelerated decline in lung function in women with chronic obstructive pulmonary disease and thus represents a sexspecific window for treatment intensification these observations also suggest that oestrogens protect from chronic obstructive pulmonary disease women exhibit greater expression of m2 over m3 muscarinic receptors and accordingly show greater improvements in lung function than men in response to the muscarinic anticholinergic bronchodilator ipatroprium80 pooled analyses of drug studies also suggest that women experience a greater improvement in quality of life than men after treatment of chronic obstructive pulmonary disease with a β2adrenoreceptor agonist combined with an anti cholinergic drug eg indacaterol and glycopyrronium81unlike chronic obstructiv | Colon_Cancer |
" 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 |
to the human gut microbiota biochem taxonomic hierarchy of the phylum firmicutes and novel firmicutes species originated from various environments in korea j microbiol front microbiol filippidou s a combination of extreme environmental conditions favor the prevalence of endosporeforming firmicutes bintsis t lactic acid bacteria as starter cultures an update in their metabolism and genetics aims microbiol verma d k bioprocessing technology in food and health potential applications and emerging scope apple academic caulier s overview of the antimicrobial compounds produced by members of the bacillus subtilis group front microbiol lopetuso l r scaldaferri f petito v gasbarrini a commensal clostridia leading players in the maintenance of gut homeopress burlington stasis gut pathogens rood j i general physiological and virulence properties of the pathogenic clostridia in clostridial diseases of animals eds uzal fa prescott jf songer jg and popoff mr wiley wells c l wilkins t d medical microbiology 4th edn university of texas medical branch galveston kho z y lal s k the human gut microbiomea potential controller of wellness and disease front microbiol koliada a association between body mass index and firmicutesbacteroidetes ratio in an adult ukrainian population bmc microbiol yuan j antibacterial compoundsmacrolactin alters the soil bacterial community and abundance of the gene encoding pks front microbiol 103389fmicb scientific reports 101038s41598020706868vol1234567890wwwnaturecomscientificreports 0c romerotabarez m 7omalonyl macrolactin a a new macrolactin antibiotic from bacillus subtilis active against methicillinresistant staphylococcus aureus vancomycinresistant enterococci and a smallcolony variant of burkholderia cepacia antimicrob agents chemother moldenhauer j chen x h borriss r piel j biosynthesis of the antibiotic bacillaene the product of a giant polyketide synthase complex of the transat family angew chem int ed engl 101002anie20070 butcher r a the identification of bacillaene the product of the pksx megacomplex in bacillus subtilis proc natl acad wu l difficidin and bacilysin from bacillus amyloliquefaciens fzb42 have antibacterial activity against xanthomonas oryzae chen xh structural and functional characterization of three polyketide synthase gene clusters in bacillus amyloliquefaciens sci rice pathogens sci rep fzb j bacteriol yang j genomicsinspired discovery of three antibacterial active metabolites aurantinins b c and d from compostassociated bacillus subtilis fmb60 j agric food chem kang h k seo c h park y marine peptides and their antiinfective activities mar drugs 103390md130 sur s romo t d grossfield a selectivity and mechanism of fengycin an antimicrobial lipopeptide from molecular dynamics j phys chem b 101021acsjpcb7b118 knight c a the first report of antifungal lipopeptide production by a bacillus subtilis subsp inaquosorum strain microbiol peypoux f bonmatin j m wallach j recent trends in the biochemistry of surfactin appl microbiol biotechnol grangemard i wallach j magetdana r peypoux f lichenysin a more efficient cation chelator than surfactin appl biores s0025 chem biotechnol zhou m bacillibactin and bacillomycin analogues with cytotoxicities against human cancer cell lines from marine bacillus sp pkuma00093 and pkuma00092 mar drugs hertlein g production of the catechol type siderophore bacillibactin by the honey bee pathogen paenibacillus larvae one e108272 gu q bacillomycin d produced by bacillus amyloliquefaciens is involved in the antagonistic interaction with the plantpathogenic fungus fusarium graminearum appl environ microbiol e0107517 barsby t kelly m t andersen r j tupuseleiamides and basiliskamides mew acyldipeptides and antifungal polyketides produced in culture by a bacillus l aterosporus isolate obtained from a tropical marine habitat j nat prod mthethwa b c comparative analyses of cytochrome p450s and those associated with secondary metabolism in bacillus species int j mol sci 103390ijms1 podust l m sherman d h diversity of p450 enzymes in the biosynthesis of natural products nat prod rep 101039c2np2 0020a greule a stok j e de voss j j cryle m j unrivalled diversity the many roles and reactions of bacterial cytochromes p450 in secondary metabolism nat prod rep 101039c7np0 0063d medema m h minimum information about a biosynthetic gene cluster nat chem biol 101038nchem bio1890 ngwenya m l blooming of unusual cytochrome p450s by tandem duplication in the pathogenic fungus conidiobolus coronatus int j mol sci 103390ijms1 matowane r g in silico analysis of cytochrome p450 monooxygenases in chronic granulomatous infectious fungus sporothrix schenckii special focus on cyp51 biochim biophys acta proteins proteom 101016jbbapa p201710003 qhanya l b genomewide annotation and comparative analysis of cytochrome p450 monooxygenases in basidiomycete biotrophic plant pathogens one e0142100 101371journ alpone01421 kgosiemang i k r syed k mashele s s comparative genomics and evolutionary analysis of cytochrome p450 monooxygenases in fungal subphylum saccharomycotina j pure appl microbiol jawallapersand p cytochrome p450 monooxygenase cyp53 family in fungi comparative structural and evolutionary analysis and its role as a common alternative antifungal drug target one e107209 101371journ alpone01072 syed k shale k pagadala n s tuszynski j systematic identification and evolutionary analysis of catalytically versatile cytochrome p450 monooxygenase families enriched in model basidiomycete fungi one e86683 101371journ alpone00866 suzuki h comparative genomics of the whiterot fungi phanerochaete carnosa and p chrysosporium to elucidate the genetic basis of the distinct wood types they colonize bmc genom 1011861471216413444 akapo o o distribution and diversity of cytochrome p450 monooxygenases in the fungal class tremellomycetes int j mol sci 103390ijms2 sello m m diversity and evolution of cytochrome p450 monooxygenases in oomycetes sci rep 101038srep1 b biol 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 furumichi m tanabe m kegg as a reference resource for gene and protein annotation nucleic acids res d457d462 101093nargkv10 nelson d r the p450 superfamily update on new sequences gene mapping accession numbers early trivial names of enzymes and nomenclature dna cell biol 101089dna1993121 nelson d r cytochrome p450 nomenclature methods mol biol clifton nj 101385089603 nelson d r cytochrome p450 nomenclature methods mol biol clifton nj 101385159259 scientific reports 101038s41598020706868vol0123456789wwwnaturecomscientificreports 0c de lima procpio r e da silva i r martins m k de azevedo j l de araºjo j m antibiotics produced by streptomyces braz j infect dis munro a w p450 bm3 the very model of a modern flavocytochrome trends biochem sci li h poulos t l the structure of the cytochrome p450bm3 haem domain complexed with the fatty acid substrate palmitoleic acid nat struct biol bm3 biochem soc trans noble m miles c reid g chapman s munro a catalytic properties of key activesite mutants of flavocytochrome p450 lee ds crystallization and preliminary xray diffraction analysis of fattyacid hydroxylase cytochrome p450bsβ from bacillus subtilis acta crystallogr sect d biol crystallogr lee ds substrate recognition and molecular mechanism of fatty acid hydroxylation by cytochrome p450 from bacillus subtilis crystallographic spectroscopic and mutational studies j biol chem girvan h m structural and catalytic properties of the peroxygenase p450 enzyme cyp152k6 from bacillus methanolicus bower s cloning sequencing and characterization of the bacillus subtilis biotin biosynthetic operon j bacteriol j in biochem cryle m j schlichting i structural insights from a p450 carrier protein complex reveal how specificity is achieved in the p450bioi acp complex proc natl acad sci cryle m j bell s g schlichting i structural and biochemical characterization of the cytochrome p450 cypx cyp134a1 from bacillus subtilis a cyclolleucyllleucyl dipeptide oxidase biochemistry cimermancic p insights 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 provide a link to the creative commons license and indicate if changes were made the images or other third party material in this article are included in the articles creative commons license unless indicated otherwise in a credit line to the material if material is not included in the articles creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this license visit httpcreat iveco mmons licen sesby40 the authors scientific reports 101038s41598020706868vol1234567890wwwnaturecomscientificreports 0c' | Colon_Cancer |
" curcumin is herbal compound that has been shown to have anticancer effects in preclinical andclinical studies the anticancer effects of curcumin include inhibiting the carcinogenesis inhibiting angiogenesisand inhibiting tumour growth this study aims to determine the clinical effects of curcumin in different types ofcancers using systematic review approachmethods a systematic review methodology is adopted for undertaking detailed analysis of the effects of curcuminin cancer therapy the results presented in this paper is an outcome of extracting the findings of the studiesselected from the s published in international databases including sid magiran iranmedex irandoc googlescholar sciencedirect scopus pubmed and web of science isi these databases were thoroughly searched andthe relevant publications were selected based on the plausible keywords in accordance with the study aims asfollows prevalence curcumin clinical features cancerresults the results are derived based on several clinical studies on curcumin consumption with chemotherapydrugs highlighting that curcumin increases the effectiveness of chemotherapy and radiotherapy which results inimproving patients survival time and increasing the expression of antimetastatic proteins along with reducingtheir side effects the comprehensive systematic review presented in this paper confirms that curcumin reduces the sideeffects of chemotherapy or radiotherapy resulting in improving patients quality of life a number of studiesreported that curcumin has increased patient survival time and decreased tumor markers levelkeywords prevalence curcumin clinical feature cancer systematic review research over the past years has significantly increased our understanding of the molecular genetic basisof cancer it is now well known that cancer is caused bya set of molecular genetic changes that lead to loss ofgrowth control and cellular differentiation resulting in correspondence nsalarikumsacir masoudmohammadi1989yahoocom5department of biostatistics school of health kermanshah university ofmedical sciences kermanshah iran7department of nursing school of nursing and midwifery kermanshahuniversity of medical sciences kermanshah iranfull list of author information is available at the end of the uncontrollable cell growth that eventually leads to tumorformation more than half of all cancers occur in developingcountries including those located in southern americaand asia nearly threequarters of people of these countries are classified into low or middleincome categoriesthe cancer survival rates in developing countries aregenerally onethird ofthe patients in the developedcountries there are million new cases of cancereach year with million new cancer cases in the developed countries and million in developing countries in the next decades cancer will be one of the leading the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cmansouri bmc cancer page of causes ofillness worldwide and the number of newcases of various types of cancer is expected to rise to million by furthermore cancer is predicted to bethe leading cause of death by given that cancerstatistics are on the rise and their treatments are costlyit is very crucial to find effective and economically viablemethods for patients in low and middle income countries therefore this study is motivated by using effectiveand relatively cheap treatments for cancer therapy asystematic review of the clinical studies on curcumin useand its effectiveness in inhabiting and treating varioustypes of cancer is carried out to obtain comprehensiveinformation aboutthe curcumin effects on cancertherefore a structured review of all published sand other relevant documents on the use of curcuminfor cancer therapy creates a more complete picture ofcurcumin effects on cancer patients from different angles in the process of this review only evidence from thestudies with highest quality are selected to gather information and derive on curcumin effects andeffectiveness at various stages of cancer therapyamong the medical herbs flavonoids are a large subgroup of the family of natural polyphenolic compoundsthat are the result of secondary metabolism in plants in recent years research has shown that flavonoids havebeen very effective in the prevention and control of common diseases complex such as cancer cardiovasculardiseases alzheimers stroke diabetes osteoporosisand rheumatoid arthritis furthermore there are robustevidence of antiviral antiinflammatory and antiallergic effects of flavonoidsin the recent years the use and effectiveness of medicinal herbs in treatment of various diseases has been received enormous attention huge research efforts weremade on extraction and examination of the properties ofthe herbal compounds in the treatment of different typesof diseases eg cancers and providing detailed mechanisms of drug performance of these compounds amongst the wide range of the medical herbs curcuminis an effective ingredient of turmeric plant with the scientific name of longa curcuma chemical name ofdiferuloylmethane and the chemical formula of c21h20 o6 as illustrated in fig curcumin makes up between to of turmericcompounds and is considered as the main cause of yellowgolden colour of turmeric and it has also been identified as responsible for many ofthe properties ofturmeric [ ] however curcumin has low inherenttoxicity and various properties with great impact and applications on a wide range of pharmacological developmentsantiinflammatoryantimicrobial and anticancer drugs []antioxidantincludingcurcumin has been shown to have preventive and therapeutic effects on various types of cancers the findingsfrom several studies suggest that curcumin compound canprevent the formation and spread of tumors or reduce theirsize it was shown that curcumin can inhibit the formationof cancer and spread the cancerous cells by exerting antiangiogenic effects inducing apoptosis and interfering withthe cell proliferation cycle [ ] curcumin exerts itsanticancer effects through a variety of mechanisms curcumin inhibits and suppresses the proliferation of a widerange of cancer cells which exerts its effects by reducingthe modulation of antiapoptotic gene products activatingcaspase and upregulating cancersuppressive genes such asp53 [] recent studies confirm the preventive andtherapeutic effects of curcumin on various types of cancersindicating that it can prevent or reduce the formation orspread of tumors curcumin inhibits tumor invasion by reducing the modification of matrix metalloproteasesmmps the cell surface adhesive molecules nfκ ap1tnfα lox and cox2 chemokines growth factorsher2 and egfr inhibits nterminal activity and tyrosine kinase protein [ ] curcumin inhibits angiogenesisin some tumors by suppressing angiogeniccytokines such as il6 il23 and il1 [] due tothe strong relationship between inflammation and cancerthe antiinflammatory effects of curcumin would well resultin its antitumor effects it was reported that curcumin hasprevented the development of several types of cancer by reducing the production of mediators of the inflammatoryprocess such as cox2 lipoxygenase inos and relatedcytokines one of the possible mechanisms for suppressing tumor proliferation is the chemical inhibitor effectof curcumin as a result topical use of curcumin considerably inhibits inflammation due to tetradecanoylphorbol13fig the chemical expansion of curcumin by coreldraw graphics suite 0cmansouri bmc cancer page of acetate 12o tpa hyperplasia cell proliferation odcactivity production of active oxygen species oxidativedna changes and papillomavirus formation [] multiple human gastrointestinal cell interactions with curcumininhibit lipid peroxidation inhibit cox2 expression inhibitpge2 production and increase glutathionestransferaseenzyme levels [ ] the other mechanism of the anticancer effects of curcumin is due to its interference in thecell cycle and reduction in cdk expression cdks are actually serine threonine kinases that control cell cycle progression furthermore curcumin inhibits the stat3phosphorylation which is responsible for signalling carcinogenic pathways given that cancer statistics are on the rise and theirtreatments are quite costly it is very crucial to find someeffective methods and economically viable for low andmiddleincome patients therefore this paper providesupto date evidence and findings of clinical studies onthe effects of curcumin contributions in tumor cells survival and metastasis using a systematic review approachmethodssystematic review approach is adopted for undertakingthis study by extracting the findings of the relevant studies selected from the s published in national andinternational databases including sid magiran iranmedex irandoc google scholar sciencedirect scopuspubmed and web of science isi these databases werethoroughly searched and the relevant publication records were selected based on the plausible keywords inaccordance with the aim of this study as follows prevalence curcumin clinical features cancerthe selection of relevant studies for the systematic review and the output quality control process involvedseveral steps first all related s were collectedbased on the search keywords mentioned in the nextstep the specifications including the name of thejournal and authors were hidden and the full text of thes were made available to the reviewers each was investigated independently by two reviewersmm shr and if an was excluded in the studyfig the flowchart on the stages of including the studies in the systematic review prisma 0cmansouri bmc cancer page of detailed rationale were give accordingly in the case ofdisagreement between the two reviewers the wasjudged by a third reviewer in this paper all studies related to clinical