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Research Articles: Therapeutics, Targets, and Development

Gambogic acid is an antagonist of antiapoptotic Bcl-2 family proteins

Burnham Institute for Medical Research, La Jolla, California

Requests for reprints: John C. Reed, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone: 858-646-3368; Fax: 858-646-3194. E-mail: reedoffice{at}burnham.org

Abstract

The natural product gambogic acid (GA) has been reported to have cytotoxic activity against tumor cells in culture and was identified as an active compound in a cell-based high-throughput screening assay for activators of caspases, proteases involved in apoptosis. Using the antiapoptotic Bcl-2 family protein, Bfl-1, as a target for screening of a library of natural products, we identified GA as a competitive inhibitor that displaced BH3 peptides from Bfl-1 in a fluorescence polarization assay. Analysis of competition for BH3 peptide binding revealed that GA inhibits all six human Bcl-2 family proteins to various extents, with Mcl-1 and Bcl-B the most potently inhibited [concentrations required for 50% inhibition (IC₅₀), <1µmol/L]. Competition for BH3 peptide binding was also confirmed using a time-resolved fluorescence resonance energy transfer assay. GA functionally inhibited the antiapoptotic Bcl-2 family proteins as shown by experiments using isolated mitochondria in which recombinant purified Bcl-2 family proteins suppress SMAC release in vitro, showing that GA neutralizes their suppressive effects on mitochondria in a concentration-dependent manner. GA killed tumor cell lines via an apoptotic mechanism, whereas analogues of GA with greatly reduced potency at BH3 peptide displacement showed little or no cytotoxic activity. However, GA retained cytotoxic activity against bax^–/–bak^–/– cells in which antiapoptotic Bcl-2 family proteins lack a cytoprotective phenotype, implying that GA also has additional targets that contribute to its cytotoxic mechanism. Altogether, the findings suggest that suppression of antiapoptotic Bcl-2 family proteins may be among the cytotoxic mechanisms by which GA kills tumor cells. [Mol Cancer Ther 2008;7(6):1639–46]

Grant support: NIH grants CA-113318 and CA-55164.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

Received 12/ 5/07; revised 4/ 3/08; accepted 4/ 7/08.