CAJ | 학술논문

目的探讨选择性PI3K抑制剂联合MEK抑制剂对携带KRAS/PTEN双突变的非小细胞肺癌细胞系NCI-H157的生长增殖和凋亡的影响及相关分子机制.方法常规培养NCI-H157细胞并分别用不同浓度的PI3K抑制剂GDC-0941和MEK抑制剂AZD6244单独或联合作用,四甲基偶氮唑盐(MTT)法初步观察对细胞生长增殖的影响.根据观察结果确定联合给药的浓度.然后分为无药对照组、PI3K抑制剂单独用药组(GDC-0941,0.5和5.0 μmol/L)、联合Ⅰ组(0.5μmol/LAZD6244 +0.5 μmol/L GDC-0941)及联合Ⅱ组(5.0 μmol/L AZD6244+ 5.0 μmol/L GDC-0941).细胞经过不同的作用后,MTT法绘制增殖抑制曲线；平板集落形成法检测集落形成能力的改变；流式细胞仪检测细胞凋亡率及周期分布；Western blot检测不同组中抑制剂靶点下游周期及凋亡相关蛋白表达的差异.结果抑制剂单独作用于NCI-H157细胞可抑制其生长增殖.联合用药组与PI3K抑制剂单独用药组相比,细胞的生长增殖抑制作用增强,联合Ⅰ组表现为两药协同作用,联合Ⅱ组表现为两药相加作用；集落形成能力下降[(77.20±1.54)个/孔比(61.50 ±2.12)个/孔,P＜0.01]和[(51.00±4.00)个/孔比(22.50 ±3.53)个/孔,P＜0.01]；凋亡率增加[(18.30±0.82)％比(21.32±0.56)％,P＜0.01]和[(27.14±1.58)％比(42.45±4.42)％,P＜0.01]；细胞周期发生改变,联合用药组G0/G1期细胞增加,S期细胞减少(P＜0.05).Western blot显示,联合用药组与PI3K单独用药组相比,cyclin D1、cyclin B1表达量减少,p21表达量和PARP剪切增加,bcl-2/bax的比值下降.结论PI3K抑制(AZD6244)可协同MEK抑制(GDC-0941)诱导NCI-H157细胞发生生长增殖抑制和凋亡,但联合用药效果受到药物剂量的影响.
목적 탐토선택성PI3K억제제연합MEK억제제대휴대KRAS/PTEN쌍돌변적비소세포폐암세포계NCI-H157적생장증식화조망적영향급상관분자궤제.방법 상규배양NCI-H157세포병분별용불동농도적PI3K억제제GDC-0941화MEK억제제AZD6244단독혹연합작용,사갑기우담서염(MTT)법초보관찰대세포생장증식적영향.근거관찰결과학정연합급약적농도.연후분위무약대조조、PI3K억제제단독용약조(GDC-0941,0.5화5.0 μmol/L)、연합Ⅰ조(0.5μmol/LAZD6244 +0.5 μmol/L GDC-0941)급연합Ⅱ조(5.0 μmol/L AZD6244+ 5.0 μmol/L GDC-0941).세포경과불동적작용후,MTT법회제증식억제곡선；평판집락형성법검측집락형성능력적개변；류식세포의검측세포조망솔급주기분포；Western blot검측불동조중억제제파점하유주기급조망상관단백표체적차이.결과억제제단독작용우NCI-H157세포가억제기생장증식.연합용약조여PI3K억제제단독용약조상비,세포적생장증식억제작용증강,연합Ⅰ조표현위량약협동작용,연합Ⅱ조표현위량약상가작용；집락형성능력하강[(77.20±1.54)개/공비(61.50 ±2.12)개/공,P＜0.01]화[(51.00±4.00)개/공비(22.50 ±3.53)개/공,P＜0.01]；조망솔증가[(18.30±0.82)％비(21.32±0.56)％,P＜0.01]화[(27.14±1.58)％비(42.45±4.42)％,P＜0.01]；세포주기발생개변,연합용약조G0/G1기세포증가,S기세포감소(P＜0.05).Western blot현시,연합용약조여PI3K단독용약조상비,cyclin D1、cyclin B1표체량감소,p21표체량화PARP전절증가,bcl-2/bax적비치하강.결론PI3K억제(AZD6244)가협동MEK억제(GDC-0941)유도NCI-H157세포발생생장증식억제화조망,단연합용약효과수도약물제량적영향.
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 Ⅰ showed 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.