title: Bufalin and cinobufagin induce apoptosis of human hepatocellular carcinoma cells via Fas- and mitochondria-mediated pathways.
abstract: Bufadienolides bufalin and cinobufagin are cardiotonic steroids isolated from the skin and parotid venom glands of the toad Bufo bufo gargarizans Cantor. They have been shown to induce a wide spectrum of cancer cell apoptosis. However, the detailed molecular mechanisms of inducing apoptosis in hepatocellular carcinoma (HCC) are still unclear. In the present study, the apoptosis-inducing effect of bufalin and cinobufagin on HCC cell line HepG(2) was investigated. We found bufalin and cinobufagin induced marked changes in apoptotic morphology and significantly increased the proportion of apoptotic cells. This apoptotic induction was associated with an increase in Fas, Bax and Bid expression, a decrease in Bcl-2 expression, disruption of the mitochondrial membrane potential, release of cytochrome c, activation of caspase-3, -8, -9 and -10, and the cleavage of poly(ADP-ribose)polymerase (PARP), which indicated that bufalin and cinobufagin induced apoptosis through both Fas- and mitochondria-mediated pathways. In addition, caspase activation during bufalin- and cinobufagin-induced apoptosis was further confirmed by caspase-3 inhibitor Z-DEVD-FMK, caspase-8 inhibitor Z-IETD-FMK, caspase-9 inhibitor Z-LEHD-FMK and caspase-10 inhibitor Z-AEVD-FMK. The results showed that bufalin- and cinobufagin-induced apoptosis was blocked by these inhibitors and particularly by caspase-10 inhibitor. Taken together, bufalin and cinobufagin induce apoptosis of HepG(2) cells via both Fas- and mitochondria-mediated pathways, and a Fas-mediated caspase-10-dependent pathway might play a crucial role.
authors.affiliation: Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Fanghua
authors.initials: F
authors.lastname: Qi
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Inagaki
authors.forename: Bo
authors.initials: B
authors.lastname: Gao
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Cui
authors.forename: Huanli
authors.initials: H
authors.lastname: Xu
authors.forename: Norihiro
authors.initials: N
authors.lastname: Kokudo
authors.forename: Anyuan
authors.initials: A
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Tang
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002018:Bufanolides / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D002018:Bufanolides; C002471:cinobufagin; C022777:bufalin