依维莫司对人非小细胞肺癌细胞系A549放射增敏作用

摘要

目的：通过使用哺乳动物雷帕霉素靶蛋白mTOR抑制药依维莫司抑制A549细胞mTOR信号通路,研究依维莫司是否具有放射增敏作用。方法单纯放射治疗(放疗)或联合依维莫司作用于人非小细胞肺癌细胞系A549,采用噻唑蓝( MTT)法测定依维莫司对A549细胞抑制率并计算半数抑制浓度( IC50)。应用药物20%抑制浓度( IC20)作用24 h后X线2,4,6,8 Gy照射。计算细胞克隆存活分数及多靶单击模型拟合生存曲线,并计算平均致死剂量( D0)、准阈剂量(Dq)、照射剂量2 Gy下细胞存活分数(SF2)和放射增敏比(SER)。采用Western blot 方法检测γ_H2AX蛋白的表达,并分析相对灰度值。结果依维莫司联合放疗可明显提高A549细胞对射线的敏感性,依维莫司+照射组D0、Dq及SF2均明显低于单纯照射组,SER为1.36。依维莫司+照射组X线照射后24 h点γ_H2AX蛋白残余量明显高于单纯照射组。结论依维莫司抑制mTOR信号通路能够提高A549细胞的放射敏感性。%Objective To exPlore the effect of mammalian target of raPamycin ( mTOR ) inhibitor eVerolimus on radiosensitiVity of human non_small cell lung cancer cell line in vitro by using eVerolimus to inhibit mTOR signaling Pathway of A549. Methods Human non_small cell lung cancer cell line A549 was subjected to radiation alone or in combination with eVerolimus treatment. The 50%inhibition concentration ( IC50 ) of eVerolimus in A549 cells was detected by methylthiazol tetrazolium ( MTT) assay in vitro. EVerolimus at the 20%inhibition concentration ( IC20 ) was used to Pretreat A549 cells for 24 h. Cells were then irradiated by X_ray with 2,4,6,8 Gy. The cell surViVal fraction was comPuted by clone formation. Cell surViVal curVe was fitted by multitarget one_hit model, and mean lethal dose ( D0 ), dose quasithreshold ( Dq ), surViVal fraction at 2 Gy ( SF2 ), and sensitization enhancement ratio (SER) were calculated. The exPression ofγ_H2AX was determined by Western blotting and then the relatiVe gray Values were analyzed. Results EVerolimus significantly imProVed the sensitiVity of A549 cells to radiation. The D0 , Dq and SF2 of eVerolimus+irradiation grouP were significantly lower than those of irradiation grouP. The SER was 1. 36. The residual amount of γ_H2AX Protein in the eVerolimus + irradiation grouP was significantly higher than that of the irradiation grouP. Conclusion EVerolimus inhibiting mTOR signaling Pathway can increase the radiosensitiVity of A549 cells.