Characterization of Cholix Toxin-induced Apoptosis in HeLa Cells
Characterization of Cholix Toxin-induced Apoptosis in HeLa Cells
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DOI：
10.1074/jbc.m111.246504
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发表时间：
2011-10-28
影响因子：
4.8
通讯作者：
Noda, Masatoshi
中科院分区：
文献类型：
--
作者：
Ogura, Kohei;Yahiro, Kinnosuke;Noda, Masatoshi
Cholix毒素（Cholix）是由霍乱弧菌产生的一种新型ADP核糖基化细胞毒素，其作用机制与白喉毒素和假单胞菌外毒素A相似，以真核细胞延伸因子2为底物。首先，发现Cholix处理的HeLa细胞表现出半胱天冬酶依赖性凋亡，而肠细胞如Caco-2，HCT 116和RKO则没有。在这里，我们研究了Cholix诱导的HeLa细胞的细胞死亡信号通路。Cholix诱导的细胞色素c释放到胞质溶胶中是由与Bax相关的促凋亡巴克的特定构象变化启动的。使用siRNA沉默巴克/bax基因或单独的巴克基因显著抑制细胞色素c释放和半胱天冬酶-7活化，但不抑制半胱天冬酶-3和-9活化。虽然用半胱天冬酶-8抑制剂（Z-IETD-FMK）预处理可减少Cholix诱导的细胞色素c释放和半胱天冬酶-3、-7和-9的活化，但细胞毒性并未降低。用抑制caspase-1、-4和-5的Z-YVAD-FMK预处理不仅抑制细胞色素c释放、caspase-3、-7、-8或-9的活化和PARP裂解，而且抑制细胞毒性，表明caspase-1、-4和-5活化在Cholix诱导的细胞凋亡的早期阶段启动，并促进caspase-8活化。这些结果表明，炎症半胱天冬酶（半胱天冬酶-1，4和-5）和半胱天冬酶-8负责线粒体信号和其他半胱天冬酶激活。总之，我们发现，Cholix诱导的caspase激活在细胞凋亡信号的产生中起着至关重要的作用，这是由两个依赖和非依赖途径介导的。
Cholix toxin (Cholix) is a novel ADP-ribosylating cytotoxin produced by Vibrio cholerae, which utilizes eukaryotic elongation factor 2 as a substrate and acts by a mechanism similar to that of diphtheria toxin and Pseudomonas exotoxin A. First it was found that Cholix-treated HeLa cells exhibited caspase-dependent apoptosis, whereas intestinal cells such as Caco-2, HCT116, and RKO did not. Here we investigated Cholix-induced cell death signaling pathways in HeLa cells. Cholix-induced cytochrome c release into cytosol was initiated by specific conformational changes of pro-apoptotic Bak associated with Bax. Silencing of bak/bax genes or bak gene alone using siRNA significantly suppressed cytochrome c release and caspase-7 activation, but not activation of caspases-3 and -9. Although pretreatment with a caspase-8 inhibitor (Z-IETD-FMK) reduced Cholix-induced cytochrome c release and activation of caspases-3, -7, and -9, cytotoxicity was not decreased. Pretreatment with Z-YVAD-FMK, which inhibits caspase-1, -4, and -5, suppressed not only cytochrome c release, activation of caspase-3, -7, -8, or -9, and PARP cleavage, but also cytotoxicity, indicating that caspase-1, -4, and -5 activation is initiated at an early stage of Cholix-induced apoptosis and promotes caspase-8 activation. These results show that the inflammatory caspases (caspase-1, -4, and -5) and caspase-8 are responsible for both mitochondrial signals and other caspase activation. In conclusion, we showed that Cholix-induced caspase activation plays an essential role in generation of apoptotic signals, which are mediated by both mitochondria-dependent and -independent pathways.