Vibrio cholerae Cholix Toxin-Induced HepG2 Cell Death is Enhanced by Tumor Necrosis Factor-Alpha Through ROS and Intracellular Signal-Regulated Kinases
Vibrio cholerae Cholix Toxin-Induced HepG2 Cell Death is Enhanced by Tumor Necrosis Factor-Alpha Through ROS and Intracellular Signal-Regulated Kinases
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DOI：
10.1093/toxsci/kfx009
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发表时间：
2017-04
影响因子：
3.8
通讯作者：
K. Ogura;Y. Terasaki;T. Miyoshi-Akiyama;Mika Terasaki;J. Moss;M. Noda;K. Yahiro
中科院分区：
文献类型：
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作者：
K. Ogura;Y. Terasaki;T. Miyoshi-Akiyama;Mika Terasaki;J. Moss;M. Noda;K. Yahiro
霍乱弧菌的Cholix毒素（Cholix）是一种强毒力因子，能抑制宿主细胞的真核延伸因子2（eEF 2），从而抑制蛋白质合成。给小鼠施用Cholix或其同系物假单胞菌外毒素A（PEA）可导致致死性肝细胞损伤。在本研究中，我们证明了在存在肿瘤坏死因子α（TNF-α）的情况下Cholix对人肝细胞的细胞毒性，据报道，TNF-α在小鼠PEA给药中起致命作用。与单独与Cholix孵育相比，TNF-α和Cholix联合处理（TNF-α/Cholix）可显著增强caspase的激活、细胞色素c从线粒体释放到细胞质以及聚ADP核糖聚合酶（PARP）的裂解，而单独与TNF-α孵育或TNF-α/无催化活性的Cholix联合处理则无此作用。在细胞死亡的早期阶段，Cholix增加有丝分裂原活化蛋白激酶的磷酸化（例如，p38、ERK、JNK）和Akt的表达均不受TNF-α单独作用的影响。MAPK抑制剂（SP 600125、SB 20852和U 0126）可抑制TNF-α/Cholix诱导的PARP裂解。蛋白激酶抑制剂Go 6976抑制TNF-α/Cholix对JNK磷酸化和PARP的切割。相比之下，在TNF-α不存在的情况下，PKC激活剂PMA促进Cholix诱导的PARP裂解。活性氧（ROS）抑制剂N-乙酰半胱氨酸（NAC）可抑制TNF-α/Cholix诱导的JNK和ERK磷酸化，从而抑制PARP裂解。这些数据表明，ROS和JNK途径是TNF-α/Cholix诱导的HepG 2细胞死亡的重要介质。
Cholix toxin (Cholix) from Vibrio cholerae is a potent virulence factor exhibiting ADP-ribosyltransferase activity on eukaryotic elongation factor 2 (eEF2) of host cells, resulting in the inhibition of protein synthesis. Administration of Cholix or its homologue Pseudomonas exotoxin A (PEA) to mice causes lethal hepatocyte damage. In this study, we demonstrate cytotoxicity of Cholix on human hepatocytes in the presence of tumor necrosis factor α (TNF-α), which has been reported to play a fatal role in PEA administered to mice. Compared with incubating HepG2 cells with Cholix alone, co-treatment with TNF-α and Cholix (TNF-α/Cholix) significantly enhanced the activation of caspases, cytochrome c release from mitochondria into cytoplasm, and poly-ADP-ribose polymerase (PARP) cleavage, while incubation with TNF-α alone or co-treatment with TNF-α/catalytically inactive Cholix did not. In the early stage of cell death, Cholix increased phosphorylation of mitogen-activated protein kinases (e.g., p38, ERK, JNK) and Akt, which was not affected by TNF-α alone. MAPK inhibitors (SP600125, SB20852, and U0126) suppressed PARP cleavage induced by TNF-α/Cholix. Protein kinase inhibitor Go6976 suppressed JNK phosphorylation and PARP cleavage by TNF-α/Cholix. In contrast, PKC activator PMA in the absence of TNF-α promoted Cholix-induced PARP cleavage. Reactive oxygen species (ROS) inhibitor, N-acetyl cysteine (NAC), suppressed TNF-α/Cholix-induced JNK and ERK phosphorylation, resulting in inhibition of PARP cleavage. These data suggest that ROS and JNK pathways are important mediators of TNF-α/Cholix-induced HepG2 cell death.