Gambogic acid (GA) is a medicinal compound derived from the gamboges resin of the tree, Garcinia hanburyi GA has documented cytotoxic activity against tumor cell lines in culture, with concentrations required for killing 50% of cells (Lethal Dose 50% [LD50]) of ~ 1 μM (1, 2). This natural product also displays anti-tumor activity in preclinical mouse models involving human tumor xenografts (3–5). In contrast, GA is reportedly well tolerated in mice and rats (2, 4, 6), suggesting that a therapeutic window might be identified at which tumor but not normal cells are killed. It would therefore be interesting to know the cytotoxic mechanism of GA.
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Gambogic Acid Is a Tissue-Specific Proteasome Inhibitor In Vitro and In Vivo.
Li X, Liu S, Huang H, Liu N, Zhao C, et al. Cell Rep. 2012 Dec 19. pii: S2211-1247(12)00420-2. doi: 10.1016/j.celrep.2012.11.023.
Gambogic acid (GA) is a natural compound derived from Chinese herbs that has been approved by the Chinese Food and Drug Administration for clinical trials in cancer patients; however, its molecular targets have not been thoroughly studied. Here, we report that GA inhibits tumor proteasome activity, with potency comparable to bortezomib but much less toxicity. First, GA acts as a prodrug and only gains proteasome-inhibitory function after being metabolized by intracellular CYP2E1. Second, GA-induced proteasome inhibition is a prerequisite for its cytotoxicity and anticancer effect without off-targets. Finally, because expression of the CYP2E1 gene is very high in tumor tissues but low in many normal tissues, GA could therefore produce tissue-specific proteasome inhibition and tumor-specific toxicity, with clinical significance for designing novel strategies for cancer treatment.

Gambogic acid is an antagonist of anti-apoptotic Bcl-2-family proteins
Dayong Zhai, Chaofang Jin, Chung-wai Shiau, et al. Mol Cancer Ther. 2008 June; 7(6): 1639–1646.. doi: 10.1158/1535-7163.MCT-07-2373
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 (HTS) assay for activators of caspases, proteases involved in apoptosis. Using the anti-apoptotic 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 fluorescent polarization assay (FPA). Analysis of competition for BH3 peptide binding revealed that GA inhibits all 6 human Bcl-2-family proteins to various extents, with Mcl-1 and Bcl-B the most potently inhibited (concentrations required for 50% inhibition [IC50] <1 μM). Competition for BH3 peptide binding was also confirmed using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. GA functionally inhibited the anti-apoptotic Bcl-2-family proteins, as demonstrated 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 analogs 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 anti-apoptotic 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 anti-apoptotic Bcl-2-family proteins may be among the cytotoxic mechanisms by which GA kills tumor cells.

Gambogic acid mediates apoptosis as a p53 inducer through down-regulation of mdm2 in wild-type p53-expressing cancer cells
Hongyan Gu, Xiaotang Wang, Shuyun Rao, et al. Mol Cancer Ther October 2008 7; 3298. doi: 10.1158/1535-7163.MCT-08-0212
Gambogic acid (GA) is a natural product with potent apoptotic activity. Here, we showed that GA broadly inhibited the growth of cancer cells that expressed wild-type p53 as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol-iumbromide assay, 3H-thymidine incorporation analysis, and an in vivo mouse xenograft model. GA induced massive cell apoptosis as judged by Annexin V and propidium iodide dual-staining experiments. Furthermore, we found that GA partially induced cancer cell growth inhibition in a p53-dependent manner because cell survival could be restored after endogenous p53 was attenuated by p53 transcriptional repressor pifithrin-α or p53 small interfering RNA. Interestingly, GA had no influence on p53 mRNA synthesis but dramatically enhanced its protein expression. This unique observation could be accounted for by the down-regulation of mdm2 at both mRNA and protein levels. It is concluded that GA enhances p53 protein level through inhibition of mdm2 expression and thereby hampers p53 harboring tumor growth.

Gambogic acid induces death inducer-obliterator 1-mediated apoptosis in Jurkat T cells
Yong Wang, Yan Chen, Zi Chen, et al. Acta Pharmacologica Sinica (2008) 29, 349–354; doi:10.1111/j.1745-7254.2008.00762.x
To explore the anticancer effects and the molecular mechanisms of gambogic acid (GA) on Jurkat cells.
Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Annexin V-fluorescein-isothiocyanate/propidium iodide, DNA defragmentation, and comet assay were used to detect apoptosis. Western blotting was used to study the expression of death inducer-obliterator-1 (DIO-1), Bcl-2, NF-κB, and procaspase 3, as well as 2 activated subunits: p17 and p20. The subcellular localization of DIO-1 was examined by immunofluorescence and Hoechst33258 staining.
GA inhibited the proliferation of Jurkat cells with 50% inhibitory concentration values of 1.51±0.09 (24 h), 0.98±0.13 (48 h), and 0.67±0.12 μmol/L (72 h). GA was able to induce apoptosis of Jurkat cells. Treated by GA, the expression of DIO-1 was upregulated, and that of Bcl-2 and NF-κB was downregulated, leading to the activation of pro-caspase 3. GA induced the translocation of DIO-1 to the nucleus.
GA suppressed the proliferation of Jurkat cells by apoptosis induction. DIO-1 triggered early-stage cell death in GA-treated Jurkat cells.

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