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Title: [Effects of 17-DMAG on non-small cell lung cancer cell lines A549 and H1975  being resistant to EGFR-TKI].
Author: Zhao L, Cao F.
Journal: Zhongguo Fei Ai Za Zhi; 2014 Nov; 17(11):778-82. PubMed ID: 25404267.
Abstract:
BACKGROUND AND OBJECTIVE: In the clinical treatment of patients with non-small cell lung cancer (NSCLC), the primary and acquired resistance of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) limits its clinical application, this need to explore new strategy or method to overcome this problem. Recently, some literatures have indicated that the antitumor role of heat shock protein 90 (HSP90) inhibitors by a variety of pathways may provide new strategy for resolving this problem. In this study, we examined the effect of 17-DMAG on NSCLC cell lines A549 and H1975 which were primary and acquired resistant to EGFR-TKI respectively, the purpose was to explore its influence on cell proliferation, apoptosis and the expression of EGFR in vitro as well as possible mechanism. METHODS: After A549 and H1975 cell lines were treated with different concentrations of 17-DMAG respectively, the inhibitory rate of cell proliferation was measured by MTT assay in 24 h, 48 h and 72 h. We investigated the effect of 17-DMAG on the cell apoptosis with flow cytometry and the expression of HSP90 and EGFR with Western blot after treated with 17-DMAG for 48 h. RESULTS: After treated with 17-DMAG, the inhibitory rate of different concentrations and time groups was significant (P<0.01), and the effect was in time- and dose-dependent manner; the apoptosis rate of both two cell lines in all treated groups were significantly higher than control group (P<0.01), and the effect was in dose-dependent manner. By Western blot analysis, there was no significant difference between all treated groups and control group for the expression of both HSP90 and EGFR protein in A549 cell line and HSP90 protein in H1975 cell line after exposed to 17-DMAG for 48 h (P>0.05), while the difference was significant for the expression of EGFR protein in H1975 cell line (P<0.01). CONCLUSIONS: 17-DMAG inhibited the proliferation of NSCLC cell lines A549 and H1975 and also induced apoptosis of both cell lines. It down-regulated the expression of mutant EGFR protein while this phenomenon was not observed in EGFR-wild type cell line. This suggested that the mechanism maybe different between A549 and H1975 cell lines with different genetic backgroud. Our study provided new strategy for treatment with NSCLC being resistant to EGFR-TKI. 背景与目的表皮生长因子受体酪氨酸激酶抑制剂（epidermal growth factor receptor-tyrosine kinase inhibitor, EGFR-TKI）在非小细胞肺癌（non-small cell lung cancer, NSCLC）患者的临床治疗中产生的原发性及获得性耐药限制了其临床应用，需要探索新的策略或方法来克服这个问题。最近有文献报道认为热休克蛋白90（heat shock protein 90, HSP90）抑制剂能从多种途径和环节发挥抗肿瘤作用，这为解决NSCLC对EGFR-TKI的耐药提供了新的思路。本研究通过观察HSP90抑制剂17-DMAG对EGFR-TKI分别原发性及获得性耐药的NSCLC细胞株A549和H1975的作用，旨在探讨它对细胞增殖、凋亡与EGFR蛋白表达的影响及其可能的机制。方法以不同浓度的17-DMAG分别作用于A549和H1975细胞株24 h、48 h、72 h，应用四甲基偶氮唑蓝（MTT）比色法检测细胞增殖；作用48 h后，应用流式细胞术PI单染法检测细胞凋亡，并应用Western blot检测细胞HSP90及EGFR蛋白表达水平。结果 17-DMAG在不同药物浓度和作用时间对A549和H1975细胞的增殖抑制率差异均有统计学意义（P<0.01），且呈时间和剂量依赖性；两种细胞不同药物浓度组和空白对照组之间的凋亡率差异均有统计学意义（P<0.01），且呈剂量依赖性； 17-DMAG作用48 h后，A549细胞的EGFR/GADPH和HSP90/GADPH及H1975细胞的HSP90/GADPH在不同药物浓度组和空白对照组之间的灰度比值差异均无统计学意义（P>0.05），而H1975细胞的EGFR/GADPH在不同药物浓度组和空白对照组之间的灰度比值差异均有统计学意义（P<0.01）。结论 17-DMAG对NSCLC细胞株A549和H1975均具有抑制增殖及促进凋亡作用，且它能降低突变型EGFR的蛋白表达水平，而对野生型EGFR的蛋白表达无明显影响。本研究为EGFR-TKI耐药的非小细胞肺癌提供了新的治疗策略。

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