Basic Research
Copyright ©2006 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Aug 28, 2006; 12(32): 5175-5181
Published online Aug 28, 2006. doi: 10.3748/wjg.v12.i32.5175
Involvement of P53 and Bax/Bad triggering apoptosis in thioacetamide-induced hepatic epithelial cells
Li-Hsuen Chen, Chia-Yu Hsu, Ching-Feng Weng
Li-Hsuen Chen, Chia-Yu Hsu, Ching-Feng Weng, Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan, China
Author contributions: All authors contributed equally to the work.
Supported by the National Science Council, Taiwan, No. 92-2317B-259-001
Correspondence to: Ching-Feng Weng, Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan, China. cfweng@mail.ndhu.edu.tw
Telephone: +886-3-8633637 Fax: +886-3-8630255
Received: February 16, 2006
Revised: March 15, 2006
Accepted: March 20, 2006
Published online: August 28, 2006
Abstract

AIM: Thioacetamide (TAA) has been used in studying liver fibrosis and cirrhosis, however, the mechanisms of TAA-induced apoptosis in liver are still unclear. The hepatic epithelial cell line clone 9 was cultured and treated with TAA to investigate the causes of cell death.

METHODS: The cell viability of TAA-induced clone 9 cells was determined using MTT assay. Total cellular GSH in TAA-induced clone 9 cells was measured using a slight modification of the Tietze assay. The activity of caspase 3 in TAA-induced clone 9 cells was monitored by the cleavage of DEVD-p-nitroanaline. TUNEL assay and flow cytometry were applied for the determination of DNA fragmentation and the proportion of apoptosis in TAA-induced clone 9 cells, respectively. The alterations of caspase 3, Bad, Bax and Phospho-P53 contents in TAA-induced clone 9 cells were measured by Western blot.

RESULTS: The experimental data indicated that TAA caused rat hepatic epithelial cell line clone 9 cell death in a dose-and time-dependent manner; 60% of the cells died (MTT assay) within 24 h after 100 mg/L TAA was applied. Apoptotic cell percentage (TUNEL assay) and caspase 3 activities were highest after 100 mg/L TAA was added for 8 h. The release of GSH and the elevation in caspase content after TAA treatment resulted in clone 9 cell apoptosis via oxidative stress and a caspase-dependent mechanism. The phospho-p53, Bax and Bad protein expressions in clone 9 cells were increased after TAA treatment.

CONCLUSION: These results reveal that TAA activates p53, increases caspase 3, Bax and Bad protein contents, perhaps causing the release of cytochrome c from mitochondria and the disintegration of membranes, leading to apoptosis of cells.

Keywords: Thioacetamide; Phospho-p53; Caspase 3; Apoptosis; Bax; Bad