investigations of curcumin use and impacts at varioustreatment weresystematically examined without any time constraintsand according to prisma guidelines fig stages of cancer selection criterias with the following characteristics were selectedfor metaanalysis original research s clinical trialstudies s that their full text and data are availableand studies that examined the clinical effects of curcumin in various types of cancers we prepared a list of s specifications based on prisma includingthe researchers name the title the year and placeof the study sample size and number of patients duration of study dosage of the drug and the result of theintervention table including review papers exclusion criteriastudiessystematic reviewmetaanalysis cohort casecontrol crosssectional descriptive and those which didnt present samples fromcancer patients and those which conducted with secondary data were excluded from the review duplicate publication and multiple publicationssamepopulation will be removed using citation managementsoftware endnote version x7 for windows thomsonreutersfrom thequality assessmentthe quality of the selected s was evaluated basedon criteria outlined by the consort checklist the lastconsort statement published in included items the consort statement has been shown to improve the scientific quality of rct reporting [ ]each was blindly assessed by two independentevaluators mm shr the result of each item wasassessed by yes point or no point and some itemswere assessed as not applicable due to the features ofstudies accordingly the maximum quality score of was considered and papers with a score of less than were considered to have low quality and thus they wereexcluded from the studycurcumin role in the prevention of cancersfree radicals and toxic products resulted from oxidativestress play an important role in the early stages of cancerformation therefore compounds that have antioxidanteffects can be helpful in preventing cancer formationcurcumin has the property of trapping free radicals andthus can play a crucial role in inhibiting the onset ofcancer several cellular and preclinical studies havereported that curcumin inhibits dna damage caused byoxidative factors such as ionizing radiation by inhibitingfree radicals and active oxygen species the nfkappab plays an important role in the formation of nitricoxide synthase and oxidative stress and as a resultcauses cancer curcumin suppresses the onset ofcancer by inhibiting nfkappab from formation [ ]curcumin was reported to be effective on liver enzymescytochrome p450 which has an imminent role in theoxidation and detoxification of toxins it also inhibits thephase i enzymes that is involved in the production oftoxic metabolites and carcinogens furthermore curcumin activates the phase ii enzymes which plays a crucialrole in detoxification of toxic metabolites curcumin prevent tumor formation and growth by inhibitingand activating these two enzymes phase i ii effect of curcumin on metastasis angiogenesis andinflammation in cancer cellsangiogenesis is the process of new blood vessel formation from preexisting vessels that is dependent on aprice equilibrium between antiangiogenic and angiogenicfactors however under pathological conditions for example tumor growth this tight regulation becomes lostwhich can result in tumor metastasis many gene products that are produced by different cells have a role inangiogenesis process hypoxia usually occurs in tumorsites in order to overcome to hypoxia tumor cells regulate and control the expression of genes related to angiogenesis cell cycle metastasis and drug resistance usinghypoxiainducible factor hif1 hif1 was first recognized as a transcription factor involved in hypoxiainduced erythropoietin expression this factor has beenpresented as a main transcription regulator for thesemolecules [ ] several studies have shown thathif1 activation of genes including vascular endothelialvegf angiopoietin1 ang1 andgrowth factorangiopoietin2 ang2 nfkb etc induced angiogenesis in the tumor cells furthermore the activation ofgenes such as insulinlike growth factor igf2 transforming growth factor a tgfa and mapk and pi3ksignalling pathway will also enable the survival proliferation and metastasis of tumor cells hif1 by activating genes involved in angiogenesis and also activatessignalling pathways associated with cell survival and proliferation plays an important role in the stability andgrowth of tumors as above mentioned hif1α is apotent activator of angiogenesis and curcumin inhibitsits expression ap1 is a transcription factor that is activated in response to hypoxia which is the principlephysiological stimulus that induces angiogenesis it isalso involved in the conversion of epithelial cells to mesenchymal cells which is the primary stage of metastasisand causes the expression of mmp and upa urokinase 0cmansouri bmc cancer page of table examines the characteristics extracted in the studiesauthors name yearcountry duration ofdosage of the drughejazi2013 belcaro2014 iranitalystudy years g per daynumber ofpatientsresultscurcumin reduces the severity of urinary symptoms mg with soy lecithin curcumin reduced side effectsbayetrobert2010 france months to mgryam2013 kanai2011 hemati2011 garcea2005 sharma2001 sharma2004 usajapaniranukukukcruzcorrea m2006 usa years months months months months months g g g to mg per day g per day g per day g per dayyu he2010 china days g per daydurgaprasad2005 india weeks g per daydhillon2008 usa g per dayide2010 japan months g per daygolombick2009 australia months g per daypolasa1992 hastak1997 indiaindia days g per day months g per daycheng2001 taiwan months g per dayrai2010 uk days g per daymarcia cruz correa2018 richard greil2018 newyorkusa month weeks mg orally twice adaydoses between and mg per minutelynne m howells2019 unitedkingdom days g per dayplasminogen activator genes that are involved in tumorangiogenesis and its invasion curcumin inhibits the expression of this transcription factor curcumin mayinhibit angiogenesis directly by regulating angiogenicgrowth factors growth factors as well as the genes including angiopoietin1 hif1 ho1 and transcription factors such as nfkappab fig [] it isknown that hypoxic stress and activation of betagrowthfactor tgf stimulate vegf expression by activatingap1 and the hypoxiainducible factors hif1 curcumin is an important inhibitor in ap1 activationand it has recently been shown that curcumin is a directinhibitor of hif1 transcription factor activity whichcauses the transcription of many genes associated withcurcumin lowers the concentration of the cea markertumorcurcumin reduces some skin complicationscurcumin increased patient survivalcurcumin reduces some skin problemscurcumin increased the effectiveness of the coloncurcumin reduced glutathione stransferase activitycurcumin reduces prostaglandin e2 productionreduces the number and size of polyps without anysignificant toxicitycurcumin has been shown to improve the overallhealth of patients with colorectal cancercurcumin reduced lipid peroxidation and increasedglutathione content in patientswelltolerated limited absorption and showedactivity in some patientsreduced the serum prostatespecific antigen contentin combination with isoflavonesdecreased para protein load and urinary n telopeptideof type i collagenreduced the urinary excretion of mutagens in smokersreduced the number of micronuclei in mucosal cellsand in circulating lymphocytesimproved the precancerous lesionsincreased vitamins c and e levels decrease dmalondialdehyde and 8hydroxy deoxy guanosine contents inthe serum and salivano difference in polyp size and number betweenplacebo and curcuminno variation in tumor size according to recist criteriacurcumin was a safe and tolerable adjunct to folfoxchemotherapy in patients with metastatic colorectal cancerangiogenesis in tumors [ ] it is also shown thatcurcumin will reduce the expression of membrane surface moleculesadhesionmolecule1 vascular cell adhesion molecule1 and eselectin which play a role in cellular adhesion fig intracellularincludingcurcumin affects a number of adhesive cellular molecules involved in tumor growth and metastasis processes curcumin caused reduction in the expression ofadhesive molecules inside the cells of icam1 vcamvcam or vascular cell adhesion molecule and mmpswhich play an important role in cellular adhesion andmetastasis furthermore curcumin results in increase ofthe expression of various antimetastatic 0cmansouri bmc cancer page of fig the effect of curcumin on angiogenesis and metastasis in cancer cells by coreldraw graphics suite including tissue inhibitor metalloproteinaseproteinstimp the nonmetastatic gene nm23 and ecadherin lack of ecadherin would increase the possibility of metastasis because ecadherin are essential tomaintain cellular adhesion angiogenesis is alsolinked with neoplasia angiogenesis means the formationof new blood vessels which is generally a major step intumor survival and growth curcumin inhibits cancer invarious ans [ ]easyto assess whetherthe antiinflammatory effects of curcumin have beenproven in many studies since oxidative stress leads tochronic inflammatory diseases antioxidant compoundscan be useful in the prevention and treatment of inflammatory disorders [ ] on the other handsince curcumin has a high antioxidant activity it willnot beantiinflammatory activity is also dependent on its antioxidant activity [ ] since many of the antioxidantsthat have been already identified do not have antiinflammatory properties it seems unlikely that the antiinflammatory effects of curcumin are due solely to itsantioxidant properties curcumin as a potent antiinflammatory factor expresses its own effects throughseveral mechanisms first curcumin inhibits the activation of the nfκ factor [ ]curcuminsthe lab based studies have revealed that curcuminneutralizes oxidative stress caused by tumor and restorestnfαnfkappabcurcuminactivityinhibitsproduction thus tcell apoptosis caused by tumor willbe minimised resultsin the initial screening of databases s wereidentified after deleting duplicate s studieswere obtained after deleting unrelated s studies were obtained17 s were also deleted due tolack of access to their fulltext or falling into the lowquality category at the end studies entered the finalphase and analysis as illustrated in fig the specifications and details of the studies considered in this systematic review are summarized in table according to the studies presented in table curcumin has reduced side effects including skin complications depending on the different doses of curcuminprescribed for the patients suffering from cancer theirsurvival rate was increased and their symptoms ofchemotherapy were reduced in studies examining theeffect of curcumin on colorectal cancer curcumin hasincreased efficacy in the large intestine reduced glutathione stransferase activity and reduced prostaglandin e2production curcumin also reduces the number and sizeof polyps without any significant toxicity curcumin inpancreatic cancer reduces lipids peroxidation and increases glutathione content in the patients with this typeof cancer in prostate cancer curcumin reduces theserum levels of prostatespecific antigen in combination 0cmansouri bmc cancer page of with isoflavones and also reduces the severity of urinarysymptoms according to the published studies the useof curcumin during radiation therapy for breast cancerpatients improved treatment outcomes for these patients such as preventing skin symptoms reducing painand suffering of patients improving their quality of lifeduring treatment and reducing delays or unwantedstops during the course of radiation therapy curcumincan regulate multiple signalling pathways and affect different moleculartargets low cost pharmacologicalsafety efficiency and multiple molecular targets makecurcumin a promising product for the prevention andtreatment of various human diseases table after collecting various s from reputable databases and deleting duplicate s and removing unrelated s to the main aim of this paper we finallyconsidered s for further investigation and analysis the main aim of this paper is to review the clinicalstudies about curcumin and its various purposeseffectson cancer the results reported from numerous clinicalstudies that have examined the effects of curcumin onthe patients who are suffering from cancer and undergoing radiotherapy and chemotherapy were very promising here we briefly describe some of these studieswhich are summarised in table garcea evaluated patients in the ukin this study each patient received to mg ofcurcumin per day at the same time that these patientsreceived curcumin they were treated with radiotherapyand chemotherapy the results of this study revealedthat curcumin increased the effectiveness of the treatment plan for colorectal cancer in the patients receivedwith curcumin importantin bayetrobert conducted a study consists offourteen patients with advanced breast cancer who werebeing treated with docetaxel chemotherapy and simultaneously received curcumin at different doses up to amaximum dose of g per day for days per each treatment cycle finally patients participated in this studywere able to complete this treatment plan nutropeniaand leukopenia were the mosttoxicitiescaused by docetaxel administration after days two patients refused to continue treatment because they received curcumintreatment continued by reducing the dosage to a maximum of g per day nine patients were screened fortumor response six weeks after completing the courseof treatment patients partially responded well butthree patients still suffered from the disease in thisstudy the ca tumor marker did not decrease butthe cea tumor marker decreased compared to the initial value prior to the treatment in patients the vegfvascular endothelial growth factor as a tomur markerwhichandcurcumin capsules howeverindicatestumrowth metastasismalignancy was reduced by compared to the baseline before treatment in the effect of curcumin on reducing the sideeffects of radiotherapy and chemotherapy in patientswith ovarian lung colon liver kidney and stomach cancers was investigated eighty patients received mg ofcurcumin simultaneously with radiotherapy the duration of this study was days the incidence of side effects such as nausea diarrhea constipation and weightloss decreased in patients who treated with both radiotherapy and curcumin in the patients who are simultaneously under radiotherapy and received curcumin theprevalence of side effects such as skin lesions mouthand throat ulcers swallowing problems nausea vomitingfatigue weakness and common medications required for treating side effects were statistically lowerthan the control group in a study conducted by hemati in iran patientsreceiving radiation therapy to the breast area due tobreast cancer from days before the start to the end ofradiotherapy mgcapsules containing curcuminwere taken orally times a day yu he evaluated patients in their study andstated that a dose of g of curcumin per day for days improved the general health of patients withcolorectal cancer through increasing the expression ofp53 molecules in tumor cells in a study conductedby cruzcorrea it was stated that a dose of g of curcumin per day will reduce the number and size of polypswithout any significant toxicity discussionin the recent years several studies have been conductedon the biological effects of curcumin in more than studies have been recently published curcumin hasshown to have various effects in cancer treatments curcumin has antioxidant antibacterial antifungal antiviralantiinflammatory antiproliferative proapoptotic effects etc curcumin has tremendous potential for treatment of neurodegenerative diseases arthritis diabetespsoriasis allergies intestinal inflammation kidney poisoning alzheimers depression aids multiple sclerosis cardiovascular disease and especially cancer [] the numerous and multifaceted effects of curcuminin determining the cellular targets and molecular mechanisms involved in curcumin pathways have attractedmuch attention from researchers curcumin is a multifaceted molecule and has many therapeutic effects themultifaceted effects of curcumin are due to its capacityto interact with different molecules and to regulate multiple molecular pathways and their targets one of the compelling properties of curcumin whichmakes it appropriate for therapeutic use is its low toxicity so that even its consumption up to a dose of g 0cmansouri bmc cancer page of per day does not cause any side effects consumption of curcumin in highdose prevents cancer cells frommultiplying although it does not damage healthy cells[ ]minimaltoxicity alongside with possessing manytherapeutic effects have led to the widespread use of natural plantderived compounds in the treatment of cancer the compounds found in nature target various cellular and molecular aspects of cancer cells the researchers have demonstrated that curcumin regulatessignalling pathways in cancer cells reduces the expression of proteins related to drug resistance and increasesthe performance of antitumor drugs at various levelscurcumin reverses drug resistance mechanisms and results in increasing the sensitivity of chemotherapyresistant cells in the research conducted by keyvanighamsari they demonstrated that curcumin is aneffective chemical in cancer treatment in laboratory studies which have been performed onthe cellular categories of colorectal cancer the derivedresults show that curcumin inhibits cell growth and alsostimulates apoptosis by interacting with several molecular targets furthermore curcumin has been used as partof dietary formulations to prevent colon cancer in vitroand in vivo these compounds have been shown to haveanticancer properties for colon cancer and its inflammation the results of this study show that curcuminwould be effective in preventing colorectal cancer in animals this property offers promising expectations inhumans due to the limited number of the human clinical studiesthe corresponding results are somehowcontradictory on the other hand there exist several unanswered questions about dosage bioavailability optimalsigns and potential toxicity which should be investigatedin future studies using sufficiently large samples inaddition curcumin can induce autophagy apoptosisand cell cycle arrest in order to reduce the survival andproliferation oflung cancer cells curcumin has thispromising capability to increase the effectiveness ofradiotherapy in the treatment of lung cancer by targetingdifferent signalling pathways such as epidermal growthfactor receptor and nf κb curcumincontaining nanocarriers increase bioavailability cell uptake and curcumin antitumor activity [ ]in a study conducted by cruzcorrea oral curcumin was prescribed to the patients with adenomatouspolyposis this research was implemented to determinethe safety and efficacy of curcumin in patients with adenomatous polyposis in this study mg of oral curcumin was administered twice per day over monthsto patients with adenomatous polyposis the resultsshowed that there was no significant difference betweenthose who received oral curcumin and those receivingplacebo in another study conducted by grell patients were subjected to receive doses between and mg per minute the main aim of their studywas to evaluate the safety of curcumin locally in patientswith advanced or metastatic cancer the results obtainedfrom their study showed that no change in tumor sizewas observed based on the recist criteria in howells evaluated patients with the age over and with metastatic colorectal cancer using the histological diagnosis quality oflife and neurotoxicity ofthese patients were assessed using questionnaires thederived results showed that curcumin is a safe and tolerable adjunct for folfox chemotherapy in patients withmetastatic colorectal cancer overall the results suggest that curcumin can be usedas an effective combination in inhibiting and controllingcancersimproving clinical symptoms and preventingtumor spread and metastasis this compound wouldaffect various molecular pathways and inhibits vasodilation cell proliferation and metastasiscurcumin is a natural product found in turmeric thathas a unique chemical structure with particular biological and medicinal properties through various cellular and molecular mechanisms curcumin inhibits thecarcinogenesis and their growth due to the fact that nospecific toxic effects of this natural product have beenreported its use has been considered as a drug supplement in therapeutic diets of cancer patients in a number of studies considered in this systematic review haveshown that taking curcumin would increase the expression of antimetastatic proteins in several other studiesit was reported that curcumin has also increased patientsurvival and decreased tumor marker concentrationabbreviationsmmps matrix metalloproteases vcam vascular cell adhesion moleculewho world health anization sid scientific information database prisma preferred reporting items for systematic reviewsacknowledgementsthe authors thank the faculty members of the faculty of nursing andmidwifery kermanshah university of medical sciencesauthorscontributionsshr and ns and km contribute | Colon_Cancer |
