Bufotalin from Venenum Bufonis inhibits growth of multidrug resistant HepG2 cells through G(2)/M cell cycle arrest and apoptosis
ABSTRACT 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.
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ABSTRACT: 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 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 PI3K/Akt/mTOR pathway by arenobufagin. Interestingly, inhibition of mTOR by rapamycin or siRNA duplexes augmented arenubufagin-induced apoptosis and autophagy. Finally, arenobufagin inhibited the growth of HepG2/ADM xenograft tumors, which were associated with PARP cleavage, LC3-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 arenubufagin that involve crosstalk 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.Carcinogenesis 02/2013; 34(6). DOI:10.1093/carcin/bgt060 · 5.27 Impact Factor
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ABSTRACT: A reservoir-type transdermal delivery system (TDS) of bufalin was designed and evaluated for various formulation variables like different penetration enhancers, formulation matrix, rate controlling membranes as well as biopharmaceutical characteristics. Hairless mouse skin was used in permeation experiments with Franz diffusion cells. In vitro skin permeation study showed that terpenes, especially D-limonene was the most effective enhancer when ethanol and PG were used as the vehicle with a synergistic effect. Among different rate controlling membranes, Ethylene vinyl acetate (EVA) membrane containing 19% vinyl acetate demonstrated a more suitable release rate for bufalin than the other membranes. In vivo pharmacokinetic study of the bufalin patch in rat showed steady-state of bufalin from 3 hr to 12 hr. In vivo release rate and accumulative amount analyzed by deconvolution method demonstrated the sustained release of bufalin as long as the patch remained on the animal for at least 12 hr. The MRT increased from 1 hr of IV administration to 9 hr of transdermal administration. In vitro permeation across mouse skin was found to have biphasic correlation with plasma AUC in the in vivo pharmacokinetic study. Current in vitro in vivo correlation (IVIVC) enabled the prediction of pharmacokinetic profile of bufalin from in vitro permeation results. In conclusion, current reservoir transdermal patch containing 10% D-limonene as a permeation enhancer, 40% ethanol, 30% PG and 15% carbopol-water gel complex provided an improved sustained release of bufalin through transdermal administration. The bufalin patch was successfully applied to biopharmaceutical study in rats and demonstrated the feasibility of this transdermal formulation for future development and clinical trials.International Journal of Pharmaceutics 03/2013; 447(1-2). DOI:10.1016/j.ijpharm.2013.02.048 · 3.79 Impact Factor
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ABSTRACT: Thorough simplification of vinca alkaloids based on pharmacophore similarity has been conducted. A concise process for the syntheses of target compounds was successfully developed with yields from poor to excellent (19-98%). Cell growth inhibitory activities of these synthesized compounds were evaluated in five cancer cell lines including MCF-7, MDA-MB-231, HepG2, HepG2/ADM and K562. Almost all compounds exhibited moderate antitumor activity with optimal IC50 value of 0.89 ± 0.07 μM in MCF-7 cells. Investigation of structure-activity relationship (SAR) indicates that electron-withdraw substituents on the ring contribute to the enhancement of the antitumor activities. The simplified vinca alkaloids are confirmed as antimitotic agents, which inhibit the polymerization of tubulin just like vinblastine.European Journal of Medicinal Chemistry 05/2013; 65C:158-167. DOI:10.1016/j.ejmech.2013.04.057 · 3.43 Impact Factor