自噬抑制剂3-甲基腺嘌呤增强鼻咽癌细胞对放射和化学治疗的敏感性

目的：探讨自噬抑制剂3-甲基腺嘌呤(3-methyladenine，3-MA)增强鼻咽癌细胞对放射治疗(放疗)和化学治疗(化疗)敏感性的作用及其相关的分子机制。方法：采用MTT法检测3-MA对细胞的增殖抑制作用；集落克隆形成法检测3-MA对细胞集落克隆形成的影响；Annexin V/PI双染法、线粒体膜电位检测试剂盒(JC-1)、DAPI染色检测细胞凋亡；Western 印迹检测内质网应激相关蛋白葡萄糖调节蛋白78(glucose-regulated protein 78，GRP78)、自噬相关蛋白beclin1和微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3，LC3)表达。结果：不同体积浓度或剂量的顺铂(cisplatin，DDP)、电离辐射(ionizing radiation，IR)、衣霉素(tunicamycin，TM)、3-MA对鼻咽癌细胞HONE-1均具有增殖抑制作用，且随着体积浓度或剂量的增加和作用时间的延长，对HONE-1细胞增殖抑制作用随之增加。经1.00mmol/L 3-MA预处理细胞后，再次给予6.00 μmol/L DDP，4.00 Gy IR，1.00 μmol/L TM处理，细胞存活率明显降低，均低于单用组，与空白对照组比较差异均有统计学意义(P<0.05)。3-MA增强DDP，IR，TM抑制细胞集落克隆形成作用。用6.00 μmol/L DDP或4.00 Gy IR处理HONE-1细胞24 h后，细胞凋亡率分别为5.8%和6.7%，与阴性对照组比较差异无统计学意义(P>0.05)。JC-1和DAPI染色显示3-MA增强DDP，IR或TM诱导的细胞凋亡作用；Western 印迹结果显示DDP，IR或TM处理组GRP78，beclin1表达呈时间依赖性上调，LC3 I向LC3 II转化，3-MA预处理组beclin1表达明显降低，LC3 I向LC3 II的转化。结论：自噬抑制剂3-MA可增强鼻咽癌细胞对放化疗的敏感性，其机制可能与3-MA抑制内质网应激诱导的自噬所产生的保护作用相关。

Autophagy inhibitor 3-methyladenine enhances the sensitivity of nasopharyngeal carcinoma cells to chemotherapy and radiotherapy

Objective: To explore the effects of 3-methyladenine (3-MA, an autophagy inhibitor) on sensitivities of nasopharyngeal carcinoma cells to radiotherapy and chemotherapy and the underlying mechanisms.Methods: Cell proliferation was examined by MTT and colony formation assay, while cell apoptosis was evaluated by annexin V/PI double staining and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining. Mitochondrial membrane potential was measured by commercial kit (JC-1). The expression of endoplasmic reticulum stress (ERS)-related protein, glucose-regulated protein 78 (GRP78) and autophagy-related protein beclin1, microtubule-associated protein 1 light chain 3 (LC3) were examined by Western blot.Results: Cisplatin (DDP), ionizing radiation (IR) or tunicamycin (TM) treatment obviously inhibited the proliferation of HONE-1 cells in a concentration-dependent and time-dependent manner. Compared with control group, pretreatment with 1 mmol/L of 3-MA significantly reduced cell viability and enhanced the apoptosis in the DDP (6.00 μmol/L), 4.00 Gy IR or TM (1.00μmol/L) groups. There was no significant difference in the apoptosis between the DDP (5.8%) and 4Gy IR (6.7%) groups. Compared with the control group, protein levels of GRP78, beclin1 and lipid-conjugated membrane-bound form (LC3-II) were significantly increased after the treatment of DDP, 4.00 Gy IR or TM, which were inhibited by pretreatment of 3-MA.Conclusion: 3-MA can sensitize HONE-1 cells to chemotherapy and radiotherapy, which is related to prevention of endoplasmic reticulum stress-induced autophagy in nasopharyngeal carcinoma cells.