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.

Acerinol, a cyclolanstane triterpenoid from Cimicifuga acerina, reverses ABCB1-mediated multidrug resistance in HepG2/ADM and MCF-7/ADR cells.

Persistent cancer chemotherapy can lead to multidrug resistance which is one of the most common reasons for failure of chemotherapy. The ABCB1 transporter is a member of the ATP-binding cassette superfamily and it is frequently over-expressed in multidrug resistant cancer cells. Active ingredients derived from traditional Chinese medicinal herbs have been reported to reverse multidrug resistance mediated by ATP-binding cassette transporters. In this study, acerinol, isolated from Cimicifuga acerina, was tested for its potential to modulate the ABCB1 transporter. Our results demonstrated that acerinol could increase the chemosensitivity of ABCB1-overexpressing HepG2/ADM and MCF-7/ADR cells to chemotherapeutic drugs, doxorubicin, vincristine and paclitaxel. Furthermore, it could also increase the retention of ABCB1 substrates doxorubicin and rhodamine 123 in HepG2/ADM and MCF-7/ADR cells. A mechanistic study showed that acerinol significantly stimulated the activity of ABCB1 ATPase without affecting the expression of ABCB1 on neither mRNA nor protein level. Acerinol was also found to reverse the resistance of MCF-7/ADR cells to vincristine, dependent partly on ABCB1. In addition, acerinol׳s action was reversible, suggesting that acerinol may act as a competitive inhibitor of ABCB1 by competing with other drug substrates like doxorubicin. Indeed, docking analysis indicated that acerinol would most likely bind to the sites on ABCB1 that partly overlapped with that of verapamil. In conclusion, the present study is the first to show that acerinol from C. acerina significantly enhances the cytotoxicity of chemotherapeutic drugs by modulating the function of ABCB1. It is hopeful to develop acerinol as a new multidrug resistance reversal agent.

Arenobufagin, a natural bufadienolide from toad venom, induces apoptosis and autophagy in human hepatocellular carcinoma cells through inhibition of PI3K/Akt/mTOR pathway.

Hepatocellular carcinoma (HCC) is a deadly form of cancer without effective chemotherapy so far. Currently, only sorafenib, a multitargeted tyrosine kinase inhibitor, slightly improves survival in HCC patients. In searching for natural anti-HCC components from toad venom, which is frequently used in the treatment of liver cancer in traditional Chinese medicine, we discovered that arenobufagin, a bufadienolide from toad venom, had potent antineoplastic activity against HCC HepG2 cells as well as corresponding multidrug-resistant HepG2/ADM cells. We found that arenobufagin induced mitochondria-mediated apoptosis in HCC cells, with decreasing mitochondrial potential, as well as increasing Bax/Bcl-2 expression ratio, Bax translocation from cytosol to mitochondria. Arenobufagin also induced autophagy in HepG2/ADM cells. Autophagy-specific inhibitors (3-methyladenine, chloroquine and bafilomycin A1) or Beclin1 and Atg 5 small interfering RNAs (siRNAs) enhanced arenobufagin-induced apoptosis, indicating that arenobufagin-mediated autophagy may protect HepG2/ADM cells from undergoing apoptotic cell death. In addition, we observed the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway by arenobufagin. Interestingly, inhibition of mTOR by rapamycin or siRNA duplexes augmented arenobufagin-induced apoptosis and autophagy. Finally, arenobufagin inhibited the growth of HepG2/ADM xenograft tumors, which were associated with poly (ADP-ribose) polymerase cleavage, light chain 3-II activation and mTOR inhibition. In summary, we first demonstrated the antineoplastic effect of arenobufagin on HCC cells both in vitro and in vivo. We elucidated the underlying antineoplastic mechanisms of arenobufagin that involve cross talk between apoptosis and autophagy via inhibition of the PI3K/Akt/mTOR pathway. This study may provide a rationale for future clinical application using arenobufagin as a chemotherapeutic agent for HCC.

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.

Anemoside A3-induced relaxation in rat renal arteries: role of endothelium and Ca2+ channel inhibition.

Anemoside A(3), a lupane-type triterpenoid saponin, exists in the roots of Pulsatilla chinensis, but its pharmacological properties are largely unknown. The present study aimed to investigate the mechanisms underlying anemoside A(3)-induced relaxation in rat renal arteries. Changes of isometric force were determined on arteries with a myograph. Anemoside A(3) caused concentration-dependent relaxation in precontracted aortas, mesenteric, left coronary, and renal arteries. Removal of endothelium or treatment with charybdotoxin plus apamin slightly but significantly attenuated the relaxation in renal arteries. TEA(+) inhibited the relaxation caused by anemoside A(3) in renal arteries with and without endothelium while glibenclamide, BaCl(2), or capsaicin had no effect on it. Anemoside A(3) produced less relaxation in rings contracted by 60 mM KCl compared with rings contracted by receptor-dependent constrictors. It further inhibited contractions induced by Ca(2+) influx through nifedipine-sensitive voltage-gated Ca(2+) channels, nifedipine-insensitive receptor-operated Ca(2+) channels, and by intracellular Ca(2+) release. Pretreatment with nifedipine attenuated anemoside A(3)-induced relaxation. Taken together, the present results indicate that anemoside A(3) produces relaxation in rat renal arteries through multiple mechanisms. The release of CTX/apamin-sensitive endothelium-derived hyperpolarizing factor, stimulation of TEA(+)-sensitive K(+) channel, and inhibition of Ca(2+) influx jointly contribute to the relaxation.