Harmine induces apoptosis in HepG2 cells via mitochondrial signaling pathway
Harmine induces apoptosis in HepG2 cells via mitochondrial signaling pathway
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DOI：
10.1016/s1499-3872(11)60102-1
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发表时间：
2011-12-01
影响因子：
3.3
通讯作者：
Jiang, Jian-Wei
中科院分区：
文献类型：
--
作者：
Cao, Ming-Rong;Li, Qiang;Jiang, Jian-Wei
背景：骆驼蓬碱具有抗肿瘤和镇痛作用，并抑制人类 DNA 拓扑异构酶。然而，尚无关于去氢骆驼蓬碱在肝细胞癌中的作用机制的详细数据。本研究旨在探讨去氢骆驼蓬碱对人肝癌细胞系HepG2增殖和凋亡的影响及其机制。方法：采用细胞计数试剂盒8（CCK-8）法和克隆形成实验测定HepG2细胞的增殖能力。 Hoechst 33258 染色后使用荧光显微镜检查 HepG2 细胞的形态。膜联蛋白V/碘化丙啶(PI)用于分析细胞凋亡，PI用于分析细胞周期。 Western blotting 用于评估凋亡调节基因 Bcl-2、Bax、Bcl-xl、Mcl-1、caspase-3 和 caspase-9 的表达。使用JC-1测定线粒体跨膜电位(Psi(m))。结果：去氢骆驼蓬碱以剂量依赖的方式抑制HepG2细胞的增殖。 Hoechst 33258 染色显示用去氢骆驼蓬碱处理的 HepG2 细胞出现核碎片和染色体浓缩、细胞皱缩和附着丧失。 sub/G1 级分的百分比以浓度依赖性方式增加，表明细胞凋亡。 PI染色显示去氢骆驼蓬碱改变了细胞周期分布，降低了G0/G1期细胞的比例，增加了S和G2/M期细胞的比例。 Harmine以浓度依赖性方式诱导细胞凋亡，诱导率分别为20.0%、32.7%和64.9%。 JC-1 显示 Psi(m) 有所下降。 HepG2细胞的凋亡与caspase-3和caspase-9的激活、Bcl-2、Mcl-1和Bcl-xl的下调有关，而Bax没有变化。结论：去氢骆驼蓬碱通过诱导细胞凋亡，具有抗HepG2细胞增殖的作用。线粒体信号通路参与细胞凋亡。本研究显示的癌症特异性选择性表明去氢骆驼蓬碱是一种有前途的治疗人类肝细胞癌的新药。
BACKGROUND: Harmine has antitumor and antinociceptive effects, and inhibits human DNA topoisomerase. However, no detailed data are available on the mechanisms of action of harmine in hepatocellular carcinoma. This study aimed to investigate the effects of harmine on proliferation and apoptosis, and the underlying mechanisms in the human hepatocellular carcinoma cell line HepG2.METHODS: The proliferation of HepG2 cells was determined by the cell counting kit-8 (CCK-8) assay and the clone formation test. The morphology of HepG2 cells was examined using fluorescence microscopy after Hoechst 33258 staining. Annexin V/propidium iodide (PI) was used to analyze apoptosis and PI to analyze the cell cycle. Western blotting was used to assess expression of the apoptosis-regulated genes Bcl-2, Bax, Bcl-xl, Mcl-1, caspase-3, and caspase-9. Mitochondrial transmembrane potential (Psi(m)) was determined using JC-1.RESULTS: Harmine inhibited the proliferation of HepG2 cells in a dose-dependent manner. Hoechst 33258 staining revealed nuclear fragmentation and chromosomal condensation, cell shrinkage, and attachment loss in HepG2 cells treated with harmine. The percentage of the sub/G1 fraction was increased in a concentration-dependent manner, indicating apoptotic cell death. PI staining showed that harmine changed the cell cycle distribution, by decreasing the proportion of cells in G0/G1 and increasing the proportion in S and G2/M. Harmine induced apoptosis in a concentration-dependent manner, with rates of 20.0%, 32.7% and 64.9%, respectively. JC-1 revealed a decrease in Psi(m). Apoptosis of HepG2 cells was associated with caspase-3 and caspase-9 activation, down-regulation of Bcl-2, Mcl-1, and Bcl-xl, and no change in Bax.CONCLUSIONS: Harmine had an anti-proliferative effect in HepG2 cells by inducing apoptosis. Mitochondrial signal pathways were involved in the apoptosis. The cancer-specific selectivity shown in this study suggested that harmine is a promising novel drug for human hepatocellular carcinoma.