Original Article
Copyright ©2012 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Apr 21, 2012; 18(15): 1753-1764
Published online Apr 21, 2012. doi: 10.3748/wjg.v18.i15.1753
Pro-apoptotic effects of tectorigenin on human hepatocellular carcinoma HepG2 cells
Chun-Ping Jiang, Hui Ding, Da-Hua Shi, Yu-Rong Wang, Er-Guang Li, Jun-Hua Wu
Chun-Ping Jiang, The First Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing 210046, Jiangsu Province, China
Chun-Ping Jiang, Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital, Medical School, Nanjing University, Nanjing 210008, Jiangsu Province, China
Hui Ding, Da-Hua Shi, Yu-Rong Wang, Er-Guang Li, Jun-Hua Wu, Jiangsu Key Laboratory of Molecular Medicine, State Key Laboratory of Pharmaceutical Biotechnology, Medical School, Nanjing University, Nanjing 210093, Jiangsu Province, China
Author contributions: Ding H, Shi DH and Wu JH designed the research; Jiang CP, Ding H, Shi DH, Wang YR and Wu JH performed the research; Jiang CP, Li EG and Wu JH did data analysis and wrote the paper; Jiang CP and Wu JH applied funds for the study.
Supported by The National Natural Science Foundation of China, No. NSFC30801417; Natural Science Foundation of Jiangsu Province, No. BK2009010 and BK2008267; Doctoral Fund of Ministry of Education of China, No. RFDP200802841004; and Science Fund of Ministry of Health of China, No. LW201008
Correspondence to: Dr. Jun-Hua Wu, Jiangsu Key Laboratory of Molecular Medicine, State Key Laboratory of Pharmaceutical Biotechnology, Medical School, Nanjing University, Nanjing 210093, Jiangsu Province, China. wujunhua@nju.edu.cn
Telephone: +86-25-83593192 Fax: +86-25-83593192
Received: June 19, 2011
Revised: September 3, 2011
Accepted: October 14, 2011
Published online: April 21, 2012
Abstract

AIM: To investigate the effects of tectorigenin on human hepatocellular carcinoma (HCC) HepG2 cells.

METHODS: Tectorigenin, one of the main components of rhizome of Iris tectorum, was prepared by simple methods, such as extraction, filtration, concentration, precipitation and recrystallization. HepG2 cells were incubated with tectorigenin at different concentrations, and their viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was detected by morphological observation of nuclear change, agarose gel electrophoresis of DNA ladder, and flow cytometry with Hoechst 33342, Annexin V-EGFP and propidium iodide staining. Generation of reactive oxygen species was quantified using DCFH-DA. Intracellular Ca2+ was monitored by Fura 2-AM. Mitochondrial membrane potential was monitored using Rhodamine 123. Release of cytochrome c from mitochondria to cytosol was detected by Western blotting. Activities of caspase-3, -8 and -9 were investigated by Caspase Activity Assay Kit.

RESULTS: The viability of HepG2 cells treated by tectorigenin decreased in a concentration- and time-dependent manner. The concentration that reduced the number of viable HepG2 cells by 50% (IC50) after 12, 24 and 48 h of incubation was 35.72 mg/L, 21.19 mg/L and 11.06 mg/L, respectively. However, treatment with tectorigenin at 20 mg/L resulted in a very slight cytotoxicity to L02 cells after incubation for 12, 24 or 48 h. Tectorigenin at a concentration of 20 mg/L greatly inhibited the viability of HepG2 cells and induced the condensation of chromatin and fragmentation of nuclei. Tectorigenin induced apoptosis of HepG2 cells in a time- and dose-dependent manner. Compared with the viability rate, induction of apoptosis was the main mechanism of the anti-proliferation effect of tectorigenin in HepG2 cells. Furthermore, tectorigenin-induced apoptosis of HepG2 cells was associated with the generation of reactive oxygen species, increased intracellular [Ca2+]i, loss of mitochondrial membrane potential, translocation of cytochrome c, and activation of caspase-9 and -3.

CONCLUSION: Tectorigenin induces apoptosis of HepG2 cells mainly via mitochondrial-mediated pathway, and produces a slight cytotoxicity to L02 cells.

Keywords: Tectorigenin; Iris tectorum maxim; Apoptosis; Hepatocellular carcinoma; HepG2; Mitochondria; Liver cancer