Background and objective The epidermal growth factor receptor (F.GFR) is an important determinant of tumor response to ionizing radiation. Elevated levels of EGFR expression and activity are frequently correlated with the radiotherapy resistance of tumors, including that of non-small cell lung cancer (NSCLC). The molecular inhibition of EGFR signaling is a promising therapeutic strategy for enhancing the cytotoxic effects of radiotherapy. The aim of the present study is to determine whether gefitinib, a selective EGFR tyrosine kinase inhibitor, can radiosensitize the NSCLC H358 and HCC827 tell lines. The study also aims to elucidate the mechanism, by which this drug restores the radiosensitivity of NSCLC cells. Methods The two cell lines were divided into two groups, the X-ray and gefitinib-interfering groups. The former was irradiated with X-rays only, and the latter was treated with 1 祄ol/L gefitinib for 24 h before irradiation under the same conditions as those for the first group. Clonogenic cell survival assay was performed to determine the radiosensitivity of both cell groups. Immunostaining for confocal microscopy was performed to observe nuclear y-H2AX repair and EGFR foci after irradiation. Nuclear EGFR expression was also detected by Western blot analysis after radiotherapy. Results The clonogenic cell survival assay revealed that the surviving fraction at 2 Gy (SF2) of the gefitinib-interfering group (0.355) was lower than that of the X-ray group (0.433) in H358 cells. There was no significant difference between the SF2 values of the two respective groups in HCC827 cells (0.223 vs 0.242). The confocal microscopy results found that gefitinib increased the average number of γ-H2AX foci after irradiation in H3S8 cells, but did not affect the foci in HCC827 cells. Based on confocal microscopy and Western blot analyses, gefitinib also inhibited the translocation of EFGR into the nuclei of H358 cells. However, EGFR was not observed in the nuclei of HCC827 cells for both treatment groups. Conclusion Gefitinib enhances the radioresponse of H358 cells, which may be attributed to the suppression of EGFR transport into the nucleus. However, gefitinib does not affect HCC827 cells, where EGFR remains in the cytoplasm after irradiation.%背景与目的 表皮生长因子受体( epidermal growth factor receptor,EGFR)是决定放疗效应的一个重要因素,其过表达或是下游通路的激活与包括非小细胞肺癌在内的肿瘤的放疗抵抗相关,因而阻断EGFR的信号通路可能会增强放疗敏感性.本研究旨在探讨小分子EGFR酪氨酸激酶抑制剂吉非替尼能否增加肺癌细胞株HCC827和H358的放疗敏感性及其能的机制.方法 选取HCC827和H358这两个非小细胞肺癌细胞株,分为单纯X线组和X线+吉非替尼两组.单纯X线组采用单纯X线照射,X线+吉非替尼组经1 μmol/L吉非替尼作用24 h后再行X线照射.克隆形成实验比较两株细胞中不同分组细胞放射敏感性,免疫荧光激光共聚焦显微镜观察X线照射后各时间点细胞核中的磷酸化H2AX(γ-H2AX)及EGFR焦点在细胞中的定位情况,Western blot法检洲放疗后胞质胞核蛋白中EGFR的表达.结果 克隆形成实验中.H358细胞实验组与对照组在各放疗剂量点的SF2值分别为0.355和0.433; HCC827细胞实验组与对照组在各放疗剂量点的SF2值分别为0.223和0.242,差别不明显.激光共聚焦显微镜观察照射4 Gy后各时间段实验组H358细胞核中g-H2AX斑点相比对照组要多,且持续时间更长.而对照组和实验组的HCC827细胞g-H2AX斑点在各时间段并无明显差异;激光共聚焦显微镜观察照射4 Gy后对照组H358的EGFR蛋白在1h内入核,而经吉非替尼处理后EGFR蛋白几乎不入核;实验组及对照组HCC827细胞的EGFR表达位置均在细胞质中,胞核中很少或者没有,可以认为并无入核现象;Western blot结果显示,H358细胞在经4Gy放射处理后有入核现象,而预先经吉非替尼处理后,EGFR蛋白几乎不存核内表达而仍位于细胞浆内.对于HCC827细胞,实验组及对照组的EGFR蛋白均在细胞质中表达,胞核中很少或没有,且两组并无明显差异.结论 吉非替尼可增加肺癌细胞株H358的放射敏感性,这可能与其阻止放疗后EGFR入核、影响放疗后双链断裂(double strand break,DSB)修复有关;而对HCC827细胞无影响,可能与其放疗后EGFR不入核相关.
展开▼
