Orignal Articles
Copyright ©2009 The WJG Press and Baishideng. All rights reserved.
World J Gastroenterol. May 14, 2009; 15(18): 2234-2239
Published online May 14, 2009. doi: 10.3748/wjg.15.2234
Induction of apoptosis in human liver carcinoma HepG2 cell line by 5-allyl-7-gen-difluoromethylenechrysin
Xiang-Wen Tan, Hong Xia, Jin-Hua Xu, Jian-Guo Cao
Xiang-Wen Tan, Department of Laboratory Animal Science, University of South China, Hengyang 421001, Hunan Province, China
Hong Xia, Institute of Cancer Research, University of South China, Hengyang 421001, Hunan Province, China
Jin-Hua Xu, Center of Biochemistry and Molecular Biology Laboratory, University of South China, Hengyang 421001, Hunan Province, China
Jian-Guo Cao, Laboratory of Medicine Engineering, Medical College, Hunan Normal University, Changsha 410006, Hunan Province, China
Author contributions: Tan XW, Xia H and Xu JH performed the majority of experiments; Tan XW wrote the manuscript; Xia H performed statistical analysis and edited the manuscript; Cao JG designed the study.
Correspondence to: Dr. Jian-Guo Cao, Laboratory of Medicine Engineering, Medical College, Hunan Normal University, Changsha 410006, Hunan Province, China. caojianguo2005@yahoo.com.cn
Telephone: +86-734-8282521
Fax: +86-734-8282521
Received: January 15, 2009
Revised: March 21, 2009
Accepted: March 28, 2009
Published online: May 14, 2009
Abstract

AIM: To investigate the effect of 5-allyl-7-gen-difluoromethylenechrysin (ADFMChR) on apoptosis of human liver carcinoma HepG2 cell line and the molecular mechanisms involved.

METHODS: HepG2 cells and L-02 cells were cultured in vitro and the inhibitory effect of ADFMChR on their proliferation was measured by MTT assay. The apoptosis of HepG2 cells was determined by flow cytometry (FCM) using propidium iodide (PI) fluorescence staining. DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of ADFMChR on the proxisome proliferator-activated receptor γ (PPARγ), NF-κB, Bcl-2 and Bax protein expression of HepG2 cells were analyzed by Western blotting.

RESULTS: MTT assay showed that ADFMChR significantly inhibited proliferation of HepG2 cells in a dose-dependent manner, with little effect on growth of L-02 cells, and when IC50 was measured as 8.45 &mgr;mol/L and 191.55 &mgr;mol/L respectively, the potency of ADFMChR to HepG2 cells, was found to be similar to 5-fluorouracil (5-FU, IC50 was 9.27 &mgr;mol/L). The selective index of ADFMChR cytotoxicity to HepG2 cells was 22.67 (191.55/8.45), higher than 5-FU (SI was 7.05 (65.37/9.27). FCM with PI staining demonstrated that the apoptosis rates of HepG2 cells treated with 3.0, 10.0 and 30.0 &mgr;mol/L ADFMChR for 48 h were 5.79%, 9.29% and 37.8%, respectively, and were significantly higher when treated with 30.0 &mgr;mol/L ADFMChR than when treated with 30.0 &mgr;mol/L ChR (16.0%) (P < 0.05) and were similar to those obtained with 30.0 &mgr;mol/L 5-FU (41.0%). DNA agarose gel electrophoresis showed that treatment of HepG2 cells with 10.0 &mgr;mol/L ADFMChR for 48 h and 72 h resulted in typical DNA ladders which could be reversed by 10.00 &mgr;mol/L GW9662, a blocker of PPARγ. Western blotting analysis revealed that after 24 h of treatment with 3.0, 10.0, 30.0 &mgr;mol/L ADFMChR, PPARγ and Bax protein expression in HepG2 cells increased but Bcl-2 and NF-κB expression decreased; however, pre-incubation with 10.0 &mgr;mol/L GW9662 could efficiently antagonize and weaken the regulatory effect of 3.0, 30.0 &mgr;mol/L ADFMChR on PPARγ and NF-κB protein expression in HepG2 cells.

CONCLUSION: ADFMChR induces apoptosis of HepG2 cell lines by activating PPARγ, inhibiting protein expression of Bcl-2 and NF-κB, and increasing Bax expression.

Keywords: Liver neoplasm, Chrysin, 5-allyl-7-gen-difluoromethylenechrysin, Apoptosis, Proxisome proliferator-activated receptor γ