DR5上调在5-烯丙基-7-二氟甲氧基白杨素增强TRAIL诱导人肺癌A549细胞凋亡中的作用
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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.
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.
引文
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[12]ZHANG T,WU M,CHEN Q,et al.Investigation into the regulation mechanisms of TRAIL apoptosis pathway by mathematical modeling[J].Acta Biochim Biophys Sin,2010,42(2):98-108.
[13]TRUNEH A,SHARMA S,SILVEMAN C,et al.Temperature-sensitive differential affinity of TRAIL for its receptors DR5 is the highest affinity receptor[J].J Biol Chem,2000,275(30):23319-23325.
[14]谢朝晖,刘飞,曹建国,等.AFMC选择性增强TRAIL对肺癌A549细胞毒性[J].湖南师范大学学报:医学版,2011,8(1):20-23.
[2]SINGH T R,SHANKAR S,CHEN X,et al.Synergistic interactions of chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand on apoptosis and on regression of breast carcinoma in vivo[J].Cancer Res,2003,63(17):5390-5400.
[3]KHOO B Y,CHUA S L,BALARAM P.Apoptotic effects of chrysin in human cancer cell lines[J].Int J Mol Sci,2010,11(5):2188-2199.
[4]HADJERI M,BARBIER M,RONOT X,et al.Modulation of P-glycopretein mediated multidrug resistance by flavonoid derivatives and analogues[J].Med Chem,2003,46(11):2125-2131.
[5]TAN X W,XIA H,XU J H,et al.Induction of apoptosis in human liver carcinoma HepG2 cell line by 5-allyl-7-gen-difluoromethylenechrysin[J].World J Gastroenterol,2009,15(18):2234-2239.
[6]LI J L,XIE W Y,CAO J G.The effect of 5-allyl-7-gen-difluoromethylenechrysin on proliferation and apoptosis in ovarian cancer cell cultured in vitro[J].Am J Chin Clin Med,2005,7:323-326.
[7]许金华,谭翔文,曹建国.ADFMChR对人肺癌A549细胞增殖活性的影响[J].南华大学学报:医学版,2009,37(6):673-676.
[8]DING J,POLIER G,KHLER R,et al.Wogonin and related natural flavones overcome tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)protein resistance of tumors by down-regulation of c-FLIP protein and up-regulation of TRAIL receptor 2 expression[J].J Biol Chem,2012,287(1):641-649.
[9]JIN C Y,PARK C,HWANG H J,et al.Naringenin up-regulates the expression of death receptor 5 and enhances TRAIL-induced apoptosis in human lung cancer A549 cells[J].Mol Nutr Food Res,2011,55(2):300-309.
[10]ZHENG X,CAO J G,LIAO D F,et al.Systhesis and anticancer effect of gem-difluoromethylenated chrysin derivatives[J].Chin Chem Lett,2006,17(11):1439-1442.
[11]WALKER S.Updates in non-small cell lung cancer[J].Clin J Oncol Nurs,2008,12(4):587-596.
[12]ZHANG T,WU M,CHEN Q,et al.Investigation into the regulation mechanisms of TRAIL apoptosis pathway by mathematical modeling[J].Acta Biochim Biophys Sin,2010,42(2):98-108.
[13]TRUNEH A,SHARMA S,SILVEMAN C,et al.Temperature-sensitive differential affinity of TRAIL for its receptors DR5 is the highest affinity receptor[J].J Biol Chem,2000,275(30):23319-23325.
[14]谢朝晖,刘飞,曹建国,等.AFMC选择性增强TRAIL对肺癌A549细胞毒性[J].湖南师范大学学报:医学版,2011,8(1):20-23.