摘要
目的:探讨选择性环氧合酶抑制剂依托度酸(etodolac)诱导肝癌SMMC7721细胞凋亡的分子机理。方法:采用流式细胞术、DNA琼脂糖凝胶电泳法测定细胞凋亡情况;Westernblotting法检测不同浓度etodolac处理后凋亡相关蛋白Bcl-2、Bax表达的变化;流式细胞术检测半胱氨酸酶-3(caspase-3)活性的变化;TransAMTMNF-κBp65/p50核转录因子活性检测试剂盒检测核因子-κB(NF-κB)活性变化。结果:流式细胞术显示etodolac(0.25、0.50、1.0、2.0mmol/L)作用SMMC7721细胞48h后,与对照组(0mmol/L)相比,出现明显凋亡峰(P<0.01vscontrol);高浓度etodolac处理后DNA琼脂糖凝胶电泳出现明显的DNALadder,凋亡相关蛋白Bcl-2表达下降,Bax表达增加;与对照组相比,低浓度组(0.25mmol/L)caspase-3活性未明显活化(P>0.05),NF-κB活性也未受明显抑制(P>0.05),随着etodolac浓度的增大(0.50、1.0、2.0mmol/L),caspase-3活性明显活化(P<0.05vscontrol);NF-κB活性明显受到抑制(P<0.05vscontrol)。经Pearson相关分析,caspase-3活性和NF-κB活性呈显著负相关(r=0.919,P<0.01)。结论:选择性COX-2抑制剂etodolac可能通过抑制NF-κB结合活性,调节Bcl-2、Bax蛋白表达,活化cas-pase-3,从而诱导肝癌SMMC7721细胞凋亡。
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 Pl 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 TransAMTM 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） （ P 〈 0.01 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 （P〈0.05, 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 （ P 〈 0.05, 0.50, 1.0, 2.0 mmol/L vs control）. Caspase- 3 activation and NF- κB activity was negatively correlated （ r = 0.919, P 〈 0.01 ）. 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.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2005年第9期1769-1774,共6页
Chinese Journal of Pathophysiology
基金
浙江省科技厅资助项目(No.021107241)
