Cytotoxic drugs lowered extracellular regulated protein kinase inhibit liver cancer SMF7721 cells pr.docx

Cytotoxic drugs lowered extracellular regulated protein kinase inhibit liver cancer SMF7721 cells pr.docx

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CytotoxicdrugsloweredextracellularregulatedproteinkinaseinhibitlivercancerSMF7721cellspr

CytotoxicdrugsloweredextracellularregulatedproteinkinaseinhibitlivercancerSMF7721cellsproliferation

[Keywords]livertumorKeywords:

livertumors;proteinkinases;cellcycle;celldivisionAbstract:

ObjectiveTostudythethreecytotoxicdrugs（Harringtoninealkali,vincristine,footleafsaponinsinhibitlivercancerSMF7721cellproliferation,intheprocessofblockingthecellcycle,extracellularregulatedproteinkinase（extracelluarregulatedproteinkinasesofERK1/2phosphorylatedproteinexpressionlevelschangetoexplorecytotoxicdrugsanti-oflivercancerSMF7721themechanismofaction.MethodsMTTassayproliferationinhibitionrateofcellcyclewasdetectedbyflowcytometry,usingWesternblotmethodobservedERK1/2phosphorylationproteinexpressionlevelschangesinresultsofthreecytotoxicdrugs（Harringtoninealkali,vincristine,footleafsaponinscanthedifferentdegreestosuppressSMF7721cellsproliferationinhibitionrateswere（28+-8%,（25+-16%and（24+-11%;HarringtoninealkalitoenabletheSMF7721cellcyclearrestattheG1phase,vincristineitInhibitioninSandG2/Mphase,Vp16sofrustratedatG2/MphasebythreecytotoxicdrugsroleintheactivationstatehepatomaSMF7721intracellularphosphorylationofERK1/2proteinlevelsdecreased.ERK1werethecontrolgroup,43%,32%,72%,ERK2,respectively29%ofthecontrolgroup,20%,61%.conclusionERKpathwaymaybecellthepoisonsthemechanismofactionofanti-hepatomacellsSMF7721one.

Keywords:

liverneoplasms;proteinkinases;cellcycle;celldivision

Abstract:

AIMTostudythechangeofphosphorylated（ac-tivatedERK1andERK2proteinexpressioninthecourseofinhibitinghepatocarcinomatouscellproliferationandarrestingcellcycleprogressionbythreekindsofcytotoxicdrugs,Har-ringtonine（HRT,Vincristine（VCR）andEtoposide（Vp16）,andtodiscussthekillingmechanismsofcytotoxicdrugstohepato-carcinomatouscell,SMF7721.METHODSProliferationinhibitionratewasdetectedbyMTTmethod.Thecellcyclewasanalyzedbyflowcytometry.ThechangeofthephosphorylatedERK1/2proteinexpressionwasob-servedbyWesternblotmethod.RESULTSThreekindsofcytotoxicdrugs（HRT,VCRandVp16inhibitedhepatocar-cinomatouscellproliferationtosomeextent.Theinhibitionratewas（28+-8%,（25+-16%and（24+-11%respective-ly.ThecellcyclewasblockedinG1phasewithHRT.ThecellcyclewasblockedinSphaseandG2/MphasewithVCR.AndthecellcyclewasblockedinG2/MphasewithVp16.Inexperimentalgroups,thecontentsofphosphorylatedERK1andERK2proteinwhichwasactiveinhepatocarcinomatouscellSMF7721obviouslydeclined.Withtheeffectsofthreecytotoxicdrugs,thecontentofERK1was43%,32%and72%respectively;thecontentofERK2was29%,20%and61%respectively.CONCLUSIONItmightbethroughERKsignaltransductionpathwayforcytotoxicdrugstoinhibithepatocarcinomatouscellSMF7721proliferationandarrestcellcycleprogression.

