Involvement of mitochondrial pathway in NCTD-induced cytotoxicity in human hepG2 cells.
Involvement of mitochondrial pathway in NCTD-induced cytotoxicity in human hepG2 cells.
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DOI：
10.1186/1756-9966-29-145
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发表时间：
2010-11-09
期刊：
影响因子：
--
通讯作者：
Luo J
中科院分区：
文献类型：
--
作者：
Chang C;Zhu YQ;Mei JJ;Liu SQ;Luo J
去甲斑蝥素（Norcantharidin）是一种去甲基化的斑蝥素类似物，从一种传统中药中提取，已被用于抗癌治疗。然而，这一过程背后的详细机制通常尚不清楚。本研究旨在探讨nctd诱导HepG2细胞凋亡的机制。采用MTT法测定细胞活力，流式细胞术测定细胞毒性。流式细胞术检测线粒体膜电位和活性氧生成。采用caspase凋亡检测试剂盒检测caspase活性的作用。Western blot检测细胞c、Bcl-2、Bax、Bid、caspase 3、-9、-8、PARP表达水平。NCTD处理后，HepG2细胞活力降低，凋亡增加。nctd诱导的细胞凋亡伴随着ROS生成的增加、线粒体膜电位的丧失、细胞色素c（cyto-c）从线粒体向细胞质的释放以及抗凋亡蛋白Bcl-2水平的下调和促凋亡蛋白Bax水平的上调。然而，另一种促凋亡分子Bid在同样的处理下没有变化。还观察到nctd增加了caspase 9、caspase 3的活性以及随后切割caspase底物PARP的活性。NCTD治疗后，前caspase-8的表达水平未发生变化。这些结果表明，NCTD通过ROS生成和线粒体途径介导的凋亡诱导HepG2细胞毒性。
Norcantharidin, the demethylated analog of cantharidin derived from a traditional Chinese medicine, Mylabris, has been used in the treatment of anti-cancer effects. However, the detailed mechanisms underlying this process are generally unclear. The aim of this study was to investigate the mechanism of NCTD-induced apoptosis in HepG2 cells. The cytotoxicity was measured by MTT assay for cellular viability and by flow cytometry. The mitochondrial membrane potential and reactive oxygen species production was evaluated by flow cytometry analysis. The role of caspase activities were assayed using caspase apoptosis detection kit . Western blot analysis was used to evaluate the level of Cyto-C, Bcl-2, Bax, Bid, caspase 3, -9, -8 and PARP expression After treatment with NCTD, a decrease in the viability of HepG2 cells and increase in apoptosis were observed. NCTD-induced apoptosis was accompanied by an increase in ROS production, loss of mitochondrial membrane potential and release of cytochrome c(cyto-c) from the mitochondria to the cytosol and down-regulation of anti-apoptotic protein Bcl-2 levels with concurrent up-regulation in pro-apoptotic protein Bax levels. However, another pro-apoptotic molecule, Bid, showed no change in such same treatment. NCTD-increased activity of caspase 9,caspase 3 and the subsequent cleavage caspase substrate PARP were also observed. The expression levels of pro-caspase-8 were not changed after NCTD treatment. These results indicate that NCTD induced cytotoxicity in HepG2 cells by apoptosis, which is mediated through ROS generation and mitochondrial pathway.
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