Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. May 28, 2015; 21(20): 6194-6205
Published online May 28, 2015. doi: 10.3748/wjg.v21.i20.6194
Gambogic acid induces apoptosis and inhibits colorectal tumor growth via mitochondrial pathways
Guang-Ming Huang, Yu Sun, Xin Ge, Xin Wan, Chun-Bo Li
Guang-Ming Huang, Yu Sun, Xin Ge, Xin Wan, Chun-Bo Li, Department of General Surgery, Heilongjiang Provincial Hospital, Harbin 150036, Heilongjiang Province, China
Author contributions: Huang GM and Sun Y contributed equally to this work; Huang GM, Sun Y and Ge X designed the research; Huang GM, Wan X and Li CB performed the research; Sun Y analyzed the data; Huang GM, Sun Y and Ge X wrote the manuscript.
Ethics approval: The study was reviewed and approved by the Heilongjiang Provincial Hospital Institutional Review Board.
Institutional animal care and use committee: All procedures involving animals were reviewed and approved by the Ethics Committee of Heilongjiang Province’s Hospital (IACUC protocol number: 2008-010).
Conflict-of-interest: The authors have no conflict of interest to declare.
Data sharing: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Xin Ge, MD, Department of General Surgery, Heilongjiang Provincial Hospital, 82 Zhongshan Road, Harbin 150036, Heilongjiang Province, China. gexin628@163.com
Telephone: +86-451-87131193 Fax: +86-451-87131195
Received: September 28, 2014
Peer-review started: September 29, 2014
First decision: October 29, 2014
Revised: November 22, 2014
Accepted: January 30, 2015
Article in press: January 30, 2015
Published online: May 28, 2015
Processing time: 244 Days and 7.6 Hours
Abstract

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 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 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 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).

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.

Keywords: Apoptosis; Death receptor pathway; Flow cytometry; Gambogic acid; Hoechst 33342; HT-29 cells; Mitochondrial pathway; MTT; Terminal deoxynucleotidyl transferase dUTP nick end labeling

Core tip: This study evaluated the effects of gambogic acid on colon cancer cells. Treatment of a human colon cancer cell line with gambogic acid inhibited proliferation via induction of apoptosis. Moreover, the growth of colon cancer cell xenograft tumors in mice was reduced by injections of gambogic acid. These anti-cancer effects were likely mediated through death receptor and mitochondrial pathways.