title: Gambogic acid induces apoptosis and inhibits colorectal tumor growth via mitochondrial pathways.
abstract: OBJECTIVE
  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 dUTP 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 mm(3), 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 2.5 μmol/L GA revealed apoptotic bodies and condensed and fragmented nuclei. Levels of caspase-8, -9 and -3 mRNAs 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, FasL, 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).
  
  
  CONCLUSIONS
  GA inhibits HT-29 proliferation via induction of apoptosis. The anti-cancer effects are likely mediated by death receptor (extrinsic) and mitochondrial (intrinsic) pathways.
authors.affiliation: Guang-Ming Huang, Yu Sun, Xin Ge, Xin Wan, Chun-Bo Li, Department of General Surgery, Heilongjiang Provincial Hospital, Harbin 150036, Heilongjiang Province, China.
authors.forename: Guang-Ming
authors.initials: GM
authors.lastname: Huang
authors.affiliation: Guang-Ming Huang, Yu Sun, Xin Ge, Xin Wan, Chun-Bo Li, Department of General Surgery, Heilongjiang Provincial Hospital, Harbin 150036, Heilongjiang Province, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Guang-Ming Huang, Yu Sun, Xin Ge, Xin Wan, Chun-Bo Li, Department of General Surgery, Heilongjiang Provincial Hospital, Harbin 150036, Heilongjiang Province, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Ge
authors.affiliation: Guang-Ming Huang, Yu Sun, Xin Ge, Xin Wan, Chun-Bo Li, Department of General Surgery, Heilongjiang Provincial Hospital, Harbin 150036, Heilongjiang Province, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Wan
authors.affiliation: Guang-Ming Huang, Yu Sun, Xin Ge, Xin Wan, Chun-Bo Li, Department of General Surgery, Heilongjiang Provincial Hospital, Harbin 150036, Heilongjiang Province, China.
authors.forename: Chun-Bo
authors.initials: CB
authors.lastname: Li
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D004305:Dose-Response Relationship, Drug; D015972:Gene Expression Regulation, Neoplastic; D019073:HT29 Cells; D006801:Humans; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D047368:Tumor Burden / Q000187:drug effects; D044004:Xanthones / Q000494:pharmacology*; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D012333:RNA, Messenger; D044004:Xanthones; C052659:gambogic acid
keywords: Apoptosis; Death receptor pathway; Flow cytometry; Gambogic acid; HT-29 cells; Hoechst 33342; MTT; Mitochondrial pathway; Terminal deoxynucleotidyl transferase dUTP nick end labeling