bufotalin and Liver-Neoplasms

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.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bufanolides; Carcinoma, Hepatocellular; Caspases; Cell Cycle Proteins; Cell Proliferation; Drug Resistance, Multiple; Enzyme Activation; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; M Phase Cell Cycle Checkpoints; Male; Membrane Potential, Mitochondrial; Mice; Proto-Oncogene Proteins c-akt; Xenograft Model Antitumor Assays

Bufotalin is one of the bufadienolides isolated from Formosan Ch'an Su, which is made of the skin and parotid glands of toads. Ingestion of toad venom results in severe morbidity and high mortality. Although Ch'an Su is clinically toxic, it has been used as an important traditional Chinese medicine for heart failure and pains. In this study, bufotalin-induced apoptosis in human hepatocellular carcinoma Hep 3B cells was investigated. The results indicate that externalization of phosphatidylserine, accumulation of sub-G(1) cells, fragmentation of DNA, and formation of apoptotic bodies were observed in bufotalin-treated Hep 3B cells. The signaling pathway might be via the activation of caspase-8, increase in mitochondrial tBid, disruption of mitochondrial membrane potential, and translocation of apoptosis-inducing factor (AIF). Active caspase-8 might activate caspase-9 and caspase-3 leading to the cleavage of nuclear PARP. Presence of AIF and cleaved PARP in the nuclei might lead to DNA fragmentation. Caspase-8 inhibitor (Z-IETD) or wide-ranging caspase inhibitor (Z-VAD) significantly suppressed the bufotalin-induced apoptosis, while the anti-Fas neutralization antibody had no effect. These data suggest that bufotalin-induced apoptosis in Hep 3B cells might involve caspases and AIF.

Topics: Apoptosis; Apoptosis Inducing Factor; Bufanolides; Carcinoma, Hepatocellular; Caspase 8; Caspases; Cell Line, Tumor; Humans; Liver Neoplasms; Signal Transduction

QSAR analysis has been used to identify the essential structural requirements for increasing the inhibitory activities of selected bufadienolides from the Chinese drug Ch'an Su (and other sources) against the primary liver carcinoma cell line PLC/PRF/5 (PLC) and the derived colchicine-resistant line (COL). The variable substituent domain of the proposed pharmacophore of the bufadienolides was investigated using a Comparative Molecular Field Analysis (CoMFA) approach. A model with considerable predictive ability was obtained. In addition, the CoMFA results agreed well with the pharmacophore bufadienolide model for the parent PLC line proposed earlier.

Topics: Antineoplastic Agents; Bufanolides; Colchicine; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Humans; Liver Neoplasms; Models, Molecular; Quantitative Structure-Activity Relationship; Tumor Cells, Cultured