联合通路抑制剂对KRAS突变型非小细胞肺癌细胞系的生长抑制作用

[Growth inhibition of combined pathway inhibitors on KRAS mutated non-small cell lung cancer cell line].

作者信息

Li Zhan-wen, Yang Zhen-li, Feng Hai-liang, Bian Xiao-cui, Liu Yan-yan, Liu Yu-qin

机构信息

Department of Pathology, Peking Union Medical College, Beijing, China.

出版信息

Zhonghua Bing Li Xue Za Zhi. 2013 May;42(5):330-5. doi: 10.3760/cma.j.issn.0529-5807.2013.05.009.

DOI:10.3760/cma.j.issn.0529-5807.2013.05.009

PMID:24004591

Abstract

OBJECTIVE

To investigate the effect of the selective PI3K inhibitor and MEK inhibitor on KRAS and PTEN co-mutated non-small cell lung cancer cell line NCI-H157 and the relevant mechanisms.

METHODS

NCI-H157 was cultured routinely and treated with different concentrations of the two inhibitors. Cell proliferation was detected by MTT cell cycle assay. Based on the MTT results 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). Colony formation assay was performed to detect colony formation efficiency. The cell cycle and apoptosis were analyzed by flow cytometry. The expression of protein related to apoptosis was tested with Western blot.

RESULTS

Cell growth was inhibited by the two inhibitors. Combination groups led to stronger cell proliferation inhibition: combination group Ishowed synergistic effect of their actions and combination group II showed an additive effect; in both groups, there were decreased colony number [(77.2 ± 1.54)/well vs (61.50 ± 2.12)/well, P < 0.01] and [(51.00 ± 4.00)/ well vs (22.50 ± 3.53)/well, P < 0.01]; and enhanced apoptotic ratios [(18.30 ± 0.82)% vs (21.32 ± 0.56)%, P < 0.01] and [(27.14 ± 1.58)% vs (42.45 ± 4.42)%, P < 0.01]. In addition, compared to the PI3K inhibitor alone group, the NCI-H157 cells in the combination groups showed increased G0/G1 phase and decreased S phase (P < 0.01). Western blotting showed that the combination groups demonstrated significantly decreased expression of cyclin D1 and cyclin B1, increased p21 and cleaved PARP and decreased bcl-2/bax ratio, compared to the PI3K inhibitor only group.

CONCLUSION

The combined inhibition of PI3K (AZD6244) and MEK (GDC-0941) has synergistic effects on the proliferation of NCI-H157 cells, but such effects appear to be in a dose-dependent manner.

摘要

目的

探讨选择性PI3K抑制剂和MEK抑制剂对KRAS和PTEN共同突变的非小细胞肺癌细胞系NCI-H157的作用及其相关机制。

方法

常规培养NCI-H157细胞，并用不同浓度的两种抑制剂进行处理。采用MTT细胞周期分析法检测细胞增殖情况。根据MTT结果将细胞分为四组：对照组、PI3K抑制剂组（GDC-0941，0.5和5.0 μmol/L）、联合组I（0.5 μmol/L AZD6244 + 0.5 μmol/L GDC-0941）和联合组II（5.0 μmol/L AZD6244 + 5.0 μmol/L GDC-0941）。进行集落形成试验以检测集落形成效率。通过流式细胞术分析细胞周期和凋亡情况。用蛋白质印迹法检测凋亡相关蛋白的表达。

结果

两种抑制剂均抑制细胞生长。联合组导致更强的细胞增殖抑制作用：联合组I显示出协同作用，联合组II显示出相加作用；两组的集落数均减少[（77.2±1.54）/孔 vs （61.50±2.12）/孔，P<0.01]和[（51.00±4.00）/孔 vs （22.50±3.53）/孔，P<0.01]；凋亡率增加[（18.30±0.82）% vs （21.32±0.56）%，P<0.01]和[（27.14±1.58）% vs （42.45±4.42）%，P<0.01]。此外，与单独使用PI3K抑制剂组相比，联合组中的NCI-H157细胞G0/G1期增加，S期减少（P<0.01）。蛋白质印迹法显示，与仅使用PI3K抑制剂组相比，联合组中细胞周期蛋白D1和细胞周期蛋白B1的表达显著降低，p21和裂解的PARP增加，bcl-2/bax比值降低。