Objective: To investigate the efficacy of h-R3 combined radiotherapy with different treatment schedules on human lung cancer cells expressing different EGFR levels.Methods: The in vitro cultured human lung cancer cell lines A549 and Calu-6 were used as research objects.Western blot assay was conducted to determine the expression of EGFR in the cell lines.The grouping of h-R3 administration was as follows: Group Ⅰ, administration 24 h before irradiation; Group Ⅱ, administration during irradiation; and Group Ⅲ, administration 24 h after irradiation.The cytotoxic and radiosensitizing effects of h-R3 on the two cell lines were determined by MTT reduction and clone formation assay.The cell apoptosis and cell-cycle distribution were detected using flow cytometer.The expression of P21, Bax, Bcl-2, phosphorylated DNA-PKcs, Akt, and pAkt were determined by Westem blot analysis.Results: The IC20and IC50 values were ( 1.13±0.37 ) and ( 24.78±1.25 ) μg/mL, respectively, 24 h after h-R3 administration in the A549 cells.Moreover,the radiosensitization ratios of the administration before, during, and after the radiation therapy were 2.02, 1.77 and 1.39, respectively.Higher apoptotic rate and G2/M blockage were observed in the group administered before radiotherapy; in addition, the changes in the expression of P21, Bax, Bcl-2, phospho-DNA-PK, and pAkt were more obvious in this group than in the others.The IC20 and IC50 values were ( 3.42±1.26 ) and ( 51.15±2.41 ) μg/mL, respectively, 24 h after h-R3 treatment in the Calu-6 cells.The radiosensitization ratios of the administration before, during, and after the radiation therapy were 1.23, 0.99 and 0.91, respectively.The apoptotic rate and G2/M blockage achieved the highest proportion in the group administered before the radiotherapy.In the group with h-R3 administration before radiotherapy, there were no apparent changes in the expressions of P21, Bax, Bcl-2, phospho-DNA-PK, and pAkt compared with the other groups.Conclusion: The best radiosensitizing effect was achieved when h-R3 was delivered before irradiation.The h-R3 differently enhanced the radiosensitizing effects in the two cells, probably via Akt/or pAkt expression.The inhibitory action of h-R3 on cell proliferation occurred both in the A549 and in the Calu-6 cells.The radiosensitizing effect was affected by different time series of administration, in which the effect of the administration before radiotherapy ranked the best.%目的:探讨尼妥珠单抗不同加药时间对A549、Calu-6人肺癌细胞系的放射增敏作用及发生机制.方法:采用人肺癌细胞系A549、Calu-6体外培养作为研究对象.Western blot法检测细胞系EGFR表达.尼妥珠单抗不同给药分组:尼妥珠单抗放疗前24 h组,放疗同时加药组,放疗后24 h加药组.MTT法及克隆形成法检测尼妥珠单抗对两种细胞系的细胞毒性作用和放射增敏作用;流式细胞仪(FCM)检测细胞凋亡和细胞周期分布;Western blot法检测P21、Bax、Bcl-2、phospho-DNA-PK、Akt和pAkt相关蛋白的表达.结果:尼妥珠单抗作用于A549细胞24h后的IC20值、IC50值分别为(1.13±0.37)、(24.78±1.25)μg/mL,放疗前、放疗中和放疗后给药的放射增敏比分别为2.02、1.77、1.39.放疗前加药组观察到更高的凋亡率和G2/M期的阻滞.同时,本组P21、Bax、Bcl-2、phospho-DNA-PK和pAkt表达的改变也较其它组明显.尼妥珠单抗作用于Calu-6细胞24 h后的IC20值、IC50值分别为(2.42±1.26)、(51.15±2.41)μg/mL.放疗前,放疗中和放疗后给药的放射增敏比分别为1.23、0.99、0.91.放疗前加药组凋亡率和G2/M期的阻滞比例最高.放疗前给药组P21、Bax、Bcl-2、phospho-DNA-PK和pAkt表达改变较其它组明显.结论:尼妥珠单抗对A549、Calu-6细胞均有抑制增殖作用.不同加药时序影响放射增敏效应,其中放疗前加药放射增敏效应最强.
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