培美曲塞对PC9肺腺癌细胞株的放射增敏作用

目的 培美曲塞是治疗非鳞癌非小细胞肺癌(non-small cell lung cancer,NSCLC)的常用药物,其对表皮生长因子受体(pithelial growth factor receptor,EGFR)敏感突变的肺腺癌是否具有放射增敏作用尚未明确.本课题研究培美曲塞在体外对EGFR 19外显子突变的人肺腺癌细胞株PC9的放射增敏作用,并初步探讨其作用机制.方法 以PC9细胞作为研究对象,采用MTT法检测培美曲塞的20%抑制浓度(20% inhibition concentration,IC20),将PC9细胞分为对照组、单独照射组、培美曲塞单药组和培美曲塞+照射组4组;采用流式细胞术检测培美曲塞联合或不联合照射对PC9细胞的凋亡率及细胞周期的影响;克隆形成实验检测培美曲塞对PC9细胞的放射效应,计算存活分数,拟合存活曲线;蛋白质印迹法检测CDC20蛋白在各组的表达.结果 MTT结果显示,PC9细胞的增殖抑制与培美曲塞呈时间-剂量依赖性,取48 h的IC20(0.084 μmol/L)的培美曲塞作为实验浓度.流式细胞术结果显示,培美曲塞单药组和单独照射组均可增加凋亡率,分别为(7.17±1.14)%和(10.78±1.52)%,而培美曲塞+照射组具有协同增效作用,凋亡率达(29.23±1.33)%,F=227.57,P<0.001;细胞周期结果显示,对照组G2/M期比例为(0.79±0.63)%,培美曲塞单药组为(0.79±0.47)%,单独照射组为(18.21±0.72)%,培美曲塞+照射组为(25.09±1.04)%,提示培美曲塞使细胞阻滞在S期,与放射线联合作用后,S期细胞比例减少,G2/M期的比例明显增多,差异有统计学意义,F=276.85,P<0.001.克隆形成实验提示,培美曲塞具有较好的放射增敏作用,其放射增敏比(sensitization enhancement ratio,SER)为1.41.培美曲塞联合照射能增加细胞周期相关蛋白CDC20的表达,F=282.12,P<0.001.结论 培美曲塞可提高EGFR 19外显子突变的PC9肺腺癌细胞的放射敏感性,其机制可能与照射引起CDC20的增加致对放射敏感的G2/M期阻滞,而照射耐受的S期比例减少有关.

Radiosensitizing effect of pemetrexed on the human lung adenocarcinoma cell line PC9

OBJECTIVE Pemetrexed is the commonly used drug on treating non-small cell lung cancer (except squamous carcinoma),whether it is sensitive to EGFR mutation of lung adenocarcinoma with radiotherapy sensitization effect is not clear yet.We explored the radiosensitization of pemetrexed on human lung adenocarcinoma cells PC9 with EGFR 19 exon mutation and its possible mechanism in this study.METHODS MTT assay was performed to measure the IC20 of pemetrexed on PC9 cell,and the PC9 cells were divided into four groups including control group,radiotherapy group,single drug pemetrexed group and the pemetrexed combined radiotherapy group.The flow cytometry was used to detect the apoptosis rate and cell cycle of PC9 cells in all groups.Clonogenic survival assay was used to determine cell survival fraction of PC9 cells received radiation alone and pemetrexed combined with irradiation.The expression of CDC20 was detected by western blotting.RESULTS The proliferation of PC9 cells was inhibited by pemetrexed,and the effect was time-dose-dependent,we took the IC20 (0.084 μmol/L) of 48h of pemetrexed as experiment concentration.Flow cytometry showed that pemetrexed and radiotherapy could increase the apoptosis rate,the cell apoptosis rates were(7.17±1.14)% and (10.78±1.52)%,and pemetrexed had synergistic effects with radiation,the cell apoptosis rate was(29.23±1.33)%,F=227.57,P<0.001.Cell cycle detection showed that G2/M phase were (0.79±0.63)% in concrol group,(0.79±0.47)% in pemetrexed group,(18.21±0.72)% in radiation group,and (25.09±1.04)% in pemetrexed combined radiation group.Pemetrexed induced S phase arrest,when combine with radiation,S phase cell proportion reduced,and the proportion of G2/M phase increased,the difference was statistically significant,F=276.85,P<0.001.Clonogenic survival assay demonstrated that pemetrexed had radiosensitization effect on PC9,the SER was 1.41.The expression of CDC20 was significantly increased after treated by pemetrexed and radiation,F=282.12,P<0.001.CONCLUSION Pemetrexed improved the radiosensitivity of lung adenocarcinoma cells PC9 with EGFR exon 19 mutation,the mechanism may be related to the increase of CDC20,the arrest of radiation sensitive of G2/M phase,and the reduction of radiation tolerance of S phase proportion.