摘要目的 观察17-丙烯胺基-17-去甲氧基格尔德霉素(17-AAG)对人肺癌A549及H446细胞凋亡的影响,初步探讨其可能的作用机制.方法 实验组细胞分别使用浓度为50、100、200、400和500 nmol/L的17-AAG进行处理,以不经药物处理组为阴性对照,通过MTT法检测12、24、48及72 h4个时间段内17-AAG对A549及H446细胞增殖的影响.以不经药物处理组为阴性对照,通过流式细胞术检测浓度为100、200和400 nmol/L的17-AAG作用48 h后,对A549及H446细胞周期的影响及其促凋亡作用.以不经药物处理组为阴性对照,通过Western blot法检测浓度为100、200和400 nmol/L的17-AAG对A549及H446细胞内热休克蛋白(Hsp) 90相关蛋白(Hsp90、Hsp70、Akt和Her-2)和凋亡相关蛋白(Bcl-2和Bax)表达的影响.本研究中结果对比类计量资料以(-x)±s表示,采用Origin 8.0统计软件,多组间均数比较采用t检验,组间两两比较用单因素方差分析(One-way ANOVA).结果 17-AAG浓度为50～ 500 nmol/L时2个细胞株的增殖受抑制,呈显著的浓度依赖性.48 h时,17-AAG对A549及H446细胞的半抑制浓度(IC50)分别为(222±13)和(189 ±7) nmol/L;细胞周期实验结果发现,17-AAG可特异性地将A549及H446细胞株的细胞周期阻滞于G2/M期;凋亡实验结果显示,17-AAG可促进A549及H446细胞凋亡;17-AAG浓度为400 nmol/L处理48 h后,A549细胞株的细胞凋亡率为46.3％,H446细胞株的细胞凋亡率为56.9％,与未加药物处理的对照组相比(A549细胞株的细胞凋亡率为11.9％,H446细胞株的细胞凋亡率为6.9％)差异有统计学意义(P＜0.01);经17-AAG处理48 h后,2个细胞株中Hsp90信号通路的相关蛋白均发生了相同趋势的变化:Akt和Her-2发生明显下调,其辅助蛋白Hsp70表达量增多,同时随着17-AAG浓度的增加Bcl-2表达下降,Bax表达上升,说明17-AAG可启动A549和H446细胞的凋亡模式.结论 17-AAG可通过Hsp90信号通路影响A549和H446细胞中Bax和Bcl-2等凋亡蛋白的表达,最终抑制细胞增殖并诱导细胞凋亡.

abstractsObjective To observe the effect of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) on the apoptosis of human lung cancer cell lines A549 and H446,and to investigate the potential mechanisms.Methods Proliferation inhibition and apoptosis assays,and the cell cycles were detected by MTT and flow cytometry respectively.Western blot was used to determine the expression level of proteins such as Hsp90,Hsp70,AKt,Her-2,Bcl-2 and Bax.Results After treated with 17-AAG,the proliferation of both A549 and H446 cells was inhibited significantly in a dose-dependent manner; as the concentration of 17-AAG was from 50 to 500 nmol/L,the IC50 values to A549 and H446 cell lines were (222 ± 13) nmol/L and (189 ±7) nmol/L respectively at 48 h.Cell cycle assays showed that 17-AAG was able to arrest cell cycles of A549 and H446 cell lines at the G2/M phase.Apoptosis assay showed that 17-AAGwas capable of inducing apoptosis in A549 and H446 cell lines.After treated with 17-AAG for 48 h,there were significant differences between the 400 nmol/L groups(46.3％ for A549 cell line and 56.9％ for H446 cell line) and the control group (11.9％ for A549 cell line and 6.9％ for H446 cell line,P ＜0.01).Western blot results showed that the relative proteins of the Hsp90 signal pathway in both A549 and H446 cell lines underwent similar changes after 17-AAG treatment:Akt and Her-2 decreased significantly while the expression of Hsp70 increased.Meanwhile,the expression of Bcl-2 decreased but that of Bax increased,indicating that 17-AAG was able to promote apoptosis mode in A549 and H446 cells.Conclusion 17-AAG can regulate the expression level of apoptosis-related proteins such as Bax and Bcl-2 by Hsp90 signaling pathway in A549 and H446 cells,and ultimately inhibit cell proliferation and induce apoptosis.