0Introduction

Theextracellularregulatedproteinkinase（extracelluarregulatedproteinkinases,ERKpathwayandmalignantphenotypetransitioniscloselyrelatedtotheblockingoftheERKpathwaywillhelptoinhibitthegrowthofcancercells.Selectedseveralcommonlyusedcytotoxicdrugs,toexploretheroleofoneofthemechanismsofregulationoftheERKpathwayiskillinglivercancercellstocytotoxicdrugs.

1Materialsandmethods

Cited1.1MaterialsSMF7721cellsfromWuhanUniversityTypicalCultivationCenter.Containing100mL·L-1FBS,1×10-5U·L-1penicillin,1×10-5g·L-1streptomycintheRRMI1640mediumat37°C,50mL·L-1CO2saturatedhumidityincubatorculturemediumwaschangedevery2~3dpassagedexperimentalselectionofcellsinthelogarithmicgrowthphasethreeharringtonine（harringtonineHRT:

BeijingUnionMedicalCollegepharmaceuticalproduct,lotnumber971191;enoughleafsaponins（etoposide,Vp16:

LianyungangHengRuiPharmaceuticalCo.,Ltd.,batchnumber990504;vincristine（vincristine,VCR:

ShanghaiHualianPharmaceuticalCo.,Ltd.,batchnumber991001;tetramethyltetrazoliumsalt（MTT）,dimethylsulfoxide（DM-SO,propidiumiodide（PI,RNaseA.areSigmaproducts.fetalcalfserum（FCSRPMI1640mediumfromGibcoCompany;anti-phosphorylatedERK1/polyclonalantibodywaspurchasedfromSantaCruz.

1.2Methodsweredividedintofourgroups:

plusdruggroup;HRTgroup;VCRgroup;Vp16groupfinalcelldensityof1×109·L-1,thetotalvolumeoftheexperimentalsystem180μL.At37°C,50mL·L-1ofCO2saturatedhumidityincubatorafter24hculture,centrifugationsupernatantwasadded（5g·L-1MTT20μLAfterculturewascontinued4h,coupledDMSO200μLterminatethereaction,usingtheBio-RadM450microplatereader（wavelength540nmmeasuredtheabsorbance（AvalueDrugfinalconcentration:

HRT5×10-4g·L-1,theofVCR2×10-4gL-1,Vp164×10-3gL-1.calculatedasfollowsdrugcellinhibitionrate[1]:

Theinhibitoryrate（%=（1-experimentalwellsAvalue÷controlholeAvalue×100%.respectivelywiththeabove-mentionedconcentrationofHRT,VCRs,,andVp16processingSMF7721cellline24htake1×10

6cells,PBSwashed3timeswith700mL·L-1ethanol,RNaseA（5×10-5g·L-1digest30min,PI（0.5g·L-130μL,darkfor30minFlowcytometricanalysisofcellularDNAcontentdistributioninformationbyModFitLTRsoftwarecollection,storageandanalysis[2]totakethedrugaction1×107cells,addingthepre-coolingofthecelllysate200μLoficecracking20mincentrifugal,thesupernatant,751spectrophotometersmeasuringproteincontent,eachsampletaken30μgprotein100mL·L-1SDS-polyacrylamidegelelectrophoresisandtransferredtonitrocellulosemembrane.thefilmby50mL·L-1nonfatdrymilkclosed,andtheantiofERK1/2phosphorylationantibodieswereincubatedovernightwithalkalinephosphataselabeledsecondaryantibodyandBCIP/NBTsubstratecolorimageacquisitionUvpgrab-itImagelsoftware,theGelworks1DAd--vancedv4.of01softwareprocessing[3].

2Results

2.1SMF7721cellproliferationHRT（5×10-3g·L-1,VCR（1×10-3g·L-1andVp16（4×10-3g·L-1role24habledegreeofinhibitionSMF7721cellproliferationinhibitionrateswereasfollows:

（28+-8%（25+-16%（24+-0.11%.

