Liver Cancer
Copyright ©2005 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Oct 28, 2005; 11(40): 6281-6287
Published online Oct 28, 2005. doi: 10.3748/wjg.v11.i40.6281
Overexpression of cyclooxygenase-2 in human HepG2, Bel-7402 and SMMC-7721 hepatoma cell lines and mechanism of cyclooxygenase-2 selective inhibitor celecoxib-induced cell growth inhibition and apoptosis
Ning-Bo Liu, Tao Peng, Chao Pan, Yu-Yu Yao, Bo Shen, Jing Leng
Ning-Bo Liu, Tao Peng, Bo Shen, Jing Leng, Laboratory of Reproductive Medicine, Department of Pathology, Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
Chao Pan, Yu-Yu Yao, Atherosclerosis Research Center, Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
Author contributions: All authors contributed equally to the work.
Supported by Medical Science Research Foundation of Jiangsu Health Bureau Grant Z200314 (to JL) and Medical Science Research Foundation of Nanjing Medical University Grant NY1999023 (to NBL) and CX2003012 (to JL)
Correspondence to: Dr. Jing Leng, Laboratory of Reproductive Medicine, Department of Pathology, Nanjing Medical University, Nanjing 210029, Jiangsu Province, China. lengjing@njmu.edu.cn
Telephone: +86-25-86862685 Fax: +86-25-86862685
Received: November 15, 2004
Revised: December 1, 2004
Accepted: December 3, 2004
Published online: October 28, 2005
Abstract

AIM: To investigate the cyclooxygenase-2 (COX-2) expression level in human HepG2, Bel-7402 and SMMC-7721 hepatoma cell lines and the molecular mechanism of COX-2 selective inhibitor celecoxib-induced cell growth inhibition and cell apoptosis.

METHODS: Hepatoma cells were cultured and treated with celecoxib. Cell in situ hybridization (ISH) and immunocytochemistry were used to detect COX-2 mRNA and protein expression. Proliferating cell nuclear antigen and phosphorylated Akt were also detected by immunocytochemistry assay. Cell growth rates were assessed by 3-(4, 5-dimethylthiazol-2-yl-2, 5-diphenylte-trazolium (MTT) bromide colorimetric assay. Celecoxib-induced cell apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and flow cytometry (FCM). The phosphorylated Akt and activated fragments of caspase-9, caspase-3 were examined by Western blotting analysis.

RESULTS: Increased COX-2 mRNA and protein expression were detected in all three hepatoma cell lines. Celecoxib could significantly inhibit cell growth and the inhibitory effect was in a dose- and time-dependent manner evidenced by MTT assays and morphological changes. The apoptotic index measured by TUNEL increased correspondingly with the increased concentration of celecoxib and the reaction time. With 50 μmol/L celecoxib treatment for 24 h, the apoptotic index of HepG2, BEL-7402 and SMMC-7721 cells was 25.01±3.08%, 26.40±3.05%, and 30.60±2.89%, respectively. Western blotting analysis showed remarkable activation of caspase-9, caspase-3 and dephosphorylation of Akt (Thr308). Immunocytochemistry also showed the reduction of PCNA expression and phosphorylation Akt (Thr308) after treatment with celecoxib.

CONCLUSION: COX-2 mRNA and protein overexpression in HepG2, Bel-7402 and SMMC-7721 cell lines correlate with the increased cell growth rate. Celecoxib can inhibit proliferation and induce apoptosis of hepatoma cell strains in a dose- and time-dependent manner.

Keywords: Apoptosis; Akt; Celecoxib; Caspase; Cell proliferation; COX-2; HCC; PCNA