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 Fasmediated caspase-10-dependent pathway might play a crucial role. (Cancer Sci 2011; 102: 951-958) H epatocellular carcinoma (HCC) is one of the most common malignancies worldwide with 600 000 deaths per year, and its incidence is still on the rise.(1) Surgical treatments, such as liver resection and transplantation, are the first-line therapeutic strategies for HCC. However, the postoperative survival rate is only 30-40% at 5 years and recurrence is quite common in patients who have had a resection.(2) In addition, because HCC is a relatively chemo-resistant tumor and highly refractory to cytotoxic chemotherapy, systemic cytotoxic chemotherapy agents are minimally effective at improving the survival of patients with advanced HCC.(3,4) Therefore, development of novel chemotherapeutic agents and more effective therapies for the treatment of HCC are urgently needed. Recently, traditional Chinese medicines and their active components have attracted a great deal of attention as candidates for HCC therapy.(5)