objective this study aimed to test the hypothesis that levobupivacaine has antitumour effects on breast cancer cellsresults colony formation and transwell assay were used to determine breast cancer cells proliferation flow cytometry annexin v and pi staining was used to investigate breast cancer cells apoptosis the effects of levobupivacaine on cellular signalling and molecular response were studied with quantitative polymerase chain reaction and western blot induction of apoptosis was confirmed by cell viability morphological changes showed cell shrinkage rounding and detachments from plates the results of the western blot and quantitative polymerase chain reaction indicated activation of active caspase and inhibition of foxo1 the results of the flow cytometry confirmed that levobupivacaine inhibited breast cancer cell proliferation and enhanced apoptosis of breast cancer cells quantitative polymerase chain reaction and western blot analysis showed increased p21 and decreased cyclin d quantitative polymerase chain reaction and western blot analysis showed that levobupivacaine significantly increased bax expression accompanied by a significant decreased bcl expression and inhibition of pi3kaktmtor signalling pathway these findings suggested that levobupivacaine inhibits proliferation and promotes breast cancer cells apoptosis in vitrokeywords levobupivacaine proliferation invasion apoptosis breast cancerintroductionbreast cancer is one of the most recorded cancer illness among women in the united states it is estimated that more than women die every year from breast cancerrelated illness despite the advance in chemotherapy and targeted treatments correspondence yanqiu63126com wqp89163com department of anaesthesiology dalian medical university dalian china department of biochemistry and molecular biology dalian medical university dalian chinafull list of author information is available at the end of the molecular signalling pathways that are involved in breast cancer transformation have become targets for treatment the mechanisms of the pi3kaktmtor signalling pathway have present some promising targets for cancer treatments this signalling pathway hinders the functions of several tumour suppressor genes such as bad gsk3 foxo transcription factors and tuberinhamartin complex which control cell survival proliferation and growth [] suppressing this signalling pathway may inhibit cancer cells proliferation and also stimulate them toward cell deaththe growing evidence of local anaesthetics inhibiting cancer cell growth seems promising though limited the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0ckwakye a0et a0al bmc res notes page of at the tissue level administration of a certain amount of local anaesthetics topical or local has shown to have a direct inhibitory effect on the action of epidermal growth factor receptor egfr which is a potential target for antiproliferation in cancer cells evidence also shows that ropivacaine and lidocaine hinder cancer cells growth invasion migration and enhance apoptosis of lung cancer cells [] to the best of our knowledge the effect of levobupivacaine on breast cancer cells is yet to be determined the present study therefore aimed to investigate the antitumour effects of levobupivacaine on breast cancer cellsmain textmaterials and a0methodsethics statementthe ethical committee of the dalian medical university first affiliated hospital approved for this study to be carried outcell culturewe purchased mcf7 and mdamb231 breast cancer cells from the atcc beijing zhongyuan limited china we maintained the mcf7 and mdamb231 cells with highglucose dmem or dmemf12 gibco usa medium the medium was supplemented with fetal bovine serum fbs gibco usa penicillin a0unitsml and streptomycin a0µgml transgen biotech china to maintain the cells the mcf7 and mdamb231 cells were then maintained in an incubator at a0ºc humidified air with co2 atmospheric condition the cells were routinely subcultured subsequentlyantibodies and a0reagentsepr17671 akt monoclonal antibody abcam china y391 mtor polyclonal antibody abcam china a2845 bcl2 polyclonal antibody abclonal technology a11550 bax polyclonal antibody abclonal technology a0265 pik3ca polyclonal antibody abclonal technology a2934 foxo1 polyclonal antibody abclonal technology epr21032 active caspase monoclonal antibody abcam china afo931 cyclin d1 polyclonal antibody affbiotech china af6290 p21 polyclonal antibody affbiotech china antimtor phospho s2448 antibody abcam china pa517387 phosphopi3k p85p55 tyr458 tyr199 polyclonal antibody themofisher scientific posphopanakt123 ser473 antibody affbiotech china peroxidaseconjugated goat antirabbit igg proteintech china prap antibodies proteintech china and gapdh antibodies proteintech chinacell viability assay and a0ic50we determined the mcf7 and mdamb cells viability using cck8 assay levobupivacaine at a concentration of or a0mm was used to treat mcf7 and mdamb cells plated in 96well plates a0cellswell and then incubated for or a0h respectively in an incubator at the atmospheric condition of a0 °c with co2 the rest of the procedures used for the cck8 assay were the same as described elsewhere flow cytometryannexin v and propidium iodide pi staining assay were used to investigate the apoptosis of mcf7 and mdamb cells following levobupivacaine treatment after treating the cells for a0h trypsin was used to harvest the treated cells and centrifugation at rcf for a0min the mcf7 and mdamb treated cells were again suspended with binding buffer and then a0 µl of fluorochromeconjugated annexin v sigmaaldrich saint louis usa was added into a0µl of the cell suspension to stain intracellular phosphatidylserine ps the cells were then incubation in a dark under room temperature the cells were again suspended and a0 µl propidium iodide staining solution sigmaaldrich saint louis usa added into a0µl of the cell suspension we detected the percentage of the apoptotic cells via flowjo software treestar ashland usa and flow cytometry facs calibur becton dickinson and sunnyvale ca usaquantitative polymerase chain reaction qpcrthe procedures used for the qpcr were the same as previously described the primers sequences were bax 5tgg cag ctg aca tgt ttt ctg3 f 5tcc cgg agg aag tcc aat g3 bcl2 5acg gtg gtg gag gag ctc tt3 f 5gcc ggt tca ggt act cag tcat3 r p21 5gcg act gtg atg cgc taa tg3 f 5gaa ggt aga gct tgg gca gg3 r gapdh ²cat gtt cgt cat ggg tgt gaa² f ²ggc atg gac tgt ggt cat gag3² rr western blotat the log phase of treated mcf7 and mdamb cells growth we harvested the cells and then washed twice with icecold pbs the rest of the procedures used for the western blot were the same as described elsewhere colony formation assaythe procedures used for the colony formation assay were the same as previously described 0ckwakye a0et a0al bmc res notes page of transwell assaythe mcf7 and mdamba231 cells that were pretreated with different dose of levobupivacaine a0mm for a0h and resuspended in culture medium with the same concentrations of levobupivacaine were seeded onto the coated membrane in the upper chamber of the transwell 24well millicell cell culture insert a0mm diameter a0μm pores merck kgaa p18p01250 china the procedures used for the transwell assay were the same as previously describe data analysisvalues were expressed as the mean ± sd statistical analysis was performed with graphpad prism version 501graphpad software la jolla ca us oneway anova was used to measure significance p dunnetts post hoc tests were used to test the difference between groupsresultslevobupivacaine decreases breast cancer cell invasiontranswell assay analysis showed significantly decreased in the invasion ability of mcf7 and mdamb231 cells in a dosedependent manner compared with the untreated cells additional file a0 fig s1a b levobupivacaine inhibits proliferation in a0breast cancer cellsthe mcf7 and mdamba231 cell viability decreased as the concentrations of levobupivacaine or a0mm increased the mcf7 cells showed a cytotoxic effect while the mdamb231 cells showed a similar cytotoxic effect of fig a01a under a fluorescence microscope cells treated with levobupivacaine showed morphological changes including cell rounding cell shrinkage and cells detachment from the plates additional file a0 fig s2a b the viability of breast cancer cells decreased in a dosedependent manner the results showed significantly decreased in the number of clones of the treated cells compared with the untreated cells fig a01b c the data showed that the mrna level of p21 significantly increased following levobupivacaine treatment fig a0 1d e western blot analysis showed a similar increased in p21 and decreased in foxo1 and cyclin d1 expressions in a dosedependent manner compared with the untreated cells fig a01f g additional file a03f glevobupivacaine promote apoptosis in a0breast cancer cellslevobupivacaine significantly reduced the number of cells showing nuclear staining when compared with the untreated cells fig a0 2a b the qpcr data showed a decreased in bcl2 and increased in bax expressions in mcf7 and mdamb231 cells compared with the untreated cells fig a0 2c d western blot analysis also showed a similar decreased in bcl2 and increased expressions of active caspase and bax compared with the untreated cells fig a02e f additional file a03e flevobupivacaine inhibits proliferation and a0promotes apoptosis in a0breast cancer through a0pi3kaktmtor signalling pathwaywestern blot analysis showed a significant decreased in the expression of the nuclear localization of ppi3k pakt and pmtor compared with the untreated cells fig a03a b additional file a03a bdiscussionbreast cancer remains a common cause of mortality among women worldwide though current orthodox drugs have demonstrated promise in breast cancer therapy its treatment options remain limited these therefore supports the concept that effective therapeutic approaches for breast cancer are critically needed several retrospective studies have demonstrated that regional anaesthesia is associated with a decreased risk of recurrence or metastasis of multiple carcinomas including breast prostate and cervical cancers [] recent growing evidence demonstrates that local anaesthetics have an antitumour effect and may suppress the motility of cellular function and invasiveness more likely via voltagegated sodium channel inhibition a study report indicates that lidocaine inhibits the growth of human hepatocellular carcinoma cells hcc by increasing the caspase activity whereas ropivacaine inhibits the growth of hcc cells by stopping the cell cycle in g2 phase lee et a0al demonstrated that local anaesthetics potentiate tnfα mediated apoptosis in hk2 cells the cellular modification of treated cells is likely dependent on the duration of exposure and the dose of the local see figure on next pagefig levobupivacaine inhibits proliferation in breast cancer cells mcf and mdamb cells were treated with different concentrations of levobupivacaine a cell viability was measured by cck assay ic50 results of levobupivacaine on mcf and mdamb cells b c colony formation of mcf and mdamb cells treated with various concentrations of levobupivacaine and stained with crystal violet d e the mrna expression levels of p21 and gapdh were analysed by qpcr f g protein expression assessment of mcf and mdamb cells by western blot against antibodies foxo1 p21 cyclin d1 and gapdh used as control the data was statistically significant at indicates p indicates p indicates p compared with untreated cells this data corresponds to the mean ± sem of three independent experiments 0ckwakye a0et a0al bmc res notes page of 0ckwakye a0et a0al bmc res notes page of fig effects of levobupivacaine on apoptosis of breast cancer cells a b mcf and mdamb cells were treated with different concentrations of levobupivacaine for h the cells were then stained with fluoresceinconjugated annexin v and pi and analysed by flow cytometry error bars represent standard error of the mean p versus the control c d relative gene expression of bax and bcl following the treatment of breast cancer cells with different concentrations of levobupivacaine for h and analysed by qpcr e f mcf and mdamb cells were treated with different concentrations of levobupivacaine for h and the activities of bax bcl and active caspase were examined by western blot analysis using specific antibodies gapdh was used as internal controls the data was statistically significant at indicates p indicates p compared with control the data correspond to the mean ± sem of three independent experiments 0ckwakye a0et a0al bmc res notes page of fig mcf and mdamb cells were treated with different concentrations of levobupivacaine for h a b the cells were lysed and subjected to sdspage and analysed by western blotting and probed with specific antibodies ppi3k pakt and pmtor the results showed a decrease in the expressions of ppi3k pakt and pmtor proteins gapdh was used as internal controls the data represent the mean ± sd of three independent experimentsanaesthetic [] in this study we employed mcf and mdamb231 cells as models and found that different concentrations of levobupivacaine could effectively inhibit breast cancer cell proliferation and promote apoptosis in a0vitro the antiproliferation and apoptosis effects observed in this study suggest that levobupivacaine may have therapeutic effects on breast cancerpi3kaktmtor signalling pathway plays a vital role in cell proliferation survival development metabolism motility and regulation of the immune response breast cancer cell resistance to therapies can result from the activation of pi3kaktmtor signalling pathway [] this has made the pi3kaktmtor signalling pathway an important object of study for understanding the development and progression of breast cancer in patients with breast cancer pi3kaktmtor signalling pathway can be a target for diagnostic prognostic and treatment purposes [] akt plays a role in the activation and inactivation of many transcription factors activation of akt correlated with the activation of mtor phosphorylation of the foxo proteins by akt may results in cytoplasmic retention by interacting with other proteins thereby isolating them from their targeted genes cyclin d1 classified as a pro oncogene is often overexpressed in several human malignancies including breast colon lung and prostate cancers reports show that overexpression of cyclin d1 and underexpression of tumour suppressor p21 is required for cancer initiation as it is confirmed that downregulation of cyclin d1 and overexpression of p21 in xenograft model discontinues the formation of cancer in the early stages datta et a0al reported that akt can phosphorylate the proapoptotic bcl2 family member bad causing its isolation from the mitochondrial membrane by other proteins local anaesthetics modify the protein levels of key members of the bcl2 family in a manner that presents an increase in the ratio of baxbcl2 which may contribute to the response of cancer cells to apoptosis in the present study the role of levobupivacaine on the expression of pi3k akt and mtor was investigated to illustrate the potential molecular mechanism we observed a significantly decreased expression of pakt ppi3k pmtor and subsequent decreased expression of foxo cyclin d1 and bcl2 following levobupivacaine treatment which correlated with decreased breast cancer cells proliferation and increased apoptosis these emerging pieces of evidence suggest that levobupivacaine may inhibit proliferation and promote apoptosis by suppressing pi3kaktmtor signalling pathway which demonstrated an antitumour effect on breast cancer cells in this studyconclusionlevobupivacaine has the potency of reducing breast cancer cell viability proliferation and also causes cell death by suppressing the pi3kaktmtor signalling pathway these findings could lead to clinical studies which will seek to examine the anticancer effects of levobupivacaine and may also increase the benefits in cancer patient as well as improve patient care 0ckwakye a0et a0al bmc res notes page of limitationsnumerous studies have reported on the antitumour effects of local anaesthetics on various cancer cells [] however our work is not without limitations in a0vivo and clinical studies on the antitumour effects of levobupivacaine are neededbiology dalian medical university dalian china department of anaesthesia and critical care school of medicine university of health and allied sciences ho ghana department of biochemistry and molecular medicine school of medicine and health sciences university for development studies tamale ghana departments of anaesthesia and critical care ridge hospital accra ghana department of medicine princefied university ho ghana received june accepted july supplementary informationsupplementary information accompanies this paper at https doi101186s1310 additional file a0 figure s1 levobupivacaine decreases breast cancer cell invasionadditional file a0 figure s2 effect of levobupivacaine on the morphology of mcf and mdamb cellsadditional file a0 original gelsblots scan used in fig 1f