Celecoxib in cervical cancer cell lines in vitro inhibition of Hela and Siha.docx
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Celecoxib in cervical cancer cell lines in vitro inhibition of Hela and Siha.docx
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CelecoxibincervicalcancercelllinesinvitroinhibitionofHelaandSiha
CelecoxibincervicalcancercelllinesinvitroinhibitionofHelaandSiha
[Abstract]Objective:
ToinvestigatecyclooxygenaseinhibitorcelecoxiboncelllinesHelaandSihacellcycleandapoptosis.Methods:
Cellculturetechniquesandflowcytometryofcyclooxygenaseinhibitorcelecoxibonhumancervicalcancercelllines(HelaandSiha)cellcycleandapoptosis.Results:
Flowcytometryanalysisshowedthat,withtheconcentrationofcelecoxibincreases,Hela,andSihacellcycleinG0/G1phasecellsincreased,Sphasecellsdecreased,G2~Mphasecellsnosignificantchanges.Withcelecoxib(≥25μmol/L)incubated72hlater,comparedwiththeblankcontrolgroupPIIndexdecreased(P<0.05),apoptosisrateincreased(P<0.05).Flowanalysisdiagramcanbeseenhypodiploidapoptoticpeak.Conclusion:
COXinhibitorcelecoxibcaninhibitinvitrohumancervicalcancercelllinesHelaandSihaproliferationandpromoteapoptosis.
[Keywords:
]cervicalcancer;celecoxib;flowcytometry
AbstractObjective:
Tostudytheinhibitoryeffectsofcelecoxib,aselectivecyclooxygenase-2inhibitor,ontwocervicalcarcinomacelllinesinvitro.Methods:
Theeffectofcellcycleandcellapoptoticrateswereanalyzedbyflowcytometry(FCM).Results:
ByFACS,itwasfoundthatthecellcyclesofHelaandSihacellswereblockedbycelecoxib,withtheincreasingofconcentration:
theG0/G1phaseratioofSiha/Helawereincreased,theSphaseratiowereinhibitedandtheG2~Mphaseratiowerenoteffectedsignificantly.Whenincubatedwithcelecoxib(≥25μmol/L)for72hours,theproliferationindexes(PI)werereduced(P<0.05),whiletheapoptoticrateswereincreased(P<0.05),wecanfindsub-G1summitinFACSgraph.Conclusion:
CelecoxibinhibitedtheproliferationandinducedtheapoptosisofSihaandHelacelllinesinvitro.
Keywords:
Cervicalcancer;Celecoxib;Flowcytometry
Cervicalcanceristhemostcommongynecologicalmalignancy,isacancercausedbysecond-leadingcauseofdeathamongwomen.Inrecentyears,studieshaveshownthatcyclooxygenase(COX),especiallytheCOX-2,withavarietyofepithelialtumorsoftheoccurrenceanddevelopmentofcloseties[1],andfoundthatCOXinhibitorsininhibitingtumoroccurrenceanddevelopmentofplayintheprocessacertainrole,butthecyclooxygenaseinhibitor,andcervicalcanceranditsgenesisanddevelopmentoftherelationshipbetweenthestudyless,soweusecellculturetechniquesandflowcytometrydetectionofselectiveCOX-2inhibitorcelecoxibonthehumancervicalcarcinomacelllines(HelaandSiha)proliferationandapoptosis,inordertofurtherexploretheanti-tumoreffectofCOXinhibitorsmechanismsandclinicalapplicationofaccumulateddata.
1Materialsandmethods
1.1Materials
HumancervicalcarcinomacelllineHelaandSihapurchasedfromXijingHospital,FourthMilitaryMedicalUniversityAffiliatedObstetricsandGynecologyLab.RPMI1640mediumwerepurchasedfromGibcoBRLInc.,fetalcalfserumwerepurchasedfromHangzhouSijiqingbiologicalcompanies,trypsinwerepurchasedfromsigmacompany.CelecoxibwerepurchasedfromShanxiWilkeTechnologyDevelopmentCorporation,anddissolvedin100%DMSO,storedin4℃spare.CellcultureandflowcytometryweretakenattheShanxiMedicalCenterlaboratories.
1.2Methods
1.2.1Cellculture:
Humancervicalcancercelllines(HelaandSiha)usingRPMI1640mediumcontaining10%inactivatedfetalcalfserum,conventionalculturedin37℃,5%CO2,saturatedhumidityincubationbox,with0.25%trypsindigestionandpassage.
1.2.2Afterincubationwithdrugsbyflowcytometryanalysis:
theelectionofthelogarithmicgrowthphasecellsaccordingtotheconcentrationof1*105/mLcultureplateswereinoculatedonsixholesthenextday,afterreplacementofculturemediumwithdifferentconcentrationsofSierraLeonetoCelebrexincubatedsothatthefinalconcentrationswere12.5μmol/L,25μmol/L,50μmol/L,andletthecorrespondingblankcontrolgroupandblankcontrolgroupandthegroupwastodotwobottlesofdifferentconcentrations.Culturedfor72hafterthetrypsindigestion(0.25%),collectingcellsfroma1*106/mLasinglecellsuspensionbyadding4℃pre-cooled70%ethanolfixedcells,PBSrinsingtwotimeswith1mLPBSsolutionre-suspendedcells,Propidiumiodine-based,darkstain,after20minbyflowcytometry,DNAcellcycleanalysissoftwareapplicationtocalculatetheDNAcontentofthesamples,analysisofcellcycleandapoptosis.Cellproliferationtoproliferationindex(proliferativeindex,PI),saidformulais:
PI(%)=(S%G2~M%)/(G0~G1%S%G2~M%)*100%.
