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1、132药学学报ActaPharmaceuticaSinica2007,42(2):132-138研究论文力达霉素经线粒体依赖通路介导细胞凋亡21△11*邱强,王真,蒋建明,李电东(1.中国医学科学院、中国协和医科大学医药生物技术研究所,北京100050;2.解放军总医院肾内科,北京100853)摘要:力达霉素(lidamycin,LDM)是具有我国自主知识产权的烯二炔类抗生素,具有较强的抗肿瘤活性,其抗肿瘤机制有待深入阐明。本研究主要利用人肝癌BEL-7402和乳腺癌MCF-7细胞研究了力达霉素对线粒体凋亡通路相关因子的影响。通过MTT法检测不同剂量力达霉素对肿瘤细胞增殖毒性;蛋白印迹技术
2、检测线粒体和细胞质细胞色素c、总Bax,Bcl-2及p53表达;RT-PCR检测p53和baxmRNA表达等。结果发现,LDM能迅速激活肿瘤细胞线粒体凋亡通路,在2h内就引起线粒体中细胞色素c释放至细胞质中。同时Bcl-2家族成员中促凋亡成员Bax持续增加,而抗凋亡成员Bcl-2先增加后减少。p53蛋白在6h显著增加。Bax蛋白表达升高与其mRNA转录水平升高有关,p53蛋白表达升高与转录后水平有关。Caspase抑制剂可以部分抑制LDM的增殖毒性。因此LDM可以通过激活细胞色素c启动的线粒体凋亡通路发挥抗肿瘤作用。关键词:力达霉素;线粒体;肿瘤;细胞凋亡中图分类号:R963;R978.1
3、文献标识码:A文章编号:0513-4870(2007)02-0132-07Effectoflidamycinonmitochondriainitiatedapoptoticpathwayinhumancancercells21△11*QIUQiang,WANGZhen,JIANGJian-ming,LIDian-dong(1.InstituteofMedicinalBiotechnology,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing100050,China;2.DepartmentofNephro
4、logy,PLAHospital,Beijing100853,China)Abstract:Althoughenediyneantibioticlidamycin(LDM)isapotentinducerofapoptosis,theunderlyingmechanismsofitsapoptoticfunctionsremaintobeexplored.Here,weaimtoelucidateitspossiblemechanismsinmitochondriainitiatedapoptoticpathwayinvolvedinhumanBEL-7402andMCF-7cells.C
5、ytochromecreleasedfrommitchondriatocytosolfractionwasdetectedbyWesternblotting.p53andBax,Bcl-2expressionsweredetectedbyWesternblottingandRT-PCR.MTTassaywasusedtodetectcytotoxicityofLDMwithorwithoutcaspaseinhibitorz-VAD-fmk.AftertheBEL-7402cellswereexposed-1to0.1μmolLLDMwithin6h,theincreaseofcytoch
6、romecinthecytosolanddecreaseinthemitochondriawereobservedwhencomparedwithuntreatedcells.TheexpressionofBax,animportantproapoptoticmemberoftheBcl-2family,increasedgraduallyintheBEL-7402cellsafterexposuretoLDM-1of0.1μmolLfor2,6,and9h,separately,whileBcl-2increasedat2and6h,anddecreasedat9hafterLDMtre
7、atmen.tEnhancedproteinexpressionswereparallelwithrespectiveincreasedmRNAlevelforBaxonly,butnotp53.CaspaseinhibitormayinhibitpartiallythekillingeffectsinducedbyLDM.Thereforeweconcludethattherapidactivationofmitoch
