Bufalin and cinobufagin induce apoptosis of human hepatocellular carcinoma cells via Fas‐ and mitochondria‐mediated pathways

• Published in: Cancer science Vol. 102; no. 5; pp. 951 - 958
• Main Authors: Qi, Fanghua, Inagaki, Yoshinori, Gao, Bo, Cui, Xiaoyan, Xu, Huanli, Kokudo, Norihiro, Li, Anyuan, Tang, Wei
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.05.2011; Blackwell
• Subjects: Antineoplastic Agents - pharmacology; Apoptosis - drug effects; Biological and medical sciences; Blotting, Western; Bufanolides - pharmacology; Carcinoma, Hepatocellular - metabolism; Cell Line, Tumor; Gastroenterology. Liver. Pancreas. Abdomen; Humans; Liver Neoplasms - metabolism; Liver. Biliary tract. Portal circulation. Exocrine pancreas; Medical sciences; Mitochondria - drug effects; Original; Signal Transduction - drug effects; Tumors; Human; Liver cancer; Mitochondria; Cancerology; Cell death; Fas Antigen; Digestive diseases; Hepatic disease; Hepatocellular carcinoma; Malignant tumor; Cancer; Apoptosis
• ISSN: 1347-9032; 1349-7006; 1349-7006
• DOI: 10.1111/j.1349-7006.2011.01900.x

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 HepG2 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 HepG2 cells via both Fas‐ and mitochondria‐mediated pathways, and a Fas‐mediated caspase‐10‐dependent pathway might play a crucial role. (Cancer Sci 2011; 102: 951–958)