毛酸浆内酯诱导人乳腺癌MCF-7细胞凋亡的机制研究

Mechanism of inducing apoptosis in human breast cancer MCF-7 cells by Physapubesin

[目的]探讨毛酸浆活性成分毛酸浆内酯(PPB)诱导人乳腺癌MCF-7细胞凋亡的机制。[方法]采用噻唑蓝法(MTT法)检测PPB对MCF-7细胞及人外周血单核细胞(hPBMC)增殖的影响;克隆形成实验考察PPB对MCF-7细胞克隆形成的影响;MTT法考察泛半胱天冬氨酸蛋白酶(Caspase)抑制剂Z-VAD-fmk对PPB诱导MCF-7细胞死亡的作用;Western Blot法考察PPB处理后凋亡相关蛋白PARP、Caspase-7/8/9、Cytochrome c、Bax、Bcl-2、Fas及FADD的表达水平。[结果]PPB时间、剂量依赖性抑制MCF-7细胞生长,且对hPBMC无明显细胞毒性;PPB呈剂量依赖性地抑制MCF-7细胞克隆形成;PPB处理后,凋亡特征性蛋白PARP、原型Caspase-7蛋白表达量显著减少,而Cleaved PARP、Cleaved Caspase-7显著增加;同时内源性凋亡途径相关蛋白Bax、Cleaved Caspase-9、Cytochrome c表达量显著上调,而Bcl-2及Caspase-9原型形式显著下调。外源性凋亡相关蛋白Fas、Cleaved Caspase-8及FADD表达量上调,而Caspase-8原型形式显著下调;加入Z-VAD-fmk可显著逆转PPB诱导的MCF-7细胞死亡。[结论]毛酸浆活性成分PPB可以诱导MCF-7细胞发生内源性和外源性凋亡。

[Objective]To investigate the anti-breast cancer effect and mechanism of Physapubesin(PPB)from Physalis pubescens L.[Methods]MTT assay was used to detect the effect of PPB on the proliferation of MCF-7 cells and the peripheral blood mononuclear cells(hPBMC).Colony formation assay was used to investigate the effect of on the colony formation of MCF-7 cells.MTT assay was used to investigate the effect of apoptosis inhibitor Z-VAD-fmk on the cell death of MCF-7 cells.The expression of apoptosis related proteins PARP,Caspase-7/8/9,cytochrome c,Bax,Bcl-2,Fas and FADD at different concentrations of PPB were detected by Western Blot.[Results]PPB inhibited the growth of MCF-7 cells in the time-and dose-dependent manners,and it has no obvious toxicity to hPBMC cells;The results of colony formation assay showed that PPB could inhibit MCF-7 cell clone formation in a dose-dependent manner;The characteristic apoptotic protein PARP,Caspase-7 was significantly reduced by PPB,while the cleaved PARP,Cleaved Caspase-7 were significantly increased.The protein of endogenous apoptotic pathway including Bax,Cleaved Caspase-9,Cytochrome c were significantly up-regulated,at the same time Bcl-2 and Caspase-9 was significantly down-regulated.In addition,the exogenous apoptotic related proteins Fas,Cleaved Caspase-8 and FADD were up-regulated by treatment with PPB,but the protein levels of Caspase-8 were down-regulated.The addition of Z-VAD-fmkcould markedly reverse the death of MCF-7 cells induced by PPB.[Conclusion]PPB can induce endogenous and exogenous apoptosis in the MCF-7 cells.