依托度酸诱导SMMC7721细胞凋亡的分子机理研究  

Molecular mechanism of etodolac-induced apoptosis in SMMC7721 cell line

作　　者：周喜乐[1] 林建江[1] 徐骁[2] 谢海洋[3] 郑树森[2] 

机构地区：[1]浙江大学医学院附属第一医院肛肠外科,浙江杭州310003 [2]浙江大学医学院附属第一医院肝胆胰外科,浙江杭州310003 [3]浙江大学医学院附属第一医院卫生部多器官联合移植研究重点实验室,浙江杭州310003

出　　处：《中国病理生理杂志》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)

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.

关 键 词：环加氧酶-2 NF-ΚB 细胞凋亡 半胱氨酸天冬氨酸蛋白酶3 

分 类 号：R363[医药卫生—病理学]