Molecular mechanism of etodolac-induced apoptosis in SMMC7721 cell line

AIM: To investigate the possible role of nuclear transcription factor kappa B (NF-κB), Bcl-2, Bax and caspase-3 in etodolac-induced apoptosis of liver tumor SMMC7721 cell line. METHODS: Cell apoptosis was determined by flow cytometry analysis with PI staining and DNA laddering. Expression of Bcl-2 and Bax protein was measured by Western blotting. Caspase-3 activity was evaluated by active caspase-3 apoptosis kit with flow cytometry. NF-κB activation was detected by ELISA-based TransAM~(TM) NF-κB p65/p50 kit. RESULTS: Etodolac, a selective COX-2 inhibitor, stimulated apoptosis in liver tumor SMMC7721 cell line significantly. Flow cytometry showed that the apoptotic rate was 16.3%±3.1%, 19.9%±3.6%, 22.9%±3.2%, 31.2%±3.3% with different concentrations of etodolac (0.25, 0.50, 1.0 or 2.0 mmol/L), while the apoptotic peak did not appear in the control group (0 mmol/L) (P0.01 vs control). Expression of Bax protein was up-regulated while Bcl-2 protein was down-regulated, and cells with caspase-3 activation was 3.61%±0.32%, 2.93%±0.15%, 10.29%±0.39%, 27.33%±1.28%, 57.40%±1.69%, respectively (P0.05, 0.50, 1.0, 2.0 mmol/L vs control). Compared with the control group, NF-κB activation was inhibited significantly as etodolac concentration increased (P0.05, 0.50, 1.0, 2.0 mmol/L vs control). Caspase-3 activation and NF-κB activity was negatively correlated (r=0.919, P0.01). CONCLUSION: Selective COX-2 inhibitor etodolac induces SMMC7721 cells apoptosis, possibly via inhibition of NF-κB activity and regulation of Bcl-2, Bax protein expression, which ultimately activate caspase-3.