Vibrio cholerae Cholix Toxin-Induced HepG2 Cell Death is Enhanced by Tumor Necrosis Factor-Alpha Through ROS and Intracellular Signal-Regulated Kinases.

Abstract

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.