论文部分内容阅读
目的 探讨糖原合成酶激酶-3β (GSK3β) 在非小细胞肺癌中对自噬的调节及调节自噬对放疗敏感性的影响.方法 通过Western blot检测正常支气管上皮细胞HBE和肺癌细胞A549, H460, H292, H1299, Calu1和SK-MES-1中GSK3β的表达情况.转染或抑制GSK3β的表达并经X射线照射后, 通过Western blot检测磷酸腺苷蛋白激酶 (AMPK), 自噬标记物微管相关蛋白轻链-3 (LC3) 和自噬降解底物p62的蛋白表达水平.应用集落形成实验检测转染或抑制GSK3β的表达并经X射线照射后的细胞增殖能力, 促进或抑制自噬并经X射线照射后的细胞增殖能力.结果 在H460细胞中分别转染野生型GSK3β-WT, 激酶激活型GSK3β-S9A, 激酶失活型GSK3β-K85R并经X射线照射, 下调AMPK和LC3表达水平, 上调P62表达水平, 抑制肺癌细胞的增殖能力, 其中转染激活型GSK3β-S9A的作用最显著.在A549细胞中抑制GSK3β并X射线照射, 上调AMPK和LC3表达水平, 促进肺癌细胞的增殖能力.在H460细胞中施加自噬抑制剂3-甲基腺嘌呤 (3-MA) 或自噬诱导剂雷帕霉素 (Rapamycin) 并经X射线照射, 3-MA抑制细胞自噬水平, 抑制细胞增殖能力, 细胞存活能力下降更显著.结论GSK3β抑制细胞自噬, 抑制细胞自噬水平可以增强非小细胞肺癌的放射敏感性.“,”Objective The goal of this study was to understand the mechanisms of GSK3β-regulated autophagy and the effects of autophagy on radiosensitivity in non-small cell lung cancer. Methods Western blot was used to detect the expression of GSK3β in HBE (normal bronchial epithelial cell line) and six lung cancer cell lines (A549, H460, H292, H1299, Calu1 and SK-MES-1), and quantitate expression changes of autophagy-related proteins after GSK3β inhibitors were added to A549 cells, or after transfecting H460 cells with GSK3β variants and subjecting them to X-ray irradiation. Clonogenic assays were used to measure the effect of autophagy on cellular proliferation. ResultsThe expression level of GSK3β in A549, H292, Calu1 and SK-MES-1 was significantly higher than that in HBE, but in H1299 and H460 was lower than that in HBE. After transfecting H460 cells with wild type GSK3β (GSK3β-WT), constitutively active GSK3β-S9A and catalytically inactive GSK3β-K85R and subjecting these cells to X-ray irradiation, the expression of AMPK and LC3 decreased, whereas p62 expression increased; these effects were particularly significant with the GSK3β-S9A mutant. In A549 cells, after GSK3β inhibition and X-ray irradiation, the expression of AMPK and LC3 proteins increased. We used 3-methyladenine (3-MA) to inhibit autophagy and the rapamycin to increase autophagy and X-ray induction. When cellular autophagy was inhibited, cell proliferation decreased. Conclusion X-rays downregulate GSK3β expression in some NSCLC cells and promote cellular autophagy. GSK3β regulates autophagy, and inhibition of this process can enhance the radiosensitivity of NSCLC cells.