telocinobufagin and bufalin

Identifying the alert groups of mechanism-based inactivators of human cytochrome P450s (hCYPs) is very helpful for early prediction of drug toxicity and for rational drug design to avoid idiosyncratic toxicity. Here, we report that a natural compound bufalin (BF) could time-dependently inactivate hCYP3A via complex CYP-catalyzed cascade oxidative metabolism. Metabolite profiling and time-dependent inhibition assays showed that 3-keto-bufalin (3-KBF), a unique nonpolar oxidative metabolite of BF, was the key substance responsible for hCYP3A inactivation. Further investigations demonstrated that 3-KBF was further metabolized by hCYPs to generate two mono-hydroxylated metabolites, which could be readily dehydrated and then covalently bound on glutathione or hCYP3A4. Collectively, this study uncovers unique CYP-catalyzed cascade oxidative pathways of BF in which two reactive intermediates bearing a Michael acceptor are finally formed as hCYP3A inactivators. These findings expand the current knowledge of CYP inactivators and suggest that some steroids bearing the 3-keto group may trigger time-dependent CYP3A inactivation.

Topics: Bufanolides; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Humans; Microsomes, Liver; Oxidative Stress

The secreted poisonin bufonids (Anura: Bufonidae) include proteins, biogenic amines, toxic bufadienolides and alkaloids. The chemical composition of the methanolic extract of parotoid gland secretions by the Amazonian toad Rhinella margaritifera was evaluated in a UFLC-DAD-micrOTOF system. Of the twenty three compounds found in the methanolic extract, eighteen were identified by the mass/charge ratio as: five arginine diacids, six bufagenins (telocinobufagin, marinobufagin, bufotalin, cinobufotalin, bufalin and cinobufagin), six bufotoxins, and an alkaloid (dehydrobufotenin).

Topics: Amphibian Venoms; Animals; Bufanolides; Bufonidae; Cell Line, Tumor; Chromatography, High Pressure Liquid; Parotid Gland

The growth inhibitory effects of bufadienolide compounds were investigated in two intractable cancer cells, a human glioblastoma cell line U-87 and a pancreatic cancer cell line SW1990. Among four bufadienolide compounds, a dose-dependent cytotoxicity was observed in these cancer cells after treatment with gamabufotalin and arenobufagin. The IC50 values of the two compounds were 3-5 times higher in normal peripheral blood mononuclear cells (PBMCs) than these values for both cancer cell lines. However, similar phenomena were not observed for two other bufadienolide compounds, telocinobufagin and bufalin. These results thus suggest that gamabufotalin and arenobufagin possess selective cytotoxic activity against tumor cells rather than normal cells. Moreover, a clear dose-dependent lactate dehydrogenase (LDH) release, a well-known hallmark of necrosis, was observed in both cancer cells treated with gamabufotalin, suggesting that gamabufotalin-mediated cell death is predominantly associated with a necrosis-like phenotype. Of most importance, treatment with as little as 8 ng/ml of gamabufotalin, even an almost non-toxic concentration to PBMCs, efficiently downregulated the percentages of CD4+CD25+Foxp3+ regulator T (Treg) cells in mitogen-activated PBMCs. Given that Treg cells play a critical role in tumor immunotolerance by suppressing antitumor immunity, these results suggest that gamabufotalin may serve as a promising candidate, as an adjuvant therapeutic agent by manipulating Treg cells to enhance the efficacy of conventional anticancer drugs and lessen their side-effects. These findings provide insights into the clinical application of gamabufotalin for cancer patients with glioblastoma/pancreatic cancer based on its cytocidal effect against tumor cells as well as its depletion of Treg cells.

Topics: Antineoplastic Agents; Bufanolides; CD4-Positive T-Lymphocytes; Forkhead Transcription Factors; Humans; Interleukin-2 Receptor alpha Subunit; Leukocytes, Mononuclear; Neoplasms; T-Lymphocytes, Regulatory

Topics: Animals; Anura; Bufanolides; Bufonidae; Digitalis; Digitalis Glycosides; Heart; Hedera; Venoms