CL-387785对非小细胞肺癌H1975细胞侵袭转移的抑制及放疗增敏作用研究

摘要

目的：探讨不可逆EGFR抑制剂CL⁃387785对EGFR T790M突变非小细胞肺癌H1975细胞侵袭转移能力的抑制及放疗增敏作用。方法分别采用0、10、25、50、100 nmol／L CL⁃387785处理H1975细胞24、48、72和96 h后用四甲基偶氮唑盐（ MTT）比色法检测增殖抑制率变化，同时用流式细胞术Annexin⁃FITC／PI双染法检测不同浓度CL⁃387785处理48、96 h后的细胞凋亡情况，Transwell法及划痕实验检测不同浓度CL⁃387785处理48、96 h后的H1975细胞侵袭及迁移能力变化，采用克隆形成实验检测CL⁃387785处理的H1975细胞经不同剂量X线（0、2、4、6、8和10 Gy）照射的存活分数（SF）并通过单击多靶模型拟合细胞存活曲线计算增敏比（ SER）。结果 CL⁃387785可抑制H1975细胞增殖，且在10～100 nmol／L范围内随作用时间延长，增殖抑制率升高，总体上抑制效应呈浓度和时间依赖方式；CL⁃387785可呈浓度依赖的方式诱导H1975细胞凋亡并缩短细胞迁移距离和减少穿膜细胞数（ P＜0�05）；在2～10 Gy照射范围内，10、25、50、100 nmol／L CL⁃387785处理后的SF均低于0 nmol／L，且SF随CL⁃387785浓度的增加而降低，以上差异均有统计学意义（ P＜0�05）；相对于0 nmol／L，10、25、50、100 nmol／L CL⁃387785处理后的 SER 依次为1�17、1�39、2�88和3�64。结论不可逆 EGFR 抑制剂 CL⁃387785可抑制 EGFR T790M突变非小细胞肺癌H1975细胞的侵袭转移能力并具有放射增敏作用。%Objective To investigate the inhibitory effect of CL⁃387785 on the invasion and metastasis of EGFR T790M mu⁃tant H1975 cells and its sensitization of radiotherapy. Methods The H1975 cell was treated with 0, 10, 25, 50, 100 nmol/L CL⁃387785. The MTT colorimetric assay was used to detect the proliferation inhibition rates at 24, 48, 72 and 96 h after treatment. Moreo⁃ver, Annexin⁃FITC/PI double staining method via flow cytometry was used to detect the apoptotic rates of different concentrations of CL⁃387785 at 48 and 96 h. Changes of invasion and migration of H1975 cells treated with CL⁃387785 were detected by Transwell and scratch assay, respectively. The clone formation assay was employed to investigate the survival fraction ( SF) of the H1975 cells after exposure to CL⁃387785 following different doses of X⁃ray ( 0, 2, 4, 6, 8 and 10 Gy) irradiation. The target model was fitted to the cell survival curve to calculate the sensitization enhancement ratio ( SER) . Results CL⁃387785 inhibited the proliferation of H1975 cells, and dose⁃and time⁃dependently increased the inhibition rates of the proliferation in range of 10⁃100 nmol/L. CL⁃387785 could induce the apoptosis and decrease the number of penetrate⁃membrane cell and migration distance of H1975 cells in a dose⁃dependent manner ( P<0�05) . In the 2⁃10 Gy irradiation range, the SF of 10, 25, 50 and 100 nmol/L CL⁃387785 were lower than that of 0 nmol/L, and SF decreased with the increase of concentration of CL⁃387785 and above differences were statistically significant ( P<0�05) . Compared with the 0 nmol/L CL⁃387785, the SER of 25, 50, 10, 100 nmol/L CL⁃387785 SER after treatment were 1�17, 1�39, 2�88 and 3�64, respectively. Conclusion The irreversible EGFR inhibitor CL⁃387785 can inhibit the invasion and metastasis of T790M EGFR mutant H1975 cells and has the effect of radiation sensitization.