PI3K和MEK联合抑制增强对K-ras基因单突变肺癌细胞的生长抑制作用

Enhanced growth inhibition by combined two pathway inhibitors on K-ras mutated non-small cell lung cancer cells

目的 观察RAS/MEK/ERK通路单独抑制或联合PI3K/AKT/mTOR通路抑制对非小细胞肺癌A549细胞增殖和凋亡的影响,并探讨其相关分子机制.方法 常规培养A549细胞,分别以不同浓度的PI3K抑制剂GDC-0941和MEK抑制剂AZD6244单独或联合处理,采用四甲基偶氮唑蓝（MTT）法检测抑制剂对细胞增殖的影响,根据MTT法检测结果确定联合给药的浓度.将A549细胞分为空白对照组、0.5 μmol/L GDC-0941处理组、低浓度联合组（0.5 μmol/L AZD6244＋ 0.5μmol/LGDC-0941）、5.0 μmol/L GDC-0941处理组和高浓度联合组（5.0 μmol/L AZD6244＋ 5.0 μmol/L GDC-0941）,采用流式细胞术检测细胞周期和凋亡,采用Western blot法检测抑制剂靶点下游与细胞周期和凋亡相关的蛋白表达.结果 AZD6244对A549细胞的增殖抑制作用呈浓度依赖性,但浓度平台出现时,增殖抑制率仅为25.5％.AZD6244联合GDC-0941对A549细胞的生长抑制作用增强,但联合用药的效果与两种抑制剂联合使用的浓度有关.0.5 μmol/L GDC-0941处理组和低浓度联合组A549细胞的凋亡率分别为（20.70±0.99）％和（18.65±0.92）％,差异无统计学意义（P＞0.05）;5.0μmol/L GDC-0941处理组和高浓度联合组A549细胞的凋亡率分别为（37.85 ±3.18）％和（52.27±4.36）％,差异有统计学意义（P＜0.01）.低浓度联合组A549细胞的细胞周期没有明显改变;高浓度联合组中G0/G1期细胞增加,S期细胞减少.单独使用AZD6244可明显抑制pERK的表达水平,但pAKT的表达增加.单独使用GDC-0941可明显抑制pAKT的表达水平,但pERK的表达增加.低浓度联合组中,细胞周期和凋亡蛋白的表达均没有发生明显改变;高浓度联合组中,cyclin D1、cyclin B1表达量受到抑制,PARP剪切增加,Bcl-2/Bax比值下降.结论 对于K-ras基因单突变的非小细胞肺癌A549细胞,单独使用一条信号通路的抑制剂会反馈性激活另一信号通路.RAS/MEK/ERK和PDK/AKT/mTOR两条通路的同时抑制呈协同作用,这种协调作用受到抑制剂浓度的影响.靶向治疗时,联合使用两条通路的抑制剂是必要的.

Objective To evaluate the effect of combined targeting of MEK and PI3K signaling pathways on K-ras mutated non-small cell lung cancer cell line A549 cells and the relevant mechanisms.Methods A549 cells were treated with different concentrations of two inhibitors.Growth inhibition was determined by MTT assay.According to the results of MTT test,the cells were divided into four groups：the control group,PI3K inhibitor group （GDC-0941,0.5 and 5.0 μmol/L）,combination group Ⅰ （0.5 μmol/L AZD6244 ＋ 0.5 μmol/L GDC-0941） and combination group Ⅱ （5.0 μmol/L AZD6244 ＋ 5.0 μmol/L GDC-0941）.The cell cycle and apoptosis were analyzed by flow cytometry.The expression of proteins related to apoptosis was tested with Western blot.Results Both GDC-0941 and AZD6244 inhibited the cell proliferation.The combination group Ⅱ led to a stronger growth inhibition.The combination group Ⅰshowed an antagonistic effect and combination group Ⅱ showed an additive or synergistic effect.Compared with the control group,the combination group Ⅰ led to reduced apoptotic rate [（20.70 ± 0.99） % vs.（18.65 ± 0.92） %,P 〉 0.05] ; Combination group Ⅱ exhibited enhanced apoptotic rate [（37.85 ± 3.18）% vs.（52.27 ±4.36）%,P〈0.01].In addition,in the combination group Ⅱ,more A549 cells were arrested in G0/G1 phase and decreased S phase （P 〈 0.01）,due to the reduced expressions of CyclinD1 and Cyclin B1,the increased cleaved PARP and the diminished ratio of Bcl-2/Bax.Conclusions For single K-ras mutated NSCLC cell line A549 cells,combination of RAS/MEK/ERK and PI3K/AKT/mTOR inhibition showed synergistic effects depending on the drug doses.Double pathways targeted therapy may be beneficial for these patients.