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17–丙烯胺基–17–去甲氧基格尔德霉素对人肺癌A549及H446细胞增殖和凋亡的影响
牛奔
蔺景双
冯涛
作者及单位信息
·
DOI： 10.3760/cma.j.issn.1001-0939.2015.04.007
Effects of 17-AAG on the proliferation and apoptosis of human lung cancer A549 and H446 cells
Niu Ben
Lin Jingshuang
Feng Tao
Authors Info & Affiliations
Niu Ben
Department of Respiratory Medicine, Central Hospital of Shengli Oilfield, Dongying 257034, China
Lin Jingshuang
Feng Tao
·
DOI： 10.3760/cma.j.issn.1001-0939.2015.04.007
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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 h 4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 ± 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(IC 50)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G 2/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。

肺肿瘤;细胞系，肿瘤;细胞凋亡;细胞增殖
ABSTRACT

ObjectiveTo 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.

MethodsProliferation 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.

ResultsAfter 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 IC 50 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 G 2/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.

Conclusion17-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.

Lung neoplasms;Cell line, tumor;Apoptosis;Cell proliferation
Feng Tao, Email: mocdef.3ab612791oatgnef
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引用本文

牛奔,蔺景双,冯涛. 17–丙烯胺基–17–去甲氧基格尔德霉素对人肺癌A549及H446细胞增殖和凋亡的影响[J]. 中华结核和呼吸杂志,2015,38(4)：267-272.

DOI:10.3760/cma.j.issn.1001-0939.2015.04.007

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肺癌分为小细胞肺癌(small cell lung cancer, SCLC)和非小细胞肺癌(non–small cell lung cancer, NSCLC)，其中NSCLC占肺癌的80%～90% [ 1 ]。由于大多数肺癌确诊时即为晚期，因此目前其总体治愈率很低。
凋亡受阻是肿瘤发生的主要原因之一，靶向治疗诱导肿瘤细胞凋亡已成为当前肿瘤治疗的热点 [ 2 ]。文献报道，与正常细胞相比，热休克蛋白90(Heat shock protein，Hsp90)在肿瘤细胞中的活跃度更高，且广泛的参与多条与肿瘤发生、发展相关的信号通路 [ 3 ]。Hsp90在肺癌细胞中(如NSCLC A549细胞株及SCLC H446细胞株)也呈现高表达状态，且当其活性被抑制时能显著影响肿瘤细胞的生长 [ 4 , 5 ]。因此，本研究选取A549和H446细胞系为研究对象，以已知的Hsp90抑制剂17–丙烯胺基–17–去甲氧基格尔德霉素(17–AAG)为材料，研究其对这2个细胞株凋亡的诱导作用。
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