柚皮素对甲状腺癌细胞凋亡、自噬的影响及其与AMPK/mTORC1通路的关系

Effects of naringenin on apoptosis and autophagy of thyroid cancer cells and its