Involvement of reactive oxygen species and caspase-dependent pathway in berberine-induced cell cycle arrest and apoptosis in C6 rat glioma cells
Involvement of reactive oxygen species and caspase-dependent pathway in berberine-induced cell cycle arrest and apoptosis in C6 rat glioma cells
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DOI：
10.3892/ijo_00000299
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发表时间：
2009-06-01
影响因子：
5.2
通讯作者：
Chung, Jing-Gung
中科院分区：
文献类型：
--
作者：
Chen, Ting-Ching;Lai, Kuang-Chi;Chung, Jing-Gung
黄连素对C6大鼠胶质瘤细胞的细胞毒作用表明，黄连素通过G2/M期阻滞和细胞凋亡引起细胞形态改变和细胞死亡。在细胞凋亡过程中，随着Weel的上调，G2/M细胞显著积聚，但也抑制了细胞周期蛋白B、CDK1和CDc25c，导致G2/M细胞停滞。在对C6细胞产生细胞毒作用的同时，还诱导了线粒体细胞色素c释放、caspase-9、-3和-8激活以及DNA片段化等一系列的细胞凋亡事件。根据Western blotting检测，黄连素增加了C6细胞中GADD153和GRP 78的水平，这是内质网(ER)应激的一个主要标志。我们还发现黄连素能促进C6细胞产生活性氧和钙离子。Western blotting结果还显示，黄连素抑制抗凋亡蛋白Bcl2的表达，而增加促凋亡蛋白Bax的表达，随后导致线粒体膜电位(Delta psi(M))的降低，随后细胞色素c的释放导致Capase-9和-3的激活，从而导致细胞凋亡的发生。在底物溶液(PhiPhiLux-G(1)D(1)，CaspaLux 8-L1D2，CaspaLux 9-M1D2分别为caspase-3，-8和-9)的基础上，用黄连素激活C6细胞中的caspase-8，-9和-3，并用流式细胞仪进行分析，caspase-8，-9和-3的每个抑制剂都能增加C6细胞的存活率。这一发现也得到了Western印迹实验的证实，结果表明黄连素促进了caspase-8、-9和-3的活性形式。这些结果表明，黄连素对C6大鼠胶质瘤细胞的细胞毒作用主要是通过诱导G2/M期阻滞细胞，并以ER依赖的方式，通过受Bax和Bcl2调控的线粒体依赖的caspase途径来实现的。
The cytotoxicity of berberine on C6 rat glioma cells indicated that berberine induced morphological changes and caused cell death through G2/M arrest and apoptosis. While undergoing apoptosis, there was a remarkable accumulation of G2/M cells with the upregulatoin of Weel but it also inhibited cyclin B, CDK1 and Cdc25c that led to G2/M arrest. Along with cytotoxicity in C6 cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3 and -8 and DNA fragmentation were induced. Berberine increased the levels of GADD153 and GRP 78 in C6 cells based on the examination of Western blotting and this is a major hallmark of endoplasmic reticulum (ER) stress. We also found that berberine promoted the production of reactive oxygen species and Ca2+ in C6 cells. Western blotting assay also showed that berberine inhibited the levels of anti-apoptotic protein Bcl-2 but increased the levels of pro-apoptotic protein Bax before leading to a decrease in the levels of mitochondrial membrane potential (Delta psi(m)) followed by cytochrome c release that caused the activations of capase-9 and -3 for apoptotic occurrence. The caspase-8, -9 and -3 were activated by berberine in C6 cells based on the substrate solution (PhiPhiLux-G(1)D(1), CaspaLux 8-L1D2, CaspaLux 9-M1D2 for caspase-3, -8 and -9, respectively) and analyzed by flow cytometer and each inhibitor of caspase-8, -9 and -3 led to increase the percentage of viable C6 cells after exposure to berberine. This finding was also confirmed by Western blot assay which showed that berberine promoted the active form of caspase-8, -9 and -3. These results demonstrate that the cytotoxicity of berberine in C6 rat glioma cells is attributable to apoptosis mainly through induced G2/M-arrested cells, in an ER-dependent manner, via a mitochondria-dependent caspase pathway regulated by Bax and Bcl-2.