自噬对人肺腺癌细胞放疗敏感性的影响

目的 探讨自噬对人肺腺癌细胞放疗敏感性的影响。 方法 采用透射电镜和荧光显微镜检测亲本人肺腺癌细胞SPC-A1和耐药细胞SPC-A1／DTX放疗前后的自噬活性。应用自噬抑制剂3-甲基腺嘌呤（3-MA）预处理经射线照射后的细胞，应用Western blotting检测自噬相关蛋白标志物LC3、p62和Beclin-1蛋白表达的变化，流式细胞术检测细胞凋亡和细胞周期分布变化。四甲基偶氮唑盐和克隆形成实验分别检测抑制自噬后细胞生存和增殖能力的改变情况。 结果 化疗耐药的人肺腺癌耐多西他赛细胞SPC-A1／DTX比亲本细胞更耐受放疗，接受相同放疗剂量后增殖能力更强；前者较后者自噬小体较多、带绿色荧光蛋白标签的LC3荧光密度明显增多，自噬相关蛋白标志物表达更加明显（P＜0.05）。放射线同时可引起两种细胞株自噬水平的增高。自噬抑制剂3-MA预先处理耐药细胞并接受放疗后，可显著抑制自噬标志物LC3和Beclin-1，上调p62表达（P＜0.05），并降低SPC-A1／DTX细胞的生存和增殖能力，放射敏感的G2／M期细胞比例和凋亡率增加，差异均有统计学意义（P＜0.05）。 结论 自噬为SPC-A1／DTX细胞耐受放射治疗提供了一个自我保护的机制。自噬能够增强人肺腺癌耐多西他赛细胞的放疗抵抗，而抑制自噬有可能是逆转人肺腺癌细胞放、化疗耐受的一个策略。

Influence of autophagy on radiosensitivity of lung adenocarcinoma cells

Objective To explore the influence of autophagy on radiosensitivity of drug resistant lung adenocarcinoma cells. Methods Transmission electron microscopy and fluorescence microscope were applied to assess autophagy activity of parent lung adenocarcinoma cells（SPC-A1） and drug resistant lung adenocarcinoma cells（SPC-A1／DTX）. After the pretreatment of autophagy inhibitor 3-methyladenine（3-MA） with SPC-A1／DTX， Western blotting analysis was used to detect autophagy related protein expression of LC3， p62 and Beclin-1 with or without 6 Gy-radiation. Apoptosis and cell cycle distribution were analyzed by flow cytometry. Cell viability and proliferation were examined by MTT and colony formation assay, respectively. Results The chemoresistant SPC-A1／DTX cells with higher autophagy activity were also radioresistant compared with the parental SPC-A1 cells（P＜0.05）. SPC-A1／DTX cells showed an increase in autophagosome and punctate localization of green fluorescent protein-LC3（characteristic of autophagy） compared to SPC-A1 cells. Irradiation elevated autophagy levels of both cells. Autophagy inhibitor 3-MA combination with irradiation suppressed the expression of autophagy markers, including LC3， p62 and Beclin-1（P＜0.05）. Irradiation-induced G2／M phase delay was increased and S phase arrest was decreased by combination of 3-MA with irradiation（P＜0.05）. The apoptotic rate was elevated when pretreatment with 3-MA before radiation. Conclusion Our results demonstrated that irradiation-induced autophagy provided a cytoprotective mechanism against radiotherapy in SPC-A1／DTX cells. Autophagy contributed to radioresistance of docetaxel-resistant human lung adenocarcinoma cells， and blocking autophagy would be a potential strategy for reversing chemoradiotherapy cross resistance of lung adenocarcinoma patients.