ActinomycinDsynergisticallyenhancestheefficacyofCDDPbyactivatingP53-PUMApathwayviadownregulatingP53-MDM2complexonKBcells

摘要： OBJECTIVELowdoseofactinomycinD(LDActD)wasreportedasapotentP53activatorandprotectednormalproliferatingcellsduringanti-mitoticchemotherapy.However,themechanismofLDActDonP53activationisstillundetermined.Inthisstudy,themechanismofLDActDonthesynergisticantitumoreffectforcisplatin(CDDP)andP53reactivationinKBcellswasstudiedindetail.METHODSCellviabilitywasdeterminedbyMTTandLDHrelease.ApoptosiswasdeterminedbyAnnexinⅤ-FITC/PIstaining.Mitochondrialmembranepotential(MMP)wasdetectedbyJC-1stain-ing.ExpressionofP53,PARP,BAX,BCL-XL,PUMA,MDM2andMDMXwasdetectedbyWesternblotting(WB)and/orimmunofluorescence(IF).P53-MDM2complexwasdetectedbyELISA.MoleculardockingofreceptorMDM2andMDMXwithactinomycinD(ACTD)wasanalyzedbyDiscoveryStudio.RESULTSComparedwithCDDPalone,P53expressionandthecytotoxicityonKBcellswassignificantlyincreasedbythecombinationtherapy.P53regulatoryproteinswereincreasedwhileMMPwasdecreased.Meanwhile,knockdownofPUMA(P53upregulatedmodulatorofapoptosis)efficientlyblockedthesynergisticeffectofLDActDtoCDDP.P53activationwasfoundtobeaccompaniedwiththeincreaseofMDMXbutnotMDM2.Meanwhile,MDM2-P53complexinKBcellswassignificantlydecreasedbyLDActD.DockingofbothreceptorMDM2andMDMXwithACTDexhibitedwellestablishedbondswithnearbyaminoacidresidues.CONCLUSIONLDActDwasprobablyaninhibitorofbothMDM2andMDMX.ThesynergisticeffectsofLDActDforCDDPonKBcellsdependedonitseffectonreactivatingP53andPUMAmediatedmitochondrialapoptosis. ... （共2頁）