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

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

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1.00 mmol/L 3-MA预处理细胞后,再次给予6.00 mol/L DDP,4.00 Gy IR,1.00 mol/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 mol/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。

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 （1.00 μmol/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.