1.2.3StatisticalMethods:
SPSSforWindow10.0packageforstatisticalanalysis,multiplecomparisonsbetweenthesamplemeanusinganalysisofvariance,P<0.05asstatisticallysignificant.Repostedelsewhereinthepaperforfreedownload
2Results
CelecoxibandSihaofHelacellcycleandcellapoptosis:
AsinTable1,Table2shows,withvariousconcentrationsofcelecoxibincubated72hlater,withtheincreaseindrugconcentration,SihaandHelachangesinthecellcycle:
G0~G1phasecellsincreased,Sphasecellsdecreased,G2~Mphasecellsnosignificantchanges.Comparedwiththecontrolgroup,SihaandHelacells,PIproliferationindexdecreased(P<0.05),apoptosisrateincreased(P<0.05),flowanalysisdiagramcanbeseenhypodiploidapoptoticpeak(seeTable3,Figure1,Figure2).Table1ofcelecoxibonSihacelllinecellcycleinTable2ofcelecoxiboncellcycleofHelacelllineofTable3ofcelecoxibonSiha/HelacelllinePIproliferationindexandapoptosisrateof*Comparedwiththecontrolgroup,thedifferencewasstatisticallysignificant(P<0.05)
3Discussion
COXinhibitorsbelongtonon-steroidalanti-inflammatorydrugs(NSAIDs),commonlyusedinclinicalanti-inflammatory,analgesic,antipyreticandothertreatment.Since100yearsago,thefirstdrugaspirinNSAIDssynthesizedbyFelixHoffmanhastheworldhavemorethan50differentcompoundsofNSAIDsonthemarket.WiththetwokindsofcyclooxygenaseCOX-1andCOX-2knowledge,NSAIDsdrugsaccordingtotheirCOX-selectivityontwodifferenthasfurtherclassification,namelynon-selectiveCOXinhibitor(COX-1andtherightinhibitionofCOX-2similartoorstrongerinhibitionofCOX-1),andthehighlyspecificCOX-2selectiveinhibitionCOX-2inhibitors.TheformeristhetraditionalNSAIDs,includingaspirin,paracetamol,anti-inflammatorypain,whichincludesthenewlydevelopedCelecoxib(celecoxib),Rofecoxib(rofecoxib),NS-398andsoon.
80years,anumberoflarge-scaleepidemiologicalsurveyfoundthatlong-termuseofCOXinhibitors(NSAIDs)treatmentofrheumatoidarthritispatients,coloncancerincidenceratehasdropped40%to60%,butalsosignificantlyreducedandthepreventionofesophagealcancer,gastriccancertheriskofsuchoccurrence.Andfamilialadenomatouspolyposispatientsafterlong-termlow-doseNSALDs,thenumberandsizeofcolonpolypsweresignificantlyreduced[2].ThismakesthepreventionandtreatmentofCOXinhibitorsasanewtargetforcancer.
InthisstudyoptedforaselectiveCOX-2inhibitorcelecoxib,usingflowcytometry,theHela(humancervicaladenocarcinomacelllinebelongsto)andSiha(belongingtothehumancervicalcarcinomacellline)twokindsofcelllinescellcycleandapoptosisanalysis.ExperimentS%reflectcellproliferation,proliferatingcellS%higher.PIproliferationindexrepresentsthecellpopulationinthenumberofproliferatingcells,toacertainextent,canreflectthestateofcellproliferation.Thestudyfoundthattogetherwithcelecoxibafter72hincubation,Hela,andSihacelllinechangedthecellcycle:
G0~G1phasecellsincreased,Sphasecellsdecreased,G2~Mphasecellsnosignificantchanges,PIproliferationindexdecreases,theapoptoticrateincreasedflowdiagramshowshypodiploidapoptoticpeak.TheseresultssuggestthatthedrugismainlythroughthecellcyclecellswerearrestedinG0~G1phase,whichinvaryingdegrees,inhibitinvitrohumancervicalcancercelllinesHelaandSihaproliferationandinduceapoptosis.Douglas,etc.,haveseparatehumancolon,rectum,skincancer,brainglialcelltumors,pancreaticcancerandothercellsNS398(aselectiveCOX-2inhibitors)invitro,theresultswereprompt,NS398caninhibitcellproliferationandinduceapoptosis[3].Somestudiesbelievethattheonehand,COXinhibitorsbyregulatingtheexpressionofbcl-2family,induceapoptosis[4];Ontheotherhand,COXinhibitors,possiblythroughproteinkinaseC(PKC)-dependentpathwayregulatingapoptosisoftumorcells[5].
Previousstudieshaveshownthat,NSAIDshavesideeffects,suchaskidneyandgastrointestinalmucosaldamage,etc.ThisisduetoCOX-1wasinhibiteddueto,anditstherapeuticeffectisduetotheinhibitionofCOX-2.ThetraditionalNSAIDsalsoblocktheCOX-1andCOX-2,thereforegastrointestinalulcersandbleedingsideeffects[6].ThecurrentstudyfoundthatselectiveCOX-2inhibitorsaseffectiveastraditionalNSAIDs,butdoesnotinhibitplateletactivity,specificCOX-2inhibitors,celecoxibandotherdrugsthathaveclinicalsymptomscausedbyulcersandulcercomplicationsshouldbelessthanthetraditionalNSAIDs[7].TherearealreadyspecificCOX-2inhibitorsappliedtotheprevention,treatmentofcoloncancertotheclinic.Therefore,developmentandutilizationofgoodefficacy,lowpriceandfewerside-effectsofCOX-2inhibitoranditsapplicationtothepreventionandtreatmentofcervicalcarcinoma,therewillbeagoodapplicationprospects.
[Reference
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