Blocking NF-kB nuclear translocation leads to p53-related autophagy activation and cell apoptosis

AIM:Toinvestigatetheanti-tumoreffectsofnuclearfactor-κB(NF-κB)inhibitorSN50andrelatedmechanismsofSGC7901humangastriccarcinomacells.METHODS:MTTassaywasusedtodeterminethecytotoxiceffectsofSN50ingastriccancercelllineSGC7901.Hoechst33258stainingwasusedtodetectapoptosismorphologicalchangesafterSN50treatment.Activationofautophagywasmonitoredwithmonodansylcadaverine(MDC)stainingafterSN50treatment.Immunofluorescencestainingwasusedtodetecttheexpressionoflightchain3(LC3).MitochondrialmembranepotentialwasmeasuredusingthefluorescentprobeJC-1.Westernblottinganalysiswereusedtodeterminetheexpressionofproteinsinvolvedinapoptosisandautophagyincludingp53,p53upregulatedmodulatorofapoptosis(PUMA),damage-regulatedautophagymodulator(DRAM),LC3andBeclin1.Wedetectedtheeffectsofp53-mediatedautophagyactivationontheapoptosisofSGC7901cellswiththep53inhibitorpifithrin-α.RESULTS:TheviabilityofSGC7901cellswasinhibitedafterSN50treatment.Inductionsintheexpressionofapoptoticproteinp53andPUMAaswellasautophagicproteinDRAM,LC3andBeclin1weredetectedwithWesternblottinganalysis.SN50-treatedcellsexhibitedpunctuatemicrotubule-associatedprotein1LC3inimmunoreactivityandMDC-labeledvesiclesincreasedaftertreatmentofSN50byMDCstaining.CollapseofmitochondrialmembranepotentialΔψweredetectedfor6to24hafterSN50treatment.SN50-inducedincreasesinPUMA,DRAM,LC3andBeclin1andcelldeathwereblockedbythep53specificinhibitorpifithrin-α.CONCLUSION:Theanti-tumoractivityofNF-κBinhibitorsisassociatedwithp53-mediatedactivationofautophagy.