Gambogic acid induces apoptosis and inhibits colorectal tumor growth via mitochondrial pathways

AIM: To investigate the effect of gambogic acid(GA) on apoptosis in the HT-29 human colon cancer cell line. METHODS: H-29 cells were used for in vitro experiments in this study. Relative cell viability was assessed using MTT assays. Cell apoptosis was detected by terminal deoxynucleotidyl transferase d UTP nick end labeling and Hoechst 33342 staining, and quantified by flow cytometry. Cellular ultrastructure was observed by transmission electron microscopy. Real-time PCR and Western blot analyses were used to evaluate gene and protein expression levels. For in vivo experiments, BALB/c nude mice received subcutaneous injections of HT-29 cells in the right armpit. When well-established xenografts were palpable with a tumor size of 75 mm3, mice were randomly assigned to a vehicle(negative) control, positive control or GA treatment group(n = 6 each). The animals in the treatment group received one of three dosages of GA(in saline; 5, 10 or 20 mg/kg) via the caudal vein twice weekly, whereas animals in the negative and positive control groups were given equal volumes of 0.9% saline or 10 mg/kg docetaxel, respectively, via the caudal vein once weekly. RESULTS: The cell viability assay showed that GA inhibited proliferation of HT-29 cells in a dose- and time-dependent manner after treatment with GA(0.00, 0.31, 0.62, 1.25, 2.50, 5.00 or 10.00 μmol/L) for 24, 48 or 72 h. After 48 h, the percentage of apoptotic cells in cells treated with 0.00, 1.25, 2.50 and 5.00 μmol/L GA was 1.4% ± 0.3%, 9.8% ± 1.2%, 25.7% ± 3.3% and 49.3% ± 5.8%, respectively. Ultrastructural analysis of HT-29 cells treated for 48 h with 2.5μmol/L GA revealed apoptotic bodies and condensed and fragmented nuclei. Levels of caspase-8,-9 and-3 m RNAs were significantly increased after treatment with GA(1.25, 2.50 or 5.00 μmol/L) for 48 h(P < 0.05 for all). Protein levels of apoptosis-related factors Fas, Fas L, FADD, cytochrome c, and Apaf-1 were increased in GA-treated cells, whereas levels of pro-caspase-8,-9 and-3 were significantly decreased(P < 0.05 for all). Furthermore, GA significantly and dose-dependently inhibited the growth of HT-29 tumors in a mouse xenograft model(P < 0.05).CONCLUSION: GA inhibits HT-29 proliferation via induction of apoptosis. The anti-cancer effects are likely mediated by death receptor(extrinsic) and mitochondrial(intrinsic) pathways.