g fig 2e f and fig 3a b for mcf and mdamb cellsabbreviationsegfr epidermal growth factor receptor hcc hepatocellular carcinoma cells nc nitrocellulose pi propidium iodide ps phosphatidylserine qpcr quantitativepolymerase chain reactionacknowledgementswe thank the first affiliated hospital and the department of biochemistry of dalian medical university for making available all the necessary materials needed for this work we also thank the key laboratory of liaoning provincial education department grant no lz2016002 and liaoning natural science foundation grant no of china for supporting this work our thanks also go to the china scholarship council and the government of the republic of ghana for giving financial aid to some of the authors to study at dalian medical universityauthors contributionsakk sk qy and qpw conceived and designed this study qpw and qy were responsible for the supervision and coordination of this study akk sk jl mnr qy and qpw conducted the data collections sk led the data analysis with inputs from akk qy and qpw akk and sk wrote the first draft of the manuscript and jl mnr sar aaf ja and ean contributed to revising and reviewing the manuscript all authors read and approved the final manuscriptfundingthis study was supported by the key laboratory of liaoning provincial education department grant no lz2016002 and liaoning natural science foundation grant no availability of data and materialsthe data used andor analysed in this study are available from the corresponding author upon reasonable requestethics approval and consent to participatethe ethical committee of the first affiliated hospital of dalian medical university approved the study protocol and because this study used breast cancer cells consent to participate was not applicable for the studyconsent for publicationsnot applicablecompeting interestsauthors declare that they have no competing interestsauthor details department of anaesthesiology dalian medical university dalian china department of anaesthesiology first affiliated hospital of dalian medical university dalian china department of biochemistry and molecular references american cancer society breast cancer facts and figures atlanta american cancer society american cancer society cancer facts and figures atlanta ameri can cancer society siegel r naishadham d jemal a cancer statistics ca cancer j clin chang yc hsu yc liu cl huang sy hu mc cheng sp local anaesthetics induce apoptosis in human thyroid cancer cells through the mitogenactivated protein kinase pathway plos one 20149e89563 gomezgutierrez jg souza v hao hy de montes ocaluna r dong yb zhou hs mcmasters km adenovirusmediated gene transfer of fkhrl1 triple mutant efficiently induces apoptosis in melanoma cells cancer biol ther sunters a de fern¡ndez mattos s stahl m brosens jj zoumpoulidou g saunders ca coffer pj medema rh coombes rc lam ew foxo3a transcriptional regulation of bim controls apoptosis in paclitaxeltreated breast cancer cell lines j biol chem fu z tindall dj foxos cancer and regulation of apoptosis oncogene barnes dm gillett ce cyclin d1 in breast cancer breast cancer res treat sherr cj roberts jm cdk inhibitors positive and negative regulators of g1phase progression genes dev pelengaris s khan m evan g cmyc more than just a matter of life and death nat rev cancer di padova m barbieri r fanciulli m arcuri e florida a effect of local anaesthetic ropivacaine on the energy metabolism of ehrlich ascites tumour cells oncol res 199810491e8 xing w chen dt pan jh chen yh yan y li q xue rf yuan yf zeng wa lidocaine induces apoptosis and suppresses tumour growth in human hepatocellular carcinoma cells in vitro and in a xenograft model in vivo anesthesiology drasner k lidocaine spinal anaesthesia a vanishing therapeutic index anesthesiology wang hw wang ly jiang l tian sm zhong td fang xm amidelinked local anaesthetics induce apoptosis in human nonsmallcell lung cancer j thorac dis https doi1021037 jtd20160966 piegeler t vottavelis eg liu g place at schwartz de beckschimmer b minshall rd beat a antimetastatic potential of amidelinked local anaesthetics inhibition of lung adenocarcinoma cell migration and inflammatory src signalling independent of sodium channel blockade anesthesiology lirk p berger r hollmann mw feigl h lidocaine time and dosedependently demethylates deoxyribonucleic acid in breast cancer cell lines in vitro br j anaesth villargarea a fraga mf espada j esteller m procaine is a dnademethylating agent with growthinhibitory effects in human cancer cells cancer res 4984e634984e9 kampo s ahmmed b zhou t owusu l anabah tw doudou nr kuugbee ed cui y lu z yan q wen qp scorpion venom analgesic peptide bmk agap inhibits stemness and epithelialmesenchymal transition by downregulating ptx3 in breast cancer front oncol hirata m sakaguchi m mochida c sotozono c kageyama k yoshihiro k munetaka h lidocaine inhibits the tyrosine kinase activity of the epidermal growth factor receptor and suppresses proliferation of corneal epithelial cells anesthesiology 0ckwakye a0et a0al bmc res notes page of grouselle m tueux o dabadie p geescaud d mazat jp effect of local anaesthetics on mitochondrial membrane potential in living cells biochem j fraser sp diss jkj chioni am mycielska me pan h yamaci rf pani f siwy z krasowska m grzywna z brackenbury wj theodorou d koyuturk m kaya h battaloglu e de tamburo bella m slade mj tolhurst r palmieri c jiang j latchman ds coombes rc djamgoz mba voltagegated sodium channel expression and potentiation of human breast cancer metastasis clin cancer res le gac g angenard g clement b laviolle b coulouarn c beloeil h local anaesthetics inhibit the growth of human hepatocellular carcinoma cells anesth analg lee ht xu h siegel cd krichevsky ie local anaesthetics induce human renal cell apoptosis am j nephrol unami a shinohara y ichikawa t baba y biochemical and microarray analyses of bupivacaineinduced apoptosis j toxicol sci villargarea a fraga mf espada j esteller m procaine is a dnademethylating agent with growthinhibitory effects in human cancer cells cancer res sakaguchi m kuroda y hirose m the antiproliferative effect of lidocaine on human tongue cancer cells with inhibition of the activity of epidermal growth factor receptor anesth analg chang yc liu cl chen mj hsu yw chen sn lin ch chen cm yang fm hu mc local anaesthetics induced apoptosis in human breast tumour cells anesth analg kawasaki c kawasaki t ogata m sata t chaudry ih lidocaine enhances apoptosis and suppresses mitochondrial functions of human neutrophil in vitro j trauma hodgkin al huxley afa quantitative description of membrane current and its application to conduction and excitation in nerve j physiol besson p driffort v bon © gradek f chevalier s roger s how do voltagegated sodium channels enhance migration and invasiveness in cancer cells biochim biophys acta roger s gillet l le guennec jy besson p voltagegated sodium channels and cancer is excitability their primary role front pharmacol roger s potier m vandier c besson p le guennec jy voltagegated sodium channels new targets in cancer therapy curr pharm des driffort v gillet l bon e marionneaulambot s oullier t joulin v collin c pag¨s jc jourdan ml chevalier s bougnoux p le guennec jy besson p roger s ranolazine inhibits nav15mediated breast cancer cell invasiveness and lung colonization mol cancer nelson m yang m dowle aa thomas jr brackenbury wj the sodium channelblocking antiepileptic drug phenytoin inhibits breast tumour growth and metastasis mol cancer yuan t li z li x yu g wang n yang x lidocaine attenuates lipopolysaccharideinduced inflammatory responses in microglia j surg res brocco mc paulo dns almeida ced carraretto ar cabral sa silveira ac gomez rs baptista jf a study of interleukin il and tumour necrosis factoralpha tnfα serum levels in rats subjected to faecal peritonitis and treated with intraperitoneal ropivacaine acta cirurgica brasileira piegeler t schl¤pfer m dull ro schwartz de beat a minshall rd beckschimmer b clinically relevant concentrations of lidocaine and ropivacaine inhibit tnfαinduced invasion of lung adenocarcinoma cells in vitro by blocking the activation of akt and focal adhesion kinase br j anaesth shankar s chen q srivastava rk inhibition of pi3kakt and mekerk pathways act synergistically to enhance antiangiogenic effects of egcg through activation of foxo transcription factor j mol signal qian j zou y rahman jsm lu b massion pp synergy between phosphatidylinositol kinaseakt pathway and bclxl in the control of apoptosis in adenocarcinoma cells of the lung mol ortega ma frailemartınez o asunsolo a bujan j garcıahonduvilla n coca s signal transduction pathways in breast cancer the important role of pi3kaktmtor j oncol royds j khan ah buggy dj update on existing ongoing prospective trials evaluating the effect of anaesthetic and analgesic techniques during primary cancer surgery on cancer recurrence or metastasis int anesthesiol clin 2016544e76 burgering bmt medema rh decision on life and death foxo forkhead transcription factors are in command when pkbakt is off duty j leukoc biol chen q ganapathy s singh kp shankar s srivastava rk resveratrol induces growth arrest and apoptosis through activation of foxo transcription factors in prostate cancer cells plos one 20105e15288 arnold a papanikolaou a cyclin d1 in breast cancer pathogenesis j clin oncol santarius t shipley j brewer d stratton mr cooper cs a census of amplified and overexpressed human cancer genes nat rev cancer datta sr brunet a greenberg me cellular survival a play in three akts genes daev lirk p hollmann mw fleischer m weber nc feigl h lidocaine and ropivacaine but not bupivacaine demethylate deoxyribonucleic acid in breast cancer cells in vitro br j anaesth 2014113suppl 1i32i3838 li k yang j han x lidocaine sensitizes the cytotoxicity of cisplatin in breast cancer cells via the upregulation of rarβ2 and rassf1a demethylation int j mol sci publishers notespringer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your ï¬eld¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold open access which fosters wider collaboration and increased citations maximum visibility for your research over 100m website views per year ¢ at bmc research is always in progresslearn more biomedcentralcomsubmissionsready to submit your research choose bmc and benefit from 0c' | Colon_Cancer |
"objective endoscopic full thickness resection eftr has shown efficacy and safety in the colorectum the aim of this analysis was to investigate whether eftr is cost effective in comparison with surgical and endoscopic treatment alternativesdesign real data from the study cohort of the prospective single arm wall resect study were used a simulated comparison arm was created based on a survey that included suggested treatment alternatives to eftr of the respective lesions treatment costs and reimbursement were calculated in euro according to the coding rules of and eftr r0 resection rate was used as a measure of effectiveness to assess cost effectiveness the average cost effectiveness ratio acer and the incremental cost effectiveness ratio icer were determined calculations were made both from the perspective of the care provider as well as of the payerresults the cost per case was ¬ for the eftr group ¬ for the standard endoscopic resection ser group ¬ for the surgical resection group and ¬ for the pooled alternative treatment to eftr from the perspective of the care provider the acer mean cost per r0 resection was ¬ for eftr ¬ for ser ¬ for surgical treatment and ¬ for all pooled and weighted alternatives to eftr the icer additional cost per r0 resection compared with eftr was ¬ for ser ¬ for surgical resection and ¬ for the pooled rate of alternatives results from the perspective of the payer were similar eftr is cost effective in comparison with surgical and endoscopic treatment alternatives in the colorectumintroductioncolorectal cancer is the third most common type of cancer and the second most common cause of cancer related deaths worldwide1 screening programmes for early detection of premalignant and malignant lesions led summary boxwhat is already known about this subject º endoscopic full thickness resection eftr has shown clinical efficacy and safety in difficult to treat lesions in the colorectum º the cost of the full thickness resection device is higher than the cost of standard endoscopic resection ser devices but lower than surgical devices º cost effectiveness analyses on treatment with eftr compared with treatment alternatives do not existwhat are the new findings º eftr leads to an almost reduction in cost per r0 resection average cost effectiveness ratio compared with surgery º to achieve an additional r0 resection by surgical treatment compared with eftr incremental cost effectiveness ratio an additional cost of ¬ is necessary º these findings are consistent both from the perspective of the care provider as well as the payerhow might it impact on clinical practice in the foreseeable future º in terms of cost effectiveness eftr should be considered first before patients with difficult to treat lesions in the colorectum are sent to surgical treatmentto a significant reduction in cancer related mortality2 with more intense screening more lesions are detected which automatically creates the need for removal standard endoscopic resections ser such as endoscopic mucosal resection emr and endoscopic submucosal dissection esd are well established and sufficient for the vast majority of lesions however ser of non lifting lesions and lesions located at difficult anatomical to cite kuellmer a0a behn a0j beyna a0t et a0al endoscopic full thickness resection and its treatment alternatives in difficult to treat lesions of the lower gastrointestinal tract a cost effectiveness analysis bmj open gastro 20207e000449 101136bmjgast2020000449received may revised july accepted 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 toarthur schmidt arthur schmidt uniklinik freiburg dekuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access locations eg appendiceal orifice is associated with increased complication rates or incomplete resection3 these types of lesions are therefore often referred to surgery which is associated with significant morbidity and mortality and higher costs6 given the high number of polypectomies performed worldwide this is not only an issue of morbidity and mortality but also a huge economic challengethe efficacy and safety of endoscopic full thickness resection eftr of non lifting and other difficult to treat lesions have been demonstrated in multiple retrospective studies and in one prospective study7 the cost of the device is markedly higher than ser devices but lower than surgical treatment the aim of the present analysis was to evaluate whether eftr is cost effective in comparison with ser as well as surgical treatmentmethodsstudy populationto calculate the cost of eftr we analysed the study cohort of the wall resect trial nct02362126 in this single arm multicentre prospective study patients with difficult to treat adenomas eg non lifting andor challenging anatomical location early adenocarcinomas and subepithelial tumours in the colorectum were treated with eftr the primary endpoints of the study en bloc and r0 resection rate were achieved in and respectively7 written informed consent was obtained from each patient included in the studysimulation second study arm based on a survey of endoscopistswith the wall resect study being single armed a second study arm was created based on treatment simulation in order to compare different treatment modalities a case report form crf was created and sent to each participating centre of the wall resect trial endoscopists at the respective location reviewed the endoscopic images and their case relevant data and decided which treatment modality they would have chosen if eftr were not available treatment alternatives included ser such as emr thermal methods and esd as well as surgical resection the crf was filled out in a pseudonymised fashioninformed consent had already been obtained within the wall resect studydetermination of case costs and reimbursementa certified online it tool the diagnosis related group drg web grouper was used to determine the reimbursement rate for each patient http drg uni muenster de index php therefore the code of the international classification of diseases icd10 and the specific code for the procedure performed operationen und prozedurenschl¼ssel ops code in each patient in both groups were put into the web grouper together with the predefined mean length of hospital stay mittlere grenzverweildauer the drg which accounts for reimbursement was calculatedin the comparison arm the drg codes of were used because this was the year the wall resect study was performed for the eftr arm the drg codes of were used as reimbursement for eftr was increased that yearto calculate the cost per case another certified online it tool g drg report browser was used www g drg de g drg system_ abschlussbericht_ zur_ weiterentwicklung_ des_ g drg systems_ und_ report_ browser this was done by filling in the respective drg icd10 and ops code into the browser the data of the g drg report browser derive from the data that were sent in to inek authority managing the german drg system by certified hospitals kalkulationshuser in grouping was performed following the rules of g drg version the main and secondary diagnoses are shown according to icd10 german modification gm version and the procedures according to ops version g drg report browser inek gmbhas reimbursement for the eftr group was taken from the cost per patient case would ideally also have been calculated from unfortunately these data will be first published by inek in to overcome this problem costs for eftr cases from the university of freiburg between and which were reported to inek were used for the analysiswith the cost of each patient case the mean cost for each treatment modality emr esd laparoscopic surgery transanal endoscopic microsurgery tem eftr could be calculated in the next step the mean cost for each treatment path ser surgical treatment and casemix alternative was determined this was done in the following fashion for ser the mean costs of emr and esd were used for calculation of the surgical treatment laparoscopic surgery and tem were taken together the mean costs of endoscopic and surgical treatments were subsumed as the casemix alternativedetermination of effectivenessthe r0 resection rate was defined as the efficacy parameter to determine cost effectiveness the r0 rate of eftr in the wall resect trial was to determine the efficacy of the therapeutic alternatives to eftr a selective literature review was performed in pubmed and cochrane databases identifying the largest studies comparing resection techniques and r0 rates the respective rates regarding ser found in the literature were for emr and for esd9 for the surgical oncological resection treatment as the gold standard a r0 resection rate was assumed for the tem a rate of had been reported10in order to compare all ser methods emresd all surgical resection methods laparoscopic surgerytem