Bufotalin from Venenum Bufonis inhibits growth of multidrug resistant HepG2 cells through G2/M cell cycle arrest and apoptosis.

Venenum Bufonis, a traditional Chinese medicine, is widely used in the treatment of liver cancer in modern Chinese medical practices. In our search for anti-hepatoma constituents in Venenum Bufonis, bufotalin, bufalin, telocinobufagin and cinobufagin were obtained. Bufotalin was the most potent active compound among these four bufadienolides, and it exerted stronger inhibitory effect on the viability of doxorubicin-induced multidrug resistant liver cancer cells (R-HepG2) than that of their parent cells HepG2. Structure-activity relationship analysis indicated that the acetyl group linked to C-16 of bufadienolides might be useful for increasing anti-hepatoma activity. Further mechanistic studies revealed that bufotalin treatment induced cell cycle arrest at G(2)/M phase through down-regulation of Aurora A, CDC25, CDK1, cyclin A and cyclin B1, as well as up-regulation of p53 and p21. Bufotalin treatment also induced apoptosis which was accompanied by decrease in mitochondrial membrane potential, increases in intracellular calcium level and reactive oxygen species production, activations of caspase-9 and -3, cleavage of poly ADP-ribose polymerase (PARP) as well as changes in the expressions of bcl-2 and bax. It was also found that the inhibition of Akt expression and phosphorylation was involved in apoptosis induction, and specific Akt inhibitor LY294002 or siRNA targeting Akt can synergistically enhanced bufotalin-induced apoptosis. In vivo study showed that bufotalin significantly inhibited the growth of xenografted R-HepG2 cells, without body weight loss or marked toxicity towards the spleen. These results indicate that bufotalin has a promising potential to become a novel anti-cancer agent for the treatment of liver cancer with multidrug resistance.

Zhang DM ，Liu JS ，Tang MK ，Yiu A ，Cao HH ，Jiang L ，Chan JY ，Tian HY ，Fung KP ，Ye WC ... -  《-》

GL-V9, a newly synthetic flavonoid derivative, induces mitochondrial-mediated apoptosis and G2/M cell cycle arrest in human hepatocellular carcinoma HepG2 cells.

We recently established that GL-V9, a newly synthetic flavonoid derivative, is an active cytotoxic component. In this study, we demonstrated that GL-V9 inhibited cells growth via inducing apoptosis and G2/M cell cycle arrest in human hepatocellular carcinoma HepG2 cells. Following the treatment of HepG2 cells with GL-V9, we observed poly (ADP-ribose) polymerase (PARP) cleavage and activation of caspase-3 and caspase-9, while caspase-8 remained unchanged. The expression ratio of Bcl-2/Bax was also decreased in GL-V9-treated cells. Meanwhile, the cell cycle-related proteins, such as cyclin B1, CDK1 and cdc25 were down-regulated in GL-V9-induced G2/M cell cycle arrest. Furthermore, we showed that GL-V9-induced apoptosis in HepG2 cells was achieved through mitochondrial pathway. It also regulated changes of mitochondrial membrane potential and increased the production of intracellular reactive oxygen species. Besides, the growth inhibitory effect of GL-V9 was examined in vivo using murine implanted tumor model. These studies indicate that GL-V9 shows promise as a therapeutic agent against human hepatoma.

Li L ，Lu N ，Dai Q ，Wei L ，Zhao Q ，Li Z ，He Q ，Dai Y ，Guo Q ... -  《-》

Anti-tumor effect of germacrone on human hepatoma cell lines through inducing G2/M cell cycle arrest and promoting apoptosis.

Germacrone is one of the main bioactive components in the traditional Chinese medicine Rhizoma curcuma. In this study, the anti-proliferative effect of germacrone on the human hepatoma cell lines and the molecular mechanism underlying the cytotoxicity of germacrone were investigated. Treatment of human hepatoma cell lines HepG2 and Bel7402 with germacrone resulted in cell cycle arrest and apoptosis in a dose-dependent manner as measured by MTT assay, flow cytometric and fluorescent microscopy analysis, while much lower effect on normal human liver cell L02 was observed. Flow cytometric analysis revealed that germacrone induced G2/M arrest in the cell cycle progression that was associated with an obvious decrease in the protein expression of cyclin B1 and its activating partner CDK1 with concomitant inductions of p21. Hoechst 33258 and Annexin V/PI staining results showed that the total cell number in apoptosis associated with a dose-dependent up-regulation of Bax and down-regulation of Bcl-2/Bcl-xl was increased. In the meantime, the up-regulation of p53 and reactive oxygen species increase were observed, which suggested that germacrone might be a new potent chemopreventive drug candidate for liver cancer via regulating the expression of proteins related to G2/M cell cycle and apoptosis, and p53 and oxidative damage may play important roles in the inhibition of human hepatoma cells growth by germacrone.

Liu Y ，Wang W ，Fang B ，Ma F ，Zheng Q ，Deng P ，Zhao S ，Chen M ，Yang G ，He G ... -  《-》

Hellebrigenin induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells through inhibition of Akt.

Hellebrigenin, one of bufadienolides belonging to cardioactive steroids, was found in skin secretions of toads and plants of Helleborus and Kalanchoe genera. In searching for natural constituents with anti-hepatoma activities, we found that hellebrigenin, isolated from traditional Chinese medicine Venenum Bufonis, potently reduced the viability and colony formation of human hepatocellular carcinoma cells HepG2, and went on to explore the underlying molecular mechanisms. Our results demonstrated that hellebrigenin triggered DNA damage through DNA double-stranded breaks and subsequently induced cell cycle G2/M arrest associated with up-regulation of p-ATM (Ser(1981)), p-Chk2 (Tyr(68)), p-CDK1 (Tyr(15)) and Cyclin B1, and down-regulation of p-CDC25C (Ser(216)). It was also found that hellebrigenin induced mitochondrial apoptosis, characterized by Bax translocation to mitochondria, disruption of mitochondrial membrane potential, release of cytochrome c into cytosol and sequential activation of caspases and PARP. In addition, Akt expression and phosphorylation were inhibited by hellebrigenin, whereas Akt silencing with siRNA significantly blocked cell cycle arrest but enhanced apoptosis induced by hellebrigenin. Activation of Akt by human insulin-like growth factor I (hIGF-I) could obviously attenuate hellebrigenin-induced cell death. In summary, our study is the first to report the efficacy of hellebrigenin against HepG2 and elucidated its molecular mechanisms including DNA damage, mitochondria collapse, cell cycle arrest and apoptosis, which will contribute to the development of hellebrigenin into a chemotherapeutic agent in the treatment of liver cancer.

Deng LJ ，Hu LP ，Peng QL ，Yang XL ，Bai LL ，Yiu A ，Li Y ，Tian HY ，Ye WC ，Zhang DM ... -  《-》

Inhibitory effect of oleanolic acid on hepatocellular carcinoma via ERK-p53-mediated cell cycle arrest and mitochondrial-dependent apoptosis.

Wang X ，Bai H ，Zhang X ，Liu J ，Cao P ，Liao N ，Zhang W ，Wang Z ，Hai C ... -  《-》