title: Characterization of Cholix toxin-induced apoptosis in HeLa cells.
abstract: 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.
authors: Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, Japan.
authors: Kohei
authors: K
authors: Ogura
authors: Kinnosuke
authors: K
authors: Yahiro
authors: Hiroyasu
authors: H
authors: Tsutsuki
authors: Sayaka
authors: S
authors: Nagasawa
authors: Shinji
authors: S
authors: Yamasaki
authors: Joel
authors: J
authors: Moss
authors: Masatoshi
authors: M
authors: Noda
mesh_terms: D020727:ADP-Ribosylation Factors / Q000737:chemistry / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D001427:Bacterial Toxins / Q000737:chemistry / Q000494:pharmacology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects*; D014734:Vibrio cholerae / Q000737:chemistry*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C494772:BAK1 protein, human; C494826:BAX protein, human; D001427:Bacterial Toxins; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D034741:RNA, Small Interfering; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; C403753:benzyloxycarbonyl-isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone; D045304:Cytochromes c; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases; D020727:ADP-Ribosylation Factors; C529269:cholix toxin, Vibrio cholerae