Wang Qwa-Fun, Chiang Chi-Wu, Wu Chun-Chi, Cheng Chi-Chih, Hsieh Shur-Jong, Chen Jung-Chou, Hsieh Yun-Chih, Hsu Shih-Lan
School of Post-baccalaureate Chinese Medicine, China Medical University, Taichung, Taiwan, Republic of China.
Planta Med. 2007 Jun;73(6):535-44. doi: 10.1055/s-2007-967200. Epub 2007 May 22.
We have previously reported that gypenosides induce apoptosis in human hepatocarcinoma Huh-7 cells through a mitochondria-dependent caspase-9 activation cascade. In order to further explore the critical events leading to apoptosis in gypenosides-treated cells, the following effects of gypenosides on components of the mitochondrial apoptotic pathway were examined: generation of reactive oxygen species (ROS), alteration of the mitochondrial membrane potential (MPT), and the subcellular distribution of Bcl-2 and Bax. We show that gypenosides-induced apoptosis was accompanied by the generation of intracellular ROS, disruption of MPT, and inactivation of ERK, as well as an increase in mitochondrial Bax and a decrease of mitochondrial Bcl-2 levels. Ectopic expression of Bcl-2 or treatment with furosemide attenuated gypenosides-triggered apoptosis. Treatment with ATA caused a drastic prevention of apoptosis and the gypenosides-mediated mitochondrial Bcl-2 decrease and Bax increase, but failed to inhibit ROS generation and MPT dysfunction. Incubation with antioxidants significantly inhibited gypenosides-mediated ROS generation, ERK inactivation, MPT and apoptosis. Moreover, an increase of the intracellular calcium ion (Ca(2+)) concentration rapidly occurred in gypenosides-treated Huh-7 cells. Buffering of the intracellular Ca(2+) increase with a Ca(2+) chelator BAMTA/AM blocked the gypenosides-elicited ERK inactivation, ROS generation, Bcl-2/Bax redistribution, mitochondrial dysfunction, and apoptosis. Based on these results, we propose that the rise in intracellular Ca(2+) concentration plays a pivotal role in the initiation of gypenosides-triggered apoptotic death.
我们之前曾报道，绞股蓝总皂苷通过线粒体依赖的半胱天冬酶-9激活级联反应诱导人肝癌Huh-7细胞凋亡。为了进一步探究绞股蓝总皂苷处理细胞后导致凋亡的关键事件，我们检测了绞股蓝总皂苷对线粒体凋亡途径各组分的以下影响：活性氧（ROS）的产生、线粒体膜电位（MPT）的改变以及Bcl-2和Bax的亚细胞分布。我们发现，绞股蓝总皂苷诱导的凋亡伴随着细胞内ROS的产生、MPT的破坏、ERK的失活，以及线粒体Bax的增加和线粒体Bcl-2水平的降低。Bcl-2的异位表达或用速尿处理可减轻绞股蓝总皂苷引发的凋亡。用ATA处理可显著预防凋亡以及绞股蓝总皂苷介导的线粒体Bcl-2降低和Bax增加，但未能抑制ROS的产生和MPT功能障碍。用抗氧化剂孵育可显著抑制绞股蓝总皂苷介导的ROS产生、ERK失活、MPT和凋亡。此外，在绞股蓝总皂苷处理的Huh-7细胞中，细胞内钙离子（Ca(2+)）浓度迅速升高。用钙离子螯合剂BAMTA/AM缓冲细胞内Ca(2+)的增加可阻断绞股蓝总皂苷引发的ERK失活、ROS产生、Bcl-2/Bax重新分布、线粒体功能障碍和凋亡。基于这些结果，我们提出细胞内Ca(2+)浓度的升高在绞股蓝总皂苷引发的凋亡死亡起始过程中起关键作用。
