Timosaponin A-III Induces Autophagy Preceding Mitochondria-Mediated Apoptosis in HeLa Cancer Cells
Timosaponin A-III Induces Autophagy Preceding Mitochondria-Mediated Apoptosis in HeLa Cancer Cells
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DOI：
10.1158/0008-5472.can-08-1983
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发表时间：
2008-12-15
期刊：
影响因子：
11.2
通讯作者：
Che, Chi-Ming
中科院分区：
文献类型：
--
作者：
Sy, Lai-King;Yan, Siu-Cheong;Che, Chi-Ming
知母皂苷A-III（TAIII）是从知母根茎中分离得到的皂苷，具有很强的细胞毒性，具有开发为抗癌剂的潜力。在这里，我们提供的证据表明，TAIII诱导HeLa细胞中的自噬，然后凋亡细胞死亡。TAIII诱导的自噬在形态学上的特点是在超微结构水平上可识别的膜结合自噬空泡的形成。表达绿色荧光蛋白（GFP）标记的微管相关蛋白1轻链3（LC 3）的TAIII处理的细胞显示点状荧光，表明LC 3募集到自噬体。这与LC 3-I（胞质形式）转化为LC 3-II（位于自噬体膜上的脂化形式）有关。TAIII治疗还诱导线粒体功能障碍，涉及活性氧物质的过度产生和线粒体膜电位的降低，伴随着诱导线粒体渗透性转变。长时间暴露于TAIII导致细胞色素c释放和半胱天冬酶-3激活，这些事件标志着凋亡性细胞死亡的发生。TAIII诱导的自噬先于细胞凋亡，如早期自噬空泡形成、GFP-LC 3易位和LC 3-II在没有caspase-3裂解的情况下增加所证明的。值得注意的是，TAIII介导的凋亡细胞死亡是通过用自噬抑制剂3-甲基腺嘌呤或针对自噬基因beclin 1的小干扰RNA处理而增强的。这些发现表明，TAIII引起的自噬反应起着阻止最终细胞死亡的保护作用。在构效关系方面，TAIII中的糖链对药物作用是不可缺少的，因为缺糖的苷元sarsasapogenin不诱导自噬并且表现出较弱的细胞毒性。[Cancer Res 2008;68（24）：10229-37]
Timosaponin A-III (TAIII), a saponin isolated from the rhizome of Anemarrhena asphodeloides, exhibits potent cytotoxicity and has the potential to be developed as an anticancer agent. Here, we provide evidence that TAIII induces autophagy in HeLa cells followed by apoptotic cell death. TAIII-induced autophagy was morphologically characterized by the formation of membrane-bound autophagic vacuoles recognizable at the ultrastructural level. TAIII-treated cells expressing green fluorescent protein (GFP)-labeled microtubule-associated protein 1 light chain 3 (LC3) displayed punctate fluorescence indicative of LC3 recruitment to the autophagosome. This was associated with the conversion of LC3-I (the cytosolic form) into LC3-II (the lipidated form located on the autophagosome membrane). TAIII treatment also induced mitochondrial dysfunction involving overproduction of reactive oxygen species and reduction of mitochondrial membrane potential accompanied by induction of mitochondrial permeability transition. Prolonged exposure to TAIII resulted in cytochrome c release and caspase-3 activation, events that signified the onset of apoptotic cell death. TAIII-induced autophagy preceded apoptosis, as evidenced by early autophagic vacuole formation, GFP-LC3 translocation, and LC3-II increase in the absence of caspase-3 cleavage. Notably, TAIII-mediated apoptotic cell death was potentiated by treatment with autophagy inhibitor 3-methyladenine or small interfering RNA against the autophagic gene beclin 1. These findings suggest that TAIII-elicited autophagic response plays a protective role that impedes the eventual cell death. In terms of structure-activity relationship, the sugar chain in TAIII is indispensable to the drug action, as the sugar-lacking aglycone sarsasapogenin did not induce autophagy and exhibited weaker cytotoxicity. [Cancer Res 2008;68(24):10229-37]