【摘要】 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。更多还原

【Abstract】 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.更多还原