and all alternative methods endoscopic and surgical kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0ctable alternative treatment strategies to eftr with their respective efficacy based on literature review and calculationtreatmentefficacy n n180surgical oncological resection laparoscopictememresdsurgical treatment laparoscopic and temser emresdcasemix alternative assumed arezzo et al fujiya et al arezzo et al calculated calculated calculatedthe combined effectiveness of surgical treatment ser and casemix alternative was calculated by multiplication of the number of patients in each modality eg emr cases for ser with the respective r0 resection rate as the first step in the second step this result would be summed up to the result of the other modalities eg esdemr result for the ser methods and divided by the number of patients in this group of resection method eg patients in the ser group overall efficacy of surgical treatment and casemix alternative was performed in the same mannereftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgerycasemix alternative with the eftr procedure a combined effectiveness of each treatment group was calculated this was done by multiplication of the number of patients in each modality eg emr cases with the respective r0 resection rate as the first step in the second step this result would be summed up to the result of the other modalities eg esdemr result for the ser methods and divided by the number of patients in this group of resection method eg patients in the ser group using this approach the overall efficacy in the ser group was calculated as overall efficacy of surgical treatment and casemix alternative was performed in the same manner and was calculated as and respectively the respective r0 rates are shown in table calculation of costeffectivenessfor assessment of cost effectiveness the average cost effectiveness ratio acer and the incremental cost effectiveness ratio icer were calculated acer expresses the mean costs for the investigated outcome11 in our study acer describes the mean costs per successful r0 resection in the different treatment modalitiesacer is calculated with the following computational formula acer mean costseffect open accessicer expresses the additional costs of a treatment alternative for improvement in the investigated outcome12 in our study these are the incremental costs for the alternative treatment to eftr required to achieve an r0 resectionicer is calculated with the following computational formula icer mean costs interventionmean costs controleffect intervention effect control the mean costs were the total costs of the respective treatment modality divided by the number of patients in each group for the calculation of cost effectiveness the ser methods emr and esd as well as the surgical resection methods laparoscopic resection and tem were taken together furthermore cost effectiveness was calculated for the casemix alternative to compare eftr with all alternativesresultscomparative study armendoscopist surveyfrom patients of the study cohort responses were included for further analysis in one patient the investigator recommended solely thermal ablation as alternative treatment of choice thus the primary endpoint r0 resection could not be evaluated from the remaining patients the endoscopists recommended surgical treatment in of of cases thereof of were laparoscopic resections and of tem in of of cases an endoscopic resection was proposed thereof of were emr and of were esdcosts from the perspective of the care providercosts per case were derived from the drg report browser which represent costs of the respective treatment alternative for the year the costs for the eftr treatment were derived from university hospital freiburg and represent the mean costs from years to mid2019 the mean cost for eftr was ¬ the cost per surgical treatment laparoscopic surgery and tem was ¬ and for ser ¬ all alternative treatment strategies casemix alternative op laparscopic surgery tem esd and emr were calculated as ¬ per case the results are shown in figure costs from the perspective of the thirdpartyaccording to the german drg system reimbursement for eftr is ¬ for surgical treatment ¬ was calculated the cost per case for ser is ¬ the cost for the casemix alternative is ¬ the results are shown in figure costeffectiveness analysis care provider viewpointaverage costeffectiveness ratiothe mean cost per r0 resection is ¬ in the eftr group and ¬ in the surgical group in the ser group the cost per r0 resection is ¬ in the casemix alternative group including all treatment kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access figure case costs ¬ for the different treatment modalities are shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgeryfigure incremental cost effectiveness ratio for the different treatment modalities compared with eftr is shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgeryalternatives except eftr the mean cost per r0 resection is ¬ the results are shown in figure the casemix alternative ¬ the results are shown in figure incremental costeffectiveness ratioin comparison with eftr the incremental cost for an additional r0 resection is ¬ if ser is performed the cost for the surgical approach is ¬ and for figure average cost effectiveness ratio ¬ for the different treatment modalities is shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgerycosteffectiveness analysis health insurance reimbursement viewpointaverage costeffectiveness ratiofrom the perspective of the health insurance the cost per r0 resection is ¬ in the eftr group in the ser group the cost is ¬ and in the surgical treatment group ¬ in the casemix alternative the cost per r0 resection is ¬ the results are shown in figure incremental costeffectiveness ratiothe icer of ser in comparison with eftr is ¬ the surgical approach costs an additional ¬ for the casemix alternative ¬ is necessary for an additional r0 resection the results are shown in figure discussionwith technical endoscopic progress patient care has constantly improved over the years however as with any technical innovation this is associated with higher costs therefore the efficacy of new methods and devices needs to be evaluated in relation to their costs13 to our knowledge this is the first cost effectiveness analysis cea for eftr our results demonstrate that eftr for difficult to treat lesions in the colorectum is cost effective in comparison with ser as well as surgical therapy furthermore the results are consistent when analysed from the perspective of the care provider as well as of the payerkuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0cfor our analysis a simulated control arm was created this was necessary as to date no randomised controlled trial rct investigating eftr versus alternative treatments has been published in our survey endoscopists proposed surgical treatment as the likely alternative to eftr in the majority of cases as opposed to ser via emr or esd all lesions within the wall resect trial were difficult to resect lesions eg non lifting adenomas exhibiting a high risk of perforation or incomplete resection when treated with ser therefore it may be surprising that ser was suggested in of cases however the suggestions were made by expert endoscopists who might have decided towards an advanced endoscopic procedure more generouslyregarding the costs for each treatment modality it was not surprising that the cost of eftr is above ser ¬ vs ¬ this is due to the cost of the device in germany ¬ plus value added tax however the cost of eftr was roughly one third of the cost of surgery ¬ vs ¬ this reflects the minimally invasive nature of eftr compared with laparoscopic or open surgical operationswhile costs for endoscopic resection and surgical therapy were taken from official and certified tools web grouper and drg report browser the factual costs of the eftr procedure for the year that are determined in a representative cross section of hospitals have not been published yet by inek the administrator of the drg system reimbursement of the procedure changed in thus these data should have been used for calculation to overcome this problem the mean case costs for eftr per case in our home institution university hospital of freiburg germany in the time between and were used as a surrogate in an economic analysis of the cost of eftr in germany presented at the annual conference of the german society for digestive diseases obtained from different endoscopic centres reported ¬ per case as this is only above our number and therefore in the same range our calculated ¬ seems to be a realistic number14in our analysis we chose the r0 rate as a means to detemine effectiveness as this is the most objective parameter to assess curative resection and treatment success furthermore the r0 rate can be compared with the treatment alternatives of eftr as high quality meta analyses and therapeutic success rates exist for those procedures10 the r0 rate for surgical colonic resection was assumed to be however the patient cohorts of these studies are not equal the wall resect study included only difficult to resect lesions mainly non lifting while the studies mentioned above included primarily treatment naive lesions larger studies on ser on non lifting lesions do not exist hence it is reasonable to assume that in these indications real r0 rates of ser would be lower and therefore cost effectiveness would be even worsefor measuring cost effectiveness acer and icer were determined the analysis was performed both open accessfrom the perspective of the care provider hospital as well as the reimbursement authority health insurance acer expresses the mean cost per r0 resection for both investigated perspectives our results reveal that costs are much lower for eftr compared with the surgical alternative although the effectiveness of the surgical approach in terms of radicality can be considered to be higher eftr is cost effective an r0 resection by eftr leads to nearly reduction in costs for the care provider ¬¬ and for the health insurance ¬¬ compared with ser eftr leads to marginally higher costs per r0 resection as explained above comparing eftr with ser has limitations as the investigated difficult lesions in the wall resect study are not well studied for emr and esd however in comparison with all treatment alternatives casemix alternative we calculated and reduction in costs similar to the surgical alternatives figure icer expresses the additional costs for an additional increase in the designated outcome in our analysis it expresses the additional costs that are necessary for an additional r0 resection as shown in figure all alternatives to eftr result in additional costs while ser results in a modest increase ¬ and ¬ additional ¬ and ¬ per r0 resection are required in the surgical group in the casemix alternative group additional costs were ¬ and ¬ respectivelyan absolute threshold at which an icer is thought to be cost effective does not exist16 in the literature the willingness to pay threshold ranges from to and is highly subjective to the investigated outcome and the healthcare system for which the cea is made17 for our analysis we assume that a more invasive treatment that produces at least ¬ more costs for an additional r0 resection cannot be regarded as being cost effectivefor our analysis we did not include costs of follow up endoscopies or further treatment arising from recurrency or from adverse events this was done due to the following reasons first reliable recurrence rates and long term follow up after eftr do not exist follow up in the wall resect trial was only weeks second the lesions of our patient cohort were heterogeneous including adenomas carcinomas and neuroendocrine tumours with different biological features and recurrent rates third treatment of recurrent lesions is not standardised and ranges from re eftr to snare polypectomy to removal with a biopsy forceps leading to highly variable costs fourth management of severe complications and consecutive morbidity differs in every patient and depends on severity of complication patients comorbidities and local expertise we do not have reliable data on costs for such treatment and a hypothetical model would have been highly speculative moreover in the wall resect trial of patients required consecutive surgery due to complications this rate is slightly kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access higher but still grossly comparable with the complication rates of emr and esd on the other hand complications after surgical resection eg anastomotic leakage are much more frequent up to and usually lead to higher morbidity hence even if costs related to complications were added icer is still likely to favour eftr compared with the group of treatment alternativesit is difficult to compare our results with other ceas as this is the first one for this indication the only previous cea on ser compared emr and esd in laterally spreading lesions irrespective of location or lifting sign in most analyses as in the study by bahin and colleagues19 a decision tree model was created to compare different outcome scenarios after each treatment path was filled with probabilities of occurrence costs per predefined outcome were calculated a potential bias of this approach is that the data for the probabilities of occurrence which influence the costs most are taken totally or at least in part from different studies19 22this harbours the risk of resulting in a very heterogeneous study population with uncontrolled confounders this risk can be minimised by deriving data from rcts with well balanced patient cohorts as recently published17 for our analysis we used a different approach than a decision tree factual variables and outcome data derived from the only prospective study on eftr treatment and not from assumptions the simulation of the control arm had to be performed due to the lack of rcts in this setting the strength of our study is that the very same clinician who actually performed the respective eftr could review the different lesions and decide on a solid basis which treatment alternative he or she would have used instead of eftr in our view this approach reflects the clinical situation more precisely than a decision tree modelin most ceas the costs per quality adjusted life years are calculated and taken for healthcare decisions neither survival nor quality of life measurements were part of the wall resect trial in line with most of the recently published cea we calculated costs per defined outcome as the primary endpoint17 our study has several limitations first the comparison arm of the study is based on simulation so there is always a risk of a bias second our analysis is specific to the german healthcare system and may therefore not be fully comparable with different healthcare systems in the world third the estimated r0 rate for the ser methods is very low and likely due to the piecemeal resection in the respective study if efficacy would have been measured as freedom of recurrence efficacy would be higher as proven in the australian colonic endoscopic study24 nonetheless we used the published r0 rate because of the possibility to match this with the endpoint of the wall resect study furthermore as described above an endpoint such as freedom of recurrence cannot be determined reliably as such data do not exist for eftr fourth costs of complications and follow up were not included this is mainly due to lack of an rct and the short follow up period in an ideal cea all treatment related and hospital stay related costs would have been calculatedin our data indicate that eftr for difficult to treat lesions of the colorectum is cost effective compared with surgical and endoscopic treatment alternatives the results are consistent both from a care provider as well as from a third party payer perspective rcts and long term follow up are needed to further assess the cost effectiveness of eftrauthor affiliations1department of medicine ii medical center university of freiburg faculty of medicine university of freiburg freiburg germany2department of gastroenterology klinikum ludwigsburg ludwigsburg baden w¼rttemberg germany3department of gastroenterology evangelisches krankenhaus d¼sseldorf dusseldorf nordrhein westfalen germany4department of internal medicine and gastroenterology elisabeth hospital essen nordrhein westfalen germany5department of medicine ii interventional and experimental endoscopy inexen university hospital wurzburg wurzburg bayern germany6department of gastroenterology university hospital augsburg augsburg bayern germany7department of gastroenterology university hospital ulm ulm baden w¼rttemberg germany8department of gastroenterology klinikum dortmund dortmund nordrhein westfalen germany9department of gastroenterology helios klinikum krefeld krefeld nordrhein westfalen germany10department of gastroenterologyoncology klinikum sindelfingen b¶blingen sindelfingen baden w¼rttemberg germanycontributors as and kc invented and planned the present study and also the underlying wall resect study as assisted with data acquisition and analysis and revised the manuscript jb was responsible for data research and acquisition ak was responsible for data analysis and writing the manuscript kc tb bs am hm hn da mb ap mf tf mg and rt took part in the online survey to create the simulation comparison arm of the study furthermore they carefully read and revised the manuscriptfunding 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 as and kc received lecture fees and study grants from ovesco endoscopy t¼bingen germany ak jb tb bs am hm hn da mb ap mf tf mg and rt have no conflicts of interest or financial ties to disclosepatient consent for publication not requiredethics approval the wall resect study was approved by the ethical board on january the study protocol conforms to the ethical guidelines of the declaration of helsinki as reflected in a prior approval by the institution's human research committee for the present study an additional approval by the institutional review board was not necessary since no additional personal data were collectedprovenance and peer review not commissioned externally peer revieweddata availability statement all data relevant to the study are included in the or uploaded as supplementary information data were derived from the wall resect trial nct02362126open access this is an open access distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial see a0http creativecommons org licenses by nc orcid idarmin a0kuellmer http orcid org kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0creferences who colorectal cancer fact sheet the global cancer observatory available http gco iarc fr today data factsheets cancers 10_ 8_ colorectum fact sheet pdf [accessed sep ] zauber ag winawer sj o'brien mj et a0al colonoscopic polypectomy and long term prevention of colorectal cancer deaths n engl j med hong sn byeon js lee b i et a0al prediction model and risk score for perforation in patients undergoing colorectal endoscopic submucosal dissection gastrointest endosc org mizushima t kato m iwanaga i et a0al technical difficulty according to location and risk factors for perforation in endoscopic submucosal dissection of colorectal tumors surg endosc agapov m dvoinikova e factors predicting clinical outcomes of endoscopic submucosal dissection in the rectum and sigmoid colon during the learning curve endosc int open 20142e235 baum p diers j lichthardt s et a0al sterblichkeit und komplikationen nach viszeralchirurgischen operationen dtsch arztebl int schmidt a beyna t schumacher b et a0al colonoscopic full thickness resection using an over the scope device a prospective multicentre study in various indications gut aepli p criblez d baumeler s et a0al endoscopic full | Colon_Cancer |
