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

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-diphenyltetrazolium (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 mu mol/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 (Thr(308)). Immunocytochemistry also showed the reduction of PCNA expression and phosphorylation Akt (Thr(308)) 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. (C) 2005 The WJG Press and Elsevier Inc. All rights reserved.