Cytotoxicdrugsfor24h2.2cellcyclearrest,thecellcyclebyblock,which,HRTrepressorcellsinG1phase,VCRInhibitorycellsinSphaseandG2/Mphase,Vp16sofrustratedattheG2/Mphase（Tab1.

Table1cytotoxicdrugsslightlyimpactofthehepatomacellSMF7721cellcycle

2.3cytotoxicdrugsinhibitionofERK1/2phosphorylationHRTVCRandVp16roleof24h,SMF7721intracellularERK1/2phosphorylationproteincontentdecreased;usingGelworks1DAdvancedv4.01softwareimagegrayvalueofeachtestgroupanalysis,ERK1,respectively,forthecontrolgroup,43%,32%,72%,ERK2,respectively,for29%ofthecontrolgroup,20%,61%,suggestingthatthereducedphosphorylatedERK1/2proteinexpression（Fig1.stateofphosphorylationofERK1/2proteinexpressionlevelsmayindirectlyreflectofERK1/2functionalactivityofhighandlow.

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Figure1slightly

3Discussion

ERKismostclosetothemembersoftheRas/Raf/MEK/ERK1/2signalpathwayandthenucleus,belongtothemitogenactivatedproteinkinase（mitogen-activatedproteinkinase,MAPKfamily,bothnormalandmalignantcellsbycertainexternalstimuli,canbeactivatedbyphosphorylationofERK1/2,translocationfromthecytoplasmtothenucleus,therebymediatedElk-1,theATF,NF-κBandAp-1,c-fosandc-juntranscriptionalactivationisinvolvedincellmultiplebiologicalresponses,suchasdifferentiationandapoptosis[4,5].acuteleukemiaandrenalcellcarcinomaandmalignantdiseasewerefoundERKbuildactivation,suggestingthatactivationoftheERKpathwaymaybeinvolvedintheprocessofmalignanttransformationofnormalcells[6].Boucheretal[7]studyconfirmedtheactivationoftheERKpathwaymaypreventapoptosisofpancreaticcancercellsandpromotecellcycleprogression.Kangetal.[8]studyconfirmedtheanti-apoptoticpropertiesoftheleukemiacelllineK562andERKbasalactivityinhibitionofERKactivityallowsK562apoptosis.visibleERKpathwayinthedevelopmentofmalignantcellsplaysanimportantroleinERKactivitycutwillhelpinhibitcancercellgrowthanddevelopment.,cytotoxicdrugskillingThetumorcellsmaybeonemechanismisthatinhibitionoftheERKpathway.

Toconfirmthisinference,weselectedexperimentshepatomacelllineSMF7721astargetcells,threekindsofinteractioncharacteristicsofdifferentcytotoxicdrugsofHRT,VCRandVp16asaninducerofERK1/2inthedestructionoflivercancercytotoxicdrugscells.,HRTcyclenon-specificdrugs;VCRMphasecyclespecificdrugs;Vp16SphaseandG2phasecyclespecificdrugsTheresultsshowthatthephosphorylationofofcytotoxicdrugsHRT,VCRandVp16abletocutSMF7721cellsstatusofERK1/2proteinexpressionlevels;alsoallowscellproliferationwasinhibitedbyblockingcellcycleprogression.Basedonthis,webelievethatthemolecularmechanismsoftheanti-hepatomacellsSMF7721ofthesethreekindsofcytotoxicdrugsmaybebyinhibitionoftheRas/Raf/MEK/ERK1/2signalpathway,reducingERKactivity,reducethetranscriptionalactivationofERK1/2targetgenesinhibitcellproliferation,arrestcellcycleprogressioninshort,inthetreatmentoflivercancerprocess,ERK/MAPKpathwaymaybecytotoxicTargetsofDrugsimportantrole.

References:

[1]ZhaoJB,CuiQ,ZhangX,WangWL,JiangYP.Experimentstudyonanticanceractiono