during the last decade green synthesized cerium oxide nanops ceo2 nps attracted remarkable interest in various fields of science and technology this review explores the vast array of biological resources such as plants microbes and other biological products being used in synthesis of ceo2 nps it also discusses their biosynthetic mechanism current understandings and trends in the green synthesis of ceo2 nps novel therapies based on green synthesized ceo2 nps are illustrated in particular their antimicrobial potential along with attempts of their mechanistic elucidation overall the main objective of this review is to provide a rational insight of the major accomplishments of ceo2 nps as novel therapeutics agents for a wide range of microbial pathogens and combating other diseases keywords nanotechnology green synthesis cerium oxide nanops antimicrobial infections biomedicalintroductionnanotechnology has got a remarkable interest in every field of science and technology and is presently considered among one of the leading research avenues it has a multitude of applications in the field of electronics imaging industry and healthcare14 mostly in healthcare it has been exploited in diseases diagnostics treatment delivery and formulations of novel drugs14 it exploits nano size structures with size ranges from nm known as nanop nps these nano scale entities have unique physiochemical properties and have been utilized in various fields of physics biology and chemistry5among other nps cerium oxide ceo2 nps have been mostly exploited due to their unique surface chemistry high stability and biocompatibility67 it is mostly used in the fabrication of sensors cells catalysis therapeutics agents drug delivery careers and antiparasitic ointments figure presently ceo2 nps is mostly synthesized via two methods such as physical and chemical79 however these methods utilize toxic reducing solvents posing several threats to the biodiversity and ecosystem moreover the nps obtained with such approaches are toxic and unstable making them less efficient910 thus recently a safe less toxic method has been used by researchers known as green synthesis this method utilizes various biological resources such as plants microbes or any other biological derivative1115 these biological extracts have a rich source of phytochemicals international of nanomedicine nadeem this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomterms php and incorporate the creative commons attribution non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0cnadeem dovepresslatter is extensively utilized for its biomedical pharmacological and food applications due to their safe and biocompatible nature9 moreover features like high yield everlasting stability and better morphologies can be obtained using a greener approach79green synthesis from plantsgreen syntheses of ceo2 nps have been reported using plant extracts microbial and other biological derivatives plants in this regard have been the most efficient source due to their abundance safe nature and rich source of reducing and stabilizing agents2629 various parts of plants such as leaves flower and stem have been used for the synthesis of ceo2 nps163031 till date the majority of green synthesis studies have been conducted on leaves extracts as it is a rich source of metabolites11163233 a broad variety of metabolitesphytochemicals in plant extracts such as ketones carboxylic acids phenols and ascorbic acid are used as reduction and stabilizing agents figure plants based ceo2 nps are produced through a simple approach in which bulk metal salt is mixed with the extract and the reaction completes in minutes to a few hours in ordinary lab conditions282934 the metallic salt solution is reduced into respective nanops via the phytochemicals whose synthesis is confirmed firstly through color change from colorless to yellowish brownish or whitish and then characterized through various spectroscopic and imaging techniques162935leaf extract of moringa oleifera l was used to synthesize ceo2 nps with spherical morphologies and size of nm the synthesized nps showed potential antimicrobial and wound healing properties36 gloriosa superba leaf extract was used as a reducing and stabilizing agent in synthesis of ceo2 nps and has shown potential antibacterial properties37 hibiscus sabdariffa natural extract yielded crystalline ceo2 with a diameter of nm30 spherical shaped nanops of size nm synthesized from gel extract of medicinally important plant aloe barbadensis38 the resultant ceo2 nps showed high antioxidant potential green synthesis of ceo2 nanops was demonstrated using jatropha curcus leaf extract and a monodispersed shape of nm10 spherical shaped cerium oxide nanops are synthesized using leaf extract of oleo europaea with a size of nm having high antimicrobial activity against both gramnegative and positive strains of bacteria16 origanum majorana extracts were used to synthesize ceo2 nps having pseudo photocatalytic activity having high figure general applications of ceo2 nanopssuch asketones amines enzymes and phenols which are believed to be responsible for the reduction and stabilization of bulk salts into respective nanops nps1619to date various applications of green synthesized ceo2 nps have been reported such as antimicrobial anti cancer antilarvicidal photocatalysis and antioxidant therapies162022 among other biomedical applications the antimicrobial potential is certainly the most exploited previously it has been reported that ceo2 nps to display their antimicrobial actions through various mechanisms9 but mostly ceo2 nps kill microbes via triggering the production of an excess of reactive oxygen species in cells916 however further studies need to be conducted to fully elucidate the complete mechanism of action here in this review we aim to focus on the following topics arrays of biological resources have been exploited to date for the synthesis of ceo2 nps moreover the synthesis mechanisms along their biomedical applications are discussed with special emphasis on the antimicrobial activitysynthesis of ceo2 npsnanops are synthesized through various physicochemical methods5 however both methods require toxic solvents high temperature and pressure which pose threats to the environment2325 moreover higher cost laborious downstream processing lesser biocompatibility instability and low yield make them further inefficient710 there is a growing need to fabricate nanostructures which have the potential to solve these problems59 presently researchers have exploited the green method to overcome all these challenges5 for instance plants microbes and other biological products have been used as reducing and or stabilizing agents in the fabrication of ecofriendly nps25 ceo2 nps have also been synthesized using various physical chemical and biological methods9 the submit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem figure biosynthesis reduction stabilization and characterization of ceo2 nanopsspherical shape nm ftir confirmed that the reduction is attributed to the presence of different phenolic and flavonoids compounds in the extract18 ceo2 was synthesized using rubia cordifolia leaf fusions spectroscopic and microscopic analysis revealed hexagonal shaped nps having a size of nm the biogenic ceo2 nps also showed excellent anticancer potential33 nano rod size ranges from nm ceo2 nps resulted when pedalium murex l was added to the aqueous solution of salt at room temperature having high antibacterial activity32 china rose petal was used as a robust bio template for the facile fabrication of novel ceria nano sheet with a size of about nm39 the deviation in size and morphology noticed among the reported studies might be due to the different influence of reaction temperature ph time concentration of salt precursor or plants extracts and part of the plant being used13272840 moreover plants based ceo2 nps showed excellent stability at diverse conditions for instance green mediated ceria nps remain stable at liquid solution changes were observed1336 similarly biogenic ceo2 nps also showed high thermal stability at high temperature and remained stable for a longer period of time which indicates their long durability and everlasting stability27283341 until now various plants have been used in the biogenic synthesis of ceo2 nps and are shown in table physiochemical and no green synthesis from microbesmicrobes also have an intrinsic potential to synthesize nanops as they are a rich source of secondary metabolites23 among other nanops ceo2 nps with various shapes and sizes have been synthesized in recent years from microbes table green synthesis of ceo2 from microbial species is a simple reliable costeffective and ecofriendly approach42 microbial metabolites such as enzymes proteins and heterocyclic derivatives play a crucial role in reducing and stabilizing of ceo2 bulk salt into respective nps4243 moreover microbiogenic ceo2 nps exhibited improved stability water dispensability and showed high fluorescent properties and were less agglomerated43aspergillus niger extract yielded cubic fluorite nps with spherical morphology and an average size of nm ftir analysis revealed the presence of an hydroxyl group carboxylic group and phenol group which are supposed to be involved in the reduction of nps21 curvularia lunata extract has also been used to synthesize spherical shaped ceo2 nps with size ranges from nm color change from white to yellow brown indicated initial reaction the nps were tested against microbial pathogens and showed excellent antibacterial potential spherical shaped ceo2 nps of size ranges from nm were made using fusarium solani extract which showed effective growth inhibition and biofilm formation of pathogenic bacterial strains42 shadab ali khan observed the biosynthesis of spherical shaped nm ceo2 nps by using a thermophilic fungus humicola capping agent43 the resultant nps were characterized by uv xps pl spectroscopy tem ftir and xrd moreover these nps showed excellent potential in treatment of neurodegenerative disorders such as alzheimers and parkinsons diseases bacterial extract has also been exploited in the fabrication of ceo2 nps for instance bacillus shaped nps with an average size of nm the bacterial mediated nps also showed excellent antioxidant potential in vitro44 despite all these applications the microbial route of synthesis has certain shortcomings such as the high probability of pathogenicity laborious culturing and contamination issues however it offers a lot of promise in the field of nanotechnology and could become a leading avenue in subtilis extract yielded spherical international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepresstable ceo2 nanops made from various plants speciesplant namepartcharacterizationshapesize nmftir groupoh ceoh hoh ocoleafxrd xps tem ftir uv vissphericalflower hrtem sem xrd xps eds ftirceohleafleafleafsem xrd ftir tgasphericalxrd sem tem uv vis ftir tgasphericaluvvis psa ftir xrd xps hrtemrtesphericalco oh hhoh ch nooh ceoseedxrd uvvis ftir fesem tgasphericalceo oceo ch co []acalypha indicaleafxrd sem tem eds ftiraloe barbadensisleafxrd tem ftir psasphericalspherical oh ceoh ceocech co ch cf ch cclaloe veraleafftir xps hrtemsphericalch ccrubia cordifolialeafuv vis xrd xps sem ftir edaxhexagonalprosopis farctaaerialuvvis pxrd tem fesem ftirsphericalchina rosepetalfesem fetem afm xrd xpsnanosheetcentella asiaticauvvis dls sem hrtem edssphericaloh ceooh ceo and no__ walnutshellleafxrd sem tem eds ftiruvvis xrd xps fesem tem hrtem eds uvdrs ftirsphericalsphericalceo and ohceoc oh chstempxrd sem tem ftir plflakyoh coo and chs nogloriosa superbahibiscus sabdariffaoliveoleo europaeaprosopis jujliflorasalvia macrosiphon boissazadirachta indicaeuphorbia tirucallipetroselinum crispummoringa oleiferaref[][][][][][][][][][][][][][][][][][][][]lemon grassgrassxrd pl tem saedleucas asperaleafpxrd sem uvvis tem saedmicrosphereuvvis ir xrd sem temsphericalpeeluv vis ftir xrd hrtemspherical watermelonfruit juicecarrageenanpxrd ftir uvvis semirregularpxrd ftir fesem uvvis and tga dtaspherical___ceo ceoceceo co co ch ohso3 ceo hoh co []continuedsubmit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem table continued plant namepartcharacterizationshapeleafxrd fesem tem ftirsphericalxrd fesem eds vsmsphericalsize nmftir group__ceratonia siliquastevia rebaudianasalvadora persicamorus nigraannona muricatajusticia adhatodas nopxrd ftir uvvis tem fesem edsspherical0h ch cc ceo co cfruitfruittem xrd uvvisxrd ftir uvdrs fesemirregular_ ___leafxrd sem tem ftir uvdrssticklikeoh ch co no cc co cncjatropha curcusleafxrd tem uvvismonodispersed _origanum majoranapedalium murexelaeagnus angustifoliaeuphorbia amygdaloidesleaftem fesem xrd ftirsphericaloh ch coleafxrd ftir uvvis drs semnanorodoh ch co co cn cn ceoleafxrd sem tem ftirsphericalch cotem sem xrd uvvissphericalorangepeelxrd tem ftir uvviscubic structurecoh or corpiper betleleafxrd ftir sem eds xps temnh no cnref[][][][][][][][][][][][][]nanomedicine but is yet to be explored particularly these microbial based nps can be used in designing novel fertilizers fabricating sterile surfaces polymers and medical accessories moreover these biogenic nps can also be exploited in disease management drug synthesis and deliverytable ceo2 nanops synthesized from various fungus speciess nomicrobe namecurvularia lunatacharacterizationshapesize nmftir grouptgdta xrd raman pl ftir uvvis and temspherical_humicola spuvvis xps pls tem ftir xrdsphericalceo ceocefusarium solaniftir pl tgdta fesem xrd edax tem xps saed clsmsphericaloh cn cocaspergillus nigeruvvis ftir xps xrd tgdta pl temsphericalhoh oco ce oref[][][][]international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepressgreen synthesis from biological productsapart from the synthesis of nanomaterial from eukaryotes and prokaryotes anisms nps have also synthesized from biological derivatives23 they also play a crucial role in the reduction and stabilization of nps2325 in contrast to plants and the microbial approach bioproduct based ceo2 are much safer scalable and have shown excellent biocompatibility4547 for instance egg white protein was used in order to synthesize ceo2 nps having size ranges from nm with spherical morphologies48 these nps were characterized and confirmed by uvs ftir tgadta and pxrd ftir analysis revealed that the phenol ether hydroxyl and amide groups were responsible for the reduction of these nps it also showed a good in vitro cytotoxicity effect towards human periodontal fibroblasts cells agarose is a natural matrix and has been used as a stabilizing and capping agent for ceo2 nps nps obtained have spherical morphologies with a diameter of nm nps were characterized using various methods including uv fesem ftir tgadta pxrd and tgadta techniques as revealed by ftir analysis it was found that the hydroxyl ether phenol and amide groups were involved in biosynthesis45 starch has also been exploited as a novel source for the synthesis of nanoceria with the results revealing spherical shape nps with a diameter of nm12spherical shaped ceo2 nps with a size of nm were synthesized from dextran the resultant nps exhibited strong anticancer potential46 gum tragacanth reported by darroudi 49 was used in the biosynthesis of ceo2 nps these nps were monodispersed shape with an average size range from nm the ceo2nps exhibited very low cytotoxic effects on neuro2a cell lines making them suitable candidates for various biomedical and pharmacological applications49 some other biological products which have been used for synthesis of ceo2 nps are listed in table despite their biological applications these biogenic nps could be used as a promising candidate in diseases treatment drug delivery and packing food some other products have also been explored for the synthesis of ceo2 nps which are shown in table biological activity of greenmediated cerium oxide nanopsantimicrobial activity of greenmediated cerium oxide nanopsin the last few years nanotechnologybased therapies have been exploited in disease diagnostics treatment and table biomediated ceo2 nanops from different sources of biological productss nonamecharacterizationshapeegg proteinuvvis fesem ftir tgadta pxrdsphericalsize nmhoneyuvvis fesem ftir tgadta eds pxrdsphericalagaroseuvvis fesem ftir tgadta pxrdsphericalstarchdextranpolyethylene glycoluvvis pxrd temtem dls xps uvvishrtem tem uvvis slsdlschitosanxrd hrtem ftir tgadta uvvispectindls fesem edss xrd ftir nmr pl fesem edss uvvissphericalsphericalsphericalsphericalspherical ¤ ftir groupoh ceo ceoce ch nooh ceo ceoce ceoc nooh ceo ceoce no____oh ch co och3 coc no ceotannic acidftir xps xrd hrtem uvvisiblepolycrystalline _ref[][][][][][][][][]submit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem figure schematic representation of antibacterial activity of ceo2 nanops cell wall disruption cell membrane disintegration free radicals productions loss of protein peptides dna fragmentation vital enzymes inhibition loss of cellular fluids and disruption in electron transportformulations of novel drugs1 for instance the antimicrobial potential of nps has been mostly exploited and has showed substantial outcomes2324 presently ceo2 nps have attracted great interest as an antimicrobial agent in particular against bacterial pathogens151650 the exact mechanism of killing microbes is yet not clearly elucidated however it is proposed that ceo2 nps mostly kill microbes via a massive production of reactive oxygen species ros in cells as shown in figure the bactericidal potential of ceo2 nps is attributed to strong electrostatic properties distinctive morphologies small size and low band energy1652 due to strong electrostatic potential ceo2 nps interact with membrane proteins thiols groups which results in protein denaturation membrane impermeability eventually leads to microbial death4253 figure exposure to ceo2 nps kills microbes via membrane collapse by attachment with mesosomes malfunctioning of cellular compartments and bio anic molecules which ultimately lead to abnormal metabolism and physiology1642 similarly green mediated nps killpathogens in a similar fashion and various biological species have been exploited and tested against a wide variety of microbes1316 table however biogenic ceo2 nps unique morphologies small size and biocompatible nature were found to be more effcient and have the potential to range of pathogenic bacterial species1113151642 