肿瘤细胞靶向性锰配合物诱导细胞死亡与抑制糖酵解的机制

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1、江苏大学硕士学位论文ABSTRACTobject：TostudytheanticanceractivityofthenovelmanganesecompoundAdpaMnAc([(Adpa)2Mn2(Ac)(H20)2](Ac))anditspossiblemechanisms；ThepossiblemechanismsoftheHypoxia-induciblefactors(HIF)prolylhydroxylase(PHD)-mimeticmanganesecompoundAdpaMnCl([(Adpa)Mn(C

2、1)(H20)])wereinvestigated，includingtheeffectsontheglycolysisandmitochondria．Methods：FourhumancancercelllinesincludingU251，HeLa，HepG2andA549cellsweretreatedbyAdpaMnAc．CancercellsproliferationsweredetectedbyMTTassayandthe50％inhibitoryconcentrations(IC50)werecalcula

3、ted．ComparetheinhibitionrateofAdpaMnAconnormallivercellsWRL一68andlivercancercellsHepG2．WesternblottodetecttheexpressionoftransferrinrecepotorI(TfRl)andDivalentmetaliontransporters-1(DMT-1)；Deferoxamine(DFO)orferriccitratewereusedtoupregulateordownregulatetheTfRIe

4、xpressionsandthenanalysedthechangeoftheanticanceractivityofAdpaMnAc．ThemorphologicalandnucleuschangesinH2B-GFP—labledHeLacellswereobservedbyalivecellsystem(LCS)．CellapoptosisWasdetectedasfollows：nucleusmorphologicalchangeswereobservedbystaining研mHoechest33342，the

5、ratesofcellapoptosisweredetectedbystaining耐mAnnexinV／PI，releasingofCytochromeC(CytoC)frommitochondriaandexpressionsofprocaspase-9：AutophagiccelldeathWasexaminedbymonodansylcadervarine(MDC)labelingandLysotrackerRedstaining；autophagy—relatedproteinsLC3andBeclin-1ev

6、aluationwimwesternblotandGFP—LC3plasmidtransfection．Themitochondriafunctionswereanalysed：Mitochondriamembranepotential(MMP)，ATPlevelsand02consumptionweremeasuredbyJC一1staining，LuciferaseassayandClarkoxygenelectroderespectively；Intracellularfreecalciumconcentratio

7、nbyFluo-3／AM；Reactiveoxygenspecies(ROS)byDCFH-DAstaining；Mitochondrialswellingwereusedtofindoutitsinteractionwitllmitochondfialoutermembrane．Ontheotherhand，weanalyzedtheinteractionbetweenAdpaMnClandhypoxia-induciblefactor-la(HIF—la)bymolecularmodeling，theactivity

8、ofHIF—labyDualLuciferaseReporterassay，themRNAlevelbyRT-PCILandtheproteinslevelbywesternblot；Detectedtheexpressionlevelsofglucosetransporterl(GLUTl)andlactate肿瘤