3-甲基腺嘌呤对人下咽癌细胞的放射增敏效应及其机制研究

Mechanism of radiosensitization of 3-methyladenine in human hypopharynx cancer cells

目的探讨自噬抑制剂3一甲基腺嘌呤（3-methyladenire，3-MA）对人下咽癌细胞（Fadu细胞）的放射增敏作用及其机制。方法用5mmol／L的3-MA联合2Gy、4Gy剂量x线照射作用于Fadu细胞，克隆形成实验和单细胞凝胶电泳技术分别检测各处理组的细胞存活率（克隆形成率）和DNA损伤程度（尾矩），采用两组独立样本的t检验分析各组间差异；用不同浓度的3-MA（1、2、5、10mmol／L）分别作用于细胞，流式细胞仪检测各组细胞周期并应用单因素方差分析比较组间差异。Westernblot检测各组p62和CyclinBl的蛋白表达水平。结果单独使用3-MA对细胞存活率和DNA损伤程度无明显影响；与不加3-MA组相比，加3-MA组细胞在2Gy和4Gy剂量的X线作用下细胞存活率显著降低（t值分别为13．41、13．98，P值均〈0．001），DNA损伤程度明显加重（t值分别为7．07、6．91，P值均〈0．001）。对照组和1、2、5、10mmol／L浓度3-MA作用后的G2／M期细胞百分数分别为（17．10±1．20）％、（23．30±2．36）％、（39．90±3．12）％、（58．47±1．65）％、（76．13±3．51）％，组间差异有统计学意义（F=278．4，P〈0．001）。Westernblot方法检测，3-MA作用于细胞后，p62蛋白表达水平呈剂量依赖性上升，CyclinBl蛋白表达水平呈剂量依赖性下降。结论自噬抑制剂3-MA能加强X线对人下咽癌细胞的致DNA损伤作用和引起G2／M期阻滞，从而具有放射增敏效应。

Objective To investigate the radiosensitizing effect and its mechanism of 3-MA in human hypopharynx cancer cells. Methods Fadu cells were treated with 5 mmol/L of 3-MA combined with 2 Gy or 4 Gy of X-ray and cell cloning efficiency and DNA lesion severity （tail moment） were examined by clonogenic survival assay and comet assay. Cell cycle distributions of Fadu cells treated with different doses of 3-MA（ 1, 2, 5, 10 mmol/L）were detected by flow cytometer. The expressions of 1362 and eyelin B1 were examined by western blot. Results The cloning efficiency and DNA lesion severity of cells treated with 3-MA alone showed no notable changes. However, 3-MA enhanced significantly the inhibitory effects of X-ray （2 Gy or 4 Gy） on cloning efficiency of the cells （t = 13.41 or 13.98, P 〈 0. 001 ）, and the cells showed more serious DNA damages （t =7.07 or 6.91 ,P 〈0.001 ）. The mean G2/M percentages of control cells and the cells treated with 1, 2, 5, 10 mmol/L of 3-MA were （ 17.10 ± 1.20） % , （23.30 ± 2.3） % , （39. 90 ± 3. 12）%, （58.47 ± 1.65）% and （76. 13 ± 3.51）%, respectively, and differences among groups were statistically significant（ F = 278.4, P 〈 0.05 ）. The expression of 1362 in cells treated with 3-MA showed a dose-dependent increase, while cyclin B1 showed a dose-dependent decrease. Conclusion The autophagy inhibitor 3-MA could enhance radiosensitivity of human hypopharynx cancer cells by inducing G2/M arrest and enhancing irradiation-induced DNA damage.