moreover it also has the potential to kill both grampositive and gramnegative bacteria but due to structural complexity of gramnegative bacterias membranes it is more sensitive against grampositive species1321374754 in antimicrobial in electrostatics between nps and the bacterial wall plant species and wall compositiondifference to differences treat a wide activity the is due international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepresstable various microbes tested against biogenic ceo2 nanopss nosourcemicrobes testedolea europaeafungus aspergillus flavus fusarium solani and aspergillus niger bacterial sps staphylococcus aureus escherichia coli pseudomonas aeruginosa and klebsiella pneumoniamoringa oleiferas aureus and e colicurvularia lunatastaphylococcus aureus streptococcus pneumoniae and bacillus subtilis pseudomonas aeruginosa proteus vulgaris and klebsiella pneumoniaeleucas asperaklebsiella aerogenes escherichia coli pseudomonas desmolyticum and staphylococcus aureusacalypha indicaescherichia coli and staphylococcus aureusannona muricataenterococcusfaecalis staphylococcus aureus klebsiella pneumonia and escherichia coligloriosa superbastaphylococcus aureus and streptococcus pneumonia ecoli proteus vulgaris klebsiella pneumonia shigella dysenteriae and pseudomonas aeruginosaaspergillus nigerstreptococcus pneumoniae bacillus subtilis proteus vulgaris and escherichia colifusarium solanistaphylococcus aureus pseudomonas aeruginosa escherichia coli and klebsiella pneumoniajusticia adhatodastaphylococcus aureus and escherichia colieuphorbia amygdaloidesp acidilacticipectine coli and b subtilisref[][][][][][][][][][][][]resistance to confer due to rapid evolution of the bacterial genome bacteria have evolved to antimicrobial agents5556 thus in quest of a new treatment biogenic ceo2 nps has shown promising results in treating multi drug resistance bacteria and could be a promising candidate against such refractory pathogenesis26 ceo2 nps along other conjugates have been amalgamated with various anic and inanic hybrids to enhance the antimicrobial response1746 similarly biomediated ceo2 nps kills fungi by producing a mass number of free radicals and ros which causes distorted structure and physiology and leads to fungus death16 however a few studies have only been conducted on fungi despite the increasing knowledge on the antimicrobial activity of ceo2 nps much remains unknown about their exact mechanism of encountering bacteria toxicity in vivo studies and environmental concerns which needs to be addressed moreover the low band energy potential of ceo2 nps could be used in fabricating sterile surfaces at hospital or lab settings and will diminish nosocomial and other acquired infectionsother potential biomedical applicationsbeside antimicrobial therapies ceo2 nps have also been used in management of other ailments3557 for instance siliqua showed high biogenic ceo2 nps have been mostly used in treatment of various cancers such as osteosarcoma colon cervical and breast cancers1820274652 results indicated that these nps exhibited strong anticancer potential and could be used as a chemotherapeutic agent thanks to their minimal toxicity capacity to induce apoptosis andor necrosis in cancer cells46 ceo2 nps synthesized from origanum majorana and ceratonia antioxidant activity183157 results showed higher expression of antioxidant enzymes which in turn eradicated free radicals and improved cellular functions31 furthermore antioxidant potential was higher when compared to commercial synthetic antioxidants18 ceo2 nps synthesized from fruit extract of morus nigra exhibited excellent antidiabetic activity on l6 cell lines the treatment was dosage dependent and nps with lesser size resulted in higher uptake of glucose in vitro35 though biogenic ceo2 nps have shown excellent pharmacological potential however the mechanism of action minimum inhibitory concentration and best possible delivery system should need to be determined moreover cytotoxicity and genotoxicity should be tested in vivo models to evaluate the compatibility in both in vivo and in vitro modelssubmit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem to their unique conclusions and future prospectsin this paper we have reviewed the recent trends and understandings of biogenic ceo2 nps and their pharmacological applications various sources such as plants microbes and other biological products have been discussed with the mechanism of synthesis and their biomedical applications due surface morphologies crystal small nature and biocompatible nature biogenic ceo2 nps have got phenomenal interest in biomedical and other fields for instance it has been used in treating various cancers antimicrobial and antioxidant therapies in particular the green synthesized nanops have shown significant antimicrobial potential against a wide range of bacterial species the mechanism of combating such pathogens have also been elucidated and supposed to be due to the mass production of reactive oxygen species and deactivation of scavenging enzymes the ros impedes the membranes disrupts the cellular compartments and disintegrates the bio anic molecules and hampers the associated functions and ultimately causes death it has also shown promising results against multidrug bacteria and could be a potential antimicrobial agent in future against such refractory pathogens however further studies should conduct in vivo models to reveal the full mechanism alongside any sideeffects moreover it has also shown excellent anticancer and antioxidant potential in vitro setups but their toxicity and dosage are yet unknown which needs to be addressed despite their role in various therapies their mechanism of synthesis needs to be optimized whereas in vivo evaluation as well as toxicity should be further screenedabbreviationsceo2 nps cerium oxide nanops dga differential thermal analysis dsl dynamic light scattering eds energydispersive xray spectroscopy fesem field emission scanning electron microscopy ftir fourier transform infrared spectroscopy hrtem highresolution transmission electron microscopy pl photoluminescence pxrd powder xray diffraction ros reactive oxygen species sls static light scattering tem transmission electron microscopy tga thermal gravimetric analysis uvvis uvvisible spectroscopy xps xray photoelectron spectrometry xrd xray diffractiondisclosurethe authors report no conflicts of interest for this workreferences kubik t boguniakubik k sugisaka m nanotechnology on duty in medical applications curr pharm biotechnol doi1021741389201053167248 bhushan b springer handbook of nanotechnology springer jianrong c yuqing m nongyue h et al nanotechnology and biosensors biotechnol adv doi101016j biotechadv200403004 smith dm simon jk baker jr jr jr applications of nanotechnology for immunology nat rev immunol doi101038nri3488 mohanraj v chen y nanopsa review trop j pharm res das s dowding jm klump ke cerium oxide nanops applications and prospects in nanomedicine nanomedicine doi102217nnm13133 he l su y lanhong j et al recent advances of cerium oxide nanops in synthesis luminescence and biomedical studies a review j rare earths doi101016s1002 walkey c das s seal s catalytic properties and biomedical applications of cerium oxide nanops environ sci doi101039c4en00138a rajeshkumar s naik p synthesis and biomedical applications of cerium oxide nanopsa review biotechnol rep doi101016jbtre201711008 magudieshwaran r ishii j raja kcn et al green and chemical synthesized ceo2 nanops for photocatalytic indoor air pollutant degradation mater lett doi101016jmatlet arunachalam t karpagasundaram m rajarathinam n ultrasound assisted green synthesis of cerium oxide nanops using prosopis juliflora leaf e | Colon_Cancer |
"what clinicians know about the diagnosis treatment and prevention of disease originates from studies mostly done on male cells male mice and men1 historically for multiple reasons including the purported safety of women and their offspring women of childbearing age were excluded from clinical trials as a result medical research and care have been centred on male physiology the assumption was that male and female cells and animals were biologically identical and evidencebased medicine was defined by clinical trials done predominantly in men1 in the us national institutes of health nih mandated the inclusion of women in nihfunded clinical trials but many investigators did not follow this mandate and many of those who did include women did not analyse the results by sex23 minimising the effectiveness of this policy preclinical research and drug development studies have also predominantly used male animal models and cells4 it is not surprising that a us government accountability office report found that eight of the ten prescription drugs withdrawn from the market between and posed greater health risks for women than for men most funding agencies from europe and north america have implemented policies to support and mandate researchers to consider sex and gender at all levels of medical research8 still the field of sexbased biology and medicine is often viewed as a specialised area of interest rather than a central consideration in medical research essential for the success of clinical care and translational science is awareness by clinicians and researchers that the diseases they are treating and studying are characterised by differences between women and men in epidemiology pathophysiology clinical manifestations psychological effects disease progression and response to treatmentthis review explores the role of sex biological constructs and gender social constructs as modifiers of the most common causes of death and morbidity and articulates the genetic biological and environmental determinants that underlie these differences we aim to guide clinicians and researchers to better understand and harness the importance of sex and gender as genetic wwwthelancetcom vol august biological and environmental modifiers of chronic disease ultimately it is a necessary and fundamental step towards precision medicine that will benefit women and mensex as a genetic modifier of biology and diseasesex differences in disease prevalence manifestation and response to treatment are rooted in the genetic differences between men and women genetic sex differences start at conception when the ovum fuses with a sperm cell carrying an x or a y chromosome resulting in an embryo carrying either xx or xy chromosomes this fundamental difference in chromosome complement eg genes outside the testisdetermining sry gene generates ubiquitous sex differences in the molecular makeup of all male and female cells9 first the y chromosome carries genes that exhibit subtle functional differences from their xlinked homologues eg zfy vs zfx and uty vs utx and also carries genes with no homologue at all eg sry in addition in men the x chromosome carries only maternal imprints ie epigenetic modifications made by the parent in generating the sex cellswhich alter the expression of genes in the offspring as women have x chromosomes from both parents they carry maternal and paternal imprints which target a different set of genes random inactivation of one of the x chromosomes in female cells which prevents sex differences in x chromosome gene dosage causes another degree of sex difference in gene expression as some of these xlinked genes escape inactivation in women those genes are often expressed at higher levels in women than in men9 sexspecific gene expression due to genomic search strategy and selection criteriawe searched pubmed for papers published in english between jan and june using sex or gender and the name of the disease of interest as search terms although we tried to cite seminal studies when necessary because of space limitation representative reviews were often selectedlancet diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine and southeast louisiana veterans health care system medical center new orleans la usa prof f mauvaisjarvis md barbra streisand womens heart center cedarssinai smidt heart institute los angeles ca usa prof n bairey merz md national heart lung institute imperial college london london uk prof p j barnes md department of pharmacology and department of neurology college of medicine center for innovation in brain science university of arizona tucson az usa prof r d brinton phd department of medical epidemiology and biostatistics and center for gender medicine karolinska institutet stockholm sweden prof jj carrero phd channing division of network medicine and the division of pulmonary and critical care medicine department of medicine brigham and womens hospital harvard medical school boston ma usa d l demeo md neuroscience institute and department of biology geia state university atlanta ga usa prof g j devries phd department of psychiatry university of colorado school of medicine anschutz medical campus aurora co usa prof c n epperson md division of oncology department of medicine washington university school of medicine st louis mo usa prof r govindan md w harry feinstone department of molecular microbiology and immunology the johns hopkins bloomberg school of public health baltimore md usa prof s l klein phd department of biomedical metabolic and neural sciences university of modena andreview 0creggio emilia azienda ospedalierouniversitaria di modena ospedale civile di baggiovara modena italy prof a lonardo md department of psychiatry department of psychology and department of obstetrics gynecology university of illinois at chicago chicago il usa prof p m maki phd department of neurology mcgovern medical school university of texas health science center houston tx usa prof l d mccullough md berlin institute of gender medicine charituniversittsmedizin berlin berlin germany prof v regitzzagrosek md department of cardiology university hospital z¼rich university of z¼rich switzerland prof v regitzzagrosek center for womens health research divisions of general internal medicine and cardiology university of colorado school of medicine aurora co usaimprinting extends to autosomes as well and these imprinted genes exhibit sexspecific and tissuespecific expression in humans10 thus fundamental sex differences deriving directly from genetic heterogeneity between the x and y chromosome complements and parentoforigin inher itance exist at the molecular level in all human cells these sex differences persist throughout life and are independent of sex hormones figure arguably the greatest source of differences between men and women comes from the y chromosomal sry gene which directs the development of a testis in men the ensuing developmental surge of testicular testosterone permanently masculinises the reproductive tract and the anisation of brain circuits affecting male behaviour at puberty1112 in humans the first surge occurs at the end of the first trimester of pregnancy because it alters cellular gene expression and tissue structure in multiple ans of men via epigenetic mechanisms this testosterone surge is also paramount in programming sex differences in physiology and susceptibility to diseases that will manifest in adulthood after this initial testicular testosterone surge gonadal hormone concentrations remain low until puberty which triggers lasting sex differences in circulating oestrogens and testosterone concentrations after puberty cells with androgen or afemale sexbrandom x chromosomeinactivation and escapexxxxxxxxxxxxxxxxxxxy chromosome complementmale sexsryctesticular testosterone surgefetal testistestosteroneohogenetic diï¬erences of male and female cellsepigenetic programming of male cellsfigure genetic causes of sex differencesa genetic sex differences start with cells carrying either xx or xy chromosome complement eg genes outside the testisdetermining sry gene which generates ubiquitous sex differences in the molecular makeup of all male and female cells b random inactivation of one x chromosome in female cells causes another level of sex differences in gene expression some xlinked genes escape inactivation in female individuals and have a higher expression in female than male individuals c the y chromosomal sry gene directs the development of a testis in male individuals which produces a surge of testicular testosterone at the end of pregnancy the testosterone surge programmes cellular gene expression and tissue structure in multiple ans of male individuals via epigenetic remodelling the combination of these genetic and developmental events programmes sex differences in physiology and susceptibility to diseases that will manifest in adulthoodoestrogen receptors will be affected differ ently in men and women the combination of all genetic and hormonal causes of sex differences aforementioned culminates in two different biological systems in men and women that translate into differences in disease predisposition manifestation and response to treatment therefore sex is an important modifier of physiology and disease via genetic epigenetic and hormonal regulations figure gender as a determinant of patients and doctors behaviour and as a modifier of health disease and medicinegender according to the global health definition refers to the socially constructed norms that impose and determine roles relationships and positional power for all people across their lifetime13 gender interacts with sex the biological and physical characteristics that define women men and those with intersex identities13 gender is not a binary term it includes the understanding that in many people traits of masculinity or femininity coexist and are expressed to different degrees gender attributes are fluid more than two thirds of women and men report genderrelated characteristics traditionally attributed to the opposite sex1415 in transgender people gender identity differs with the sex they were assigned at birth so far transgender people have generally been underrepresented in clinical studies to date although this underrepresentation is changing gender is an equally important variable as biological sex in human health and influences the behaviour of communities clinicians and patients1415 gender roles represent the behavioural norms applied to men and women in society which influence individuals everyday actions expectations and experiences including diet perceived stress smoking and physical activity and affect health and disease susceptibility gender identity describes the fluidity of how a person perceives oneself as a woman or a man which affects feelings and behaviours gender relations refer to how we interact with or are treated by people on the basis of our ascribed gender institutionalised gender reflects the distribution of power between men and women in the political educational and social institutions in society and shapes social norms that define perpetuate and often justify different expectations and opportunities for women and men1617 as such the distribution of genderrelated charac teristics within populations of men and women can influence health differently than biological sex together these gender constructs determine access to health care helpseeking behaviours and