摘要
目的:观察5-烯丙基-7-二氟甲氧基白杨素(AFMC))是否协同肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导人肺腺癌A549细胞凋亡.并探讨其机制是否涉及死亡受体5(DR5)的上调.方法:体外培养人肺腺癌A549细胞和人胚肺WI-38细胞.碘化丙啶(PI)染色流式细胞术(FCM)定量分析细胞凋亡率;DNA琼脂糖凝胶电泳观察细胞DNA梯形条带;Western Blotting检测DR5蛋白表达.结果:单独用亚毒性浓度的AFMC(1.0μmol/L)或TRAIL(30μg/L)处理A549细胞,细胞凋亡率不超过5%.AFMC(1.0μmol/L)联合TRAIL(30μg/L)的A549细胞凋亡率增高(37.80%,P<0.01);而WI-38细胞仅有极少量的细胞凋亡(P>0.05).DNA琼脂糖凝胶电泳显示:两者合用A549细胞呈现典型的DNA梯形条带图谱,而WI-38细胞未出现DNA梯形条带;Western Blotting分析发现:AFMC呈浓度和时间依赖性上调A549细胞DR5的表达,DR5特异性抑制剂DR5/Fc嵌合蛋白能有效降低两者合用诱导的A549细胞凋亡率(P<0.05).然而,AFMC对WI-38细胞DR5表达无明显影响.结论:亚毒性浓度的AFMC选择性增强TRAIL诱导人肺癌A549细胞凋亡.其机制可能与其上调DR5表达有关.
Aim: To investigate the synergistic action of 5-allyl-7-gen-difluoromethoxychrysin( AFMC) combined with Tumor necrosis factor-related apoptosis-inducing ligand( TRAIL) in apoptosis of human lung cancer A549 cell line and explore whether the potential internal mechanism is refered to the up-regulation of DR5 protein. Methods: Human lung cancer A549 cells and human immortalized embryonic lung WI-38 cells were cultured in vitro. Cell apoptotic rate was determined by flow cytometry( FCM) using PI staining. DNA ladder bands were observed by DNA agarose gel electrophoresis. The expression of DR5 protein was analyzed using Western blotting. Results: Flow cytometry( FCM) analysis indicated that the apoptosis rate of A549 cells by 1. 0 μmol /L AFMC( at suboptimal concentrations) or 30 μg / L TRAIL alone were less than 5%. Combined treatment of A549 cells with AFMC and TRAIL,the apoptosis rate significantly increased( 37. 80%,P < 0. 01). But the same treatment to WI-38 cells had no apparent effect( P > 0. 05). The typical ladder bands could be shown in DNA agarose gel electrophoresis in A549 cells but not in WI-38 cells by the combined use of AFMC and TRAIL. Western blotting analysis indicated that AFMC markedly induced the expression of death receptor 5( DR5) in A549 cells,in a time-and concentration-dependent manner. DR5 / Fc chimera protein,a specific inhibitor for DR5,could dramatically reduce the apoptotic rate of A549 cells by the combination of AFMC and TRAIL( P < 0. 05). On the other hand,AFMC-mediated induction of DR5 expression is not observed in WI-38 cells. Conclusion: AFMC at subtoxic concentration selectively enhances susceptibility to TRAIL-induced apoptosis in human lung cancer A549 cells through up-regulating DR5 expression.
出处
《晓庄学院自然科学学报》
CAS
北大核心
2014年第1期25-30,共6页
Journal of Natural Science of Hunan Normal University
基金
湖南省卫生厅科研基金资助项目(B2010-104)
湖南省教育厅重点资助项目(11A073)
湖南省科技厅科研基金资助项目(2010SK3008)
