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]
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