individual use of the healthcare system being perceived as a man or a woman triggers different responses from clinicians who might diagnose and suggest interventions differently according to gender as such gender largely determines the use of preventive measures and referral for or acceptance of invasive therapeutic strategies genderrelated behaviours contribute to risk exposure and preventive behaviour in several wwwthelancetcom vol august review 0cdiseases this postulation is well exemplified in the cardiovascular field in which women often underestimate their risk compared with men and seek consultation later than men in the clinic for treatment of myocardial infarction1819 in the genesispraxy prospective study mortality year after an acute coronary event was more strongly associated with gender than with biological sex1415 similarly control of cardio vascular risk factors hypertension diabetes depres sive symptoms was better predicted by gender than by biological sex1415 therefore including a gender dimension in clinical studies and practice will contribute to the understanding of different clinical manifestations and outcomes of diseases in women and men1617 although beyond the scope of this review it is also important to consider that regarding health and disease gender intersect with race or ethnicity and age20 sex and gender are fundamentally and frequently reciprocally interrelated in biology and disease24 sex influences behaviours eg towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and pain and nutritional habits might produce epigenetic modifications that modulate gene expression and biological phenotypes figure summarises how sex and gender are interrelated in biology and diseasesex and gender differences in major chronic diseaseshaving established the importance of sex and gender in disease we will summarise their influences on the most common causes of death and debilitating diseases in the usa as an example figure note that these sex and gender disparities are relevant to other highincome countries as well as lowincome and middleincome countries where the burden of these diseases becomes increasingly like those in highincome countries26 in most diseases efforts to separate the effects of sex and gender are still incomplete so that we just refer to the differences among women and men because current knowledge about pathophysiology diagnosis and treatment of disease is primarily based on men as representative of the human species this review focuses on how women differ from men we discuss some key aspects regarding the dimensions of men in a dedicated sectionheart diseaseepidemiology pathogenesis manifestations and diagnosisheart disease is the leading cause of death in the usa in heart disease accounted for · of all deaths for men and for · of all deaths for women figure ischaemic heart disease and heart failure are major contributors to heart disease mortality and have important sex and gender differences for example heart failure disproportionately contributes to coronary heart disease mortality in women27 potentially due to undiagnosed ischaemic heart disease in women the strength of the association with cardiovascular risk factors differ by sex biological sexsex chromosomesepigeneticeï¬ectssex hormonesohohohobehaviour of patients and doctorssocietygender constructslifestylenutritional habitsexerciseperceived stresssmokingdiseasepathophysiologymanifestationresponse to treatmentdisease perceptionhelpseeking behaviouruse of health caredecision makingtherapeutic responsesex and gender diï¬erences in health disease and medicinefigure interrelation between sex and gender in health diseases and medicinebiological sex causes sex differences through genetic and hormonal influences in disease pathophysiology clinical manifestations and response to treatment sex also influences behaviours towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and nutritional habits produce epigenetic modifications that modulate the expression of biological sex gender constructs determine patients perception of disease helpseeking behaviour and individual use of health care gender constructs also influence decision making and trigger different therapeutic responses from providers biased by gendersystolic blood pressure and hypertension smoking and diabetes are associated with higher hazard ratios for myocardial infarction in women than in men28ischaemic heart disease is the most recognised example for integrating the concept of gender and sex which shape divergent or distinct disease outcomes compared with men women suffering from ischaemic heart disease are older this difference is historically believed to be due to the protection of endogenous oestrogens29 although contemporary study refutes this simplistic explanation30 and associations cannot be inferred to be causation still women suffering from ischaemic heart disease are underdiagnosed3132 and less likely to have a prehospital diagnosis of myocardial infarction33 the reasons for this disparity reflect the intersection between sex and gender first biological sex differences exist in the pathogenesis of ischaemic heart disease whereas men are more likely to be affected by obstructive coronary artery disease of large vessels than women coronary microvascular dysfunction36 leading to chronic myocardial ischaemia without obstructive coronary artery disease has a higher prevalence in women than men37 a metaanalysis reported that following acute myocardial infarction both sexes most often presented with chest pain but compared with men women were more likely to present with pain between the shoulder blades nausea or vomiting and shortness of breathprof j g regensteiner phd department of medicine department of paediatrics and department of neuroscience washington university school of medicine st louis mo usa prof j b rubin md center for the study of sex differences in health aging and disease geetown university washington dc usa prof k sandberg phd division of gastroenterology duke university medical center durham nc usa a suzuki md and durham va medical center durham nc usa a suzukicorrespondence to prof franck mauvaisjarvis diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine new orleans la usa fmauvaistulaneeduwwwthelancetcom vol august review 0cmale individualsother·heart disease·protection might disappear after menopause45 by contrast testosterone induces adverse cardiac remod elling in the male heart44chronic liver disease ·inï¬uenza and pneumonia ·suicide ·alzheimers disease ·type diabetes ·stroke ·cpd ·injuries ·female individualsother·septicaemia ·chronic kidney disease ·inï¬uenza and pneumonia ·type diabetes ·injuries ·alzheimers disease ·stroke ·cpd ·cancer·heart disease·cancer·figure percent distribution of the ten leading causes of death by sex usa adapted from heron25 cpdchronic pulmonary diseasesecond a gender bias appears to be responsible for the absence of recognition of ischaemic heart disease presentation in women38 men and women with ischaemic heart disease who score high on feminine roles and personality traits on questionnaires designed to ascertain aspects of gender are at an increased risk of recurrent ischaemic heart disease independent of female sex39heart failure affects of adults aged years and older and more women than men in absolute numbers4041 heart failure occurs at an older age and with less ischaemic causes in women than in men however hypertension and diabetes predispose older women to heart failure to a greater extent than men heart failure with preserved ejection fraction a form of heart failure with normal systolic function is twice as prevalent in women as in men by contrast heart failure with reduced ejection fraction affects more men than women women who have heart failure with preserved ejection fraction have smaller and stiffer hearts than men inflammation and the resulting fibrosis play a sexspecific role in the pathogenesis of heart failure with preserved ejection fraction under stress premenopausal womens hearts develop less inflammation resulting in less fibrosis than mens hearts4243 this difference is partially driven by sex hormones as oestrogens produce antiinflammatory actions on endothelial and immune cells and promote cardioprotective effects in premenopausal women44 this response to treatmentcompared with men women suffering from ischaemic heart disease are less likely to receive evidencebased treatment3132 and when suffering from acute myocardial infarction they are less likely to receive reperfusion33 an stelevation myocardial infarction registry revealed that compared with men women exhibit delayed reperfusion leading to higher mortality46 women suffering from acute myocardial infarction treated by male emergency physicians have a higher mortality rate than those treated by female physicians38 additionally male physicians are more effective at treating female patients with acute myocardial infarction when they work with female colleagues and when they have experience in treating female patients38 this treatment disparity between women and men can be corrected by improving emergency recognition of stelevation myocardial infarction in women and acceleration of percutaneous coronary intervention which equilibrates gender mortality47guidelines for the treatment of heart failure are similar for women and men24 however evidence suggests that optimal survival in women occurs with lower doses of β blockers angiotensin receptor blockers and angiotensin converting enzyme inhibitors than in men48 finally fewer women undergo heart transplantation than men although women are more frequently donors suggesting a referral bias could exist41cancersepidemiology pathogenesis manifestations and diagnosiscancers are the second leading cause of death dominated by lung cancer accounting for · of deaths in men and · of deaths in women figure more men develop cancer than women49 with few exceptions eg meningioma thyroid cancer lung cancer in nonsmokers nonreproductive cancers exhibit a male predominance though for some cancers oropharynx larynx oesophagus and bladder the male versus female incidence ratios can be higher than a male predominance in cancers that affect both sexes is evident around the world in all races and at all ages50 survival is also shorter for men than women across multiple cancer types the higher cancer risk in men is partially explained by gender constructs like dietary habits or risk behaviours such as smoking and alcohol consump tion51 it is unlikely to be the only cause after appropriate adjustment for these risk factors adult men still have a higher cancer risk than women52 moreover a similar male bias in incidence and survival is seen in paediatric cancers before puberty and the adoption of highrisk cancerpromoting behaviours eg smoking53the universal male predominance in cancer incidence and differential outcomes argues for a fundamental role wwwthelancetcom vol august review 0cin inutero tumour suppressors of sex in addition to gender in cancer biology sexspecific biology includes genetic differences xx vs xy chromosomes the incomplete xinactivation in female individuals which results in biallelic expression of xencoded female cells54 y chromosomeencoded oncogenes such as the rnabinding motif on y chromosome in male cells55 and the chromatin remodelling effects of testicular testosterone in male cells56 these mechanisms have an influence on several of the hallmarks of cancer57 including metabolism growth regulation58 angiogenesis and immunity which all contribute to cancer predisposition59 a crucial example is the male predisposition to glioblastoma which is the most common form of brain cancer in glioblastoma there is a cellintrinsic predisposition of male astrocytes a subtype of glial cell to malignant transformation60 after puberty sex hormones produce additional epigenetic and acute effects on cells that further influence sex disparities in cancer for example the increased frequency and aggressive phenotype of hepatocellular carcinoma in male individuals has been linked to the stimulatory effects of androgens in male individuals and the protective effects of oestrogens in female individuals61 importantly the biology of cancer is not the same across histological and genetic diagnostic groups or even within single histol ogical subtypes thus the interaction between sex gender and cancer mechanisms cannot be expected to be constant take colon cancer the second leading cause of cancerrelated death for example although women have a lower overall incidence of colon cancer than men they have a higher incidence of rightsided colon cancers which have the worst outcomes62 tumours from women with rightsided colon cancers exhibit a distinct molecular signature of energy metabolism compared with those of women with leftsided colon cancers63 this molecular signature is not observed when comparing tumours from men with rightsided colon cancers to men with leftsided colon cancers thus overall the male predisposition to cancer is probably the consequence of genetic program ming of male cells and the effect of sex hormones after puberty interacting with genderspecific behaviours to establish cancer risksresponse to treatmentin the future cancer prevention and treatment will be improved by sexspecific and genderspecific approaches for example immune checkpoint inhibitors can improve survival for men with advanced melanomas and nonsmallcell lung cancers more than for women64 the molecular subtyping of glioblastomas based on sexspecific transcriptomes has the potential to enhance chemotherapy in a sexspecific manner65 in colon cancer sex differences in xenobiotic metabolism regulatory networks might underlie greater treatment response in women than men and require modification of approaches for men with colon cancer66 other elements of cancer metabolism are also ripe for novel sexspecific targeting in treatment cellular nutrient partitioning is sexually dimorphic so approaches such as ketogenic diets or glutaminase inhibition might be associated with substantial sexspecific responses67 furthermore data from models of development ageing and cancer all indicate that molecular pathways such as the enzyme phosphatidylinositol 3kinase and tumour suppressors such as p53 and retinoblastoma protein are sexually dimorphic and require sexspecific targeting545968chronic pulmonary diseaseepidemiology pathogenesis manifestations and diagnosischronic pulmonary disease is the third leading cause of death for women · of deaths and the fourth for men · figure it is mostly accounted for by chronic obstructive pulmonary disease and to a lesser extent by asthma chronic obstructive pulmonary disease is characterised by irreversible airflow limitation and is associated with previous exposure to smoking or air pollutants women are overrepresented among individuals with chronic obstructive pulmonary disease especially among those with earlyonset disease or those who have never smoked69 women are also twice as likely to have chronic obstructive pulmonary disease with chronic bronchitis and women with severe chronic obstructive pulmonary disease have as much emphysema as men countering the misconception of emphysema as a male form of chronic obstructive pulmonary disease70 the female lung is more susceptible to chronic obstructive pulmonary disease than the male lung and women develop symptoms of the disease at a younger age with less tobacco exposure than men71 the genetic epidemiology of chronic obstructive pulmonary disease copdgene study72 suggests that earlyonset chronic obstructive pulmonary disease might originate in utero in susceptible women from alterations in lung development potentiated by maternal asthma and smoking genetic factors or hormonal influences future studies should focus on the contribution of maternally inherited factors such as mitochondrial and x chromosome genes to understand disease pathogenesis it is important to consider gender constructs as well smoking advertising campaigns targeting women rose in the 1960s and the resulting higher smoking rates influenced womens risk for developing chronic obstructive pulmonary disease73 from a disease severity vantage point chronic obstructive pulmonary disease exacerbation rates are also higher in women than men especially at a younger age74 additionally despite the burden of symptomatology and increased rates of hospitalisations and deaths women with chronic obstructive pulmonary disease are often misdiagnosed and disproportionally suffer from comorbid conditions including anxiety and depression therefore physicians should consider chronic obstructive pulmonary disease in the differential diagnosis of women with pulmonary symptoms regardless of tobacco or pollutant exposure historieswwwthelancetcom vol august review 0casthma characterised by variable airflow obstruction and chronic airway inflammation also affects men and women differently asthma is more prevalent in prepubertal boys than girls regarding asthma both male biological sex lung development and atopy and male gender constructs related to outdoor play and indoor pet exposure are factors contributing to the devel opment of asthma and sex versus gender contributions could be difficult to separate75 from puberty onwards more female than male individuals have asthma with an increased severity in middleaged women and a higher mortality rate76 this phe nomenon could be secondary to gender differences eg symptoms perception or healthseeking behaviours however biological sex plays a crucial role in asthma and sex hormones have a major impact on female asthma symptoms and severity after puberty75 worsening of asthma occurs in women before menstruation and is known as premenstrual asthma premenstrual asthma is more common in women with severe rather than mild asthma obesity rather than normal weight and a long rather than a short duration of asthma77 premenstrual asthma is hypoth esised to be due to a fall in progesterone and patients with a severe disease respond to progestogens78 during pregnancy approximately a third of asthmatic women exhibit a worsening of asthma a third show an improvement and the remainder are unaffected79response to treatmentthe menopausal transition represents a pivotal time of accelerated decline in lung function in women with chronic obstructive pulmonary disease and thus represents a sexspecific window for treatment intensification these observations also suggest that oestrogens protect from chronic obstructive pulmonary disease women exhibit greater expression of m2 over m3 muscarinic receptors and accordingly show greater improvements in lung function than men in response to the muscarinic anticholinergic bronchodilator ipatroprium80 pooled analyses of drug studies also suggest that women experience a greater improvement in quality of life than men after treatment of chronic obstructive pulmonary disease with a β2adrenoreceptor agonist combined with an anti cholinergic drug eg indacaterol and glycopyrronium81unlike chronic obstructiv | Colon_Cancer |
" 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 |
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