旁路信号通路激活介导的EML4-ALK融合基因阳性肺癌细胞株H3122对alectinib继发耐药的研究

发布时间：2018-04-22 12:07

本文选题：EML-ALK融合基因 + Alectinib　；参考：《中国病理生理杂志》2017年05期

【摘要】：目的:本研究旨在探讨肝细胞生长因子(HGF)、表皮生长因子(EGF)及转化生长因子α(TGF-α)是否以旁路激活的方式诱导EML4-ALK融合基因阳性肺癌细胞株H3122对alectinib的耐药,并进一步探讨旁路信号激活在alectinib耐药中的作用。方法:用不同浓度的alectinib、克唑替尼(crizotinib)、17-DMAG或(和)HGF(50μg/L)、EGF(100μg/L)、TGF-α(100μg/L)处理EML4-ALK阳性肺癌细胞株H3122,采用CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,应用Western blot技术检测细胞中ALK、c-Met、EGFR及相应磷酸化蛋白的表达,观察其下游通路关键蛋白AKT、ERK、p-AKT和p-ERK水平。结果:Alectinib作用72 h后,H3122细胞株的活力随着alectinib药物浓度的增加而逐渐下降,呈剂量依赖性。HGF、EGF和TGF-α诱导后,alectinib抑制H3122细胞的生长曲线往右移,HGF、EGF和TGF-α处理能够降低alectinib对肺癌细胞活力的抑制作用。0.05μmol/L alectinib作用H3122细胞株48 h后的凋亡率为(20.12±1.36)%,而alectinib联合HGF、EGF和TGF-α后的凋亡率分别为(7.85±1.03)%、(5.60±0.79)%和(4.58±1.00)%,显著低于alectinib单药处理(P0.05)。Alectinib单药成功抑制p-ALK及其下游信号通路,HGF明显增加细胞中p-Met及其下游p-AKT、p-ERK的蛋白水平,EGF和TGF-α明显增加细胞中p-EGFR及其下游p-AKT、p-ERK的表达,alectinib抑制p-ALK,但不能抑制HGF、EGF和TGF-α诱导的pAKT和p-ERK的蛋白表达。此外,联合应用crizotinib和17-DMAG可以抑制因HGF和EGFR配体而导致的H3122耐药细胞的活力。结论:HGF、EGF和TGF-α可通过旁路激活的方式诱导EML4-ALK阳性肺癌细胞H3122对alectinib耐药,其机制可能与HGF激活c-Met磷酸化、EGF和TGF-α激活EGFR磷酸化有关。
[Abstract]:Objective: to investigate whether EML4-ALK fusion gene positive lung cancer cell line H3122 can be induced to be resistant to alectinib by bypass activation of hepatocyte growth factor (HGF), epidermal growth factor (EGF) and transforming growth factor 伪 (TGF- 伪). The role of bypass signal activation in drug resistance of alectinib was further investigated. Methods: EML4-ALK positive lung cancer cell line H3122 was treated with different concentrations of alectinib, crizotinib, 17-DMAG or (and HGF50 渭 g / L). The activity of H3122 was detected by CCK-8 assay, apoptosis was detected by flow cytometry, and the expression of ALKFc-Metagro EGFR and phosphorylated protein in H3122 cells was detected by Western blot technique. The levels of p-AKT and p-ERK were observed. Results the activity of H3122 cell line was decreased with the increase of alectinib concentration after 72 h treatment with 1: Alectinib. HGF-EGF and TGF- 伪 induce H3122 cell growth curve to the right of H3122 cells induced by EGF and TGF- 伪 in a dose-dependent manner. HGF- EGF and TGF- 伪 treatment can reduce the inhibitory effect of alectinib on the viability of lung cancer cells. The apoptosis rate of H3122 cell line induced by 0.05 渭 mol/L alectinib for 48 h is 20.12 卤1.36%, while that of alectinib combined with HGF- EGF is 20.12 卤1.36%. The apoptotic rates were 5.60 卤0.79% and 4.58 卤1.00% after TGF- 伪 and 7.85 卤1.03%, respectively, which were significantly lower than that of P0.05N. Alectinib treated with alectinib alone. HGF significantly increased the protein levels of p-Met and its downstream p-AKTp-ERK protein. EGF and TGF- 伪 increased the expression of p-EGFR and TGF- 伪 in the cells. The expression of p-AKTtinib inhibited the expression of p-ALK, but could not inhibit the protein expression of pAKT and p-ERK induced by HGF EGF and TGF- 伪. In addition, the combination of crizotinib and 17-DMAG inhibited the activity of H3122 cells induced by HGF and EGFR ligands. Conclusion EML4-ALK positive lung cancer cell H3122 can be induced to be resistant to alectinib by the way of bypass activation, which may be related to the activation of c-Met phosphorylation by HGF and EGFR phosphorylation by TGF- 伪.
【作者单位】：广西医科大学研究生院;广西医科大学附属肿瘤医院呼吸肿瘤内科;
【基金】：国家自然科学基金资助项目(No.81260357;No.81060188) 广西自然科学基金资助项目(No.2015GXNSFAA139162)
【分类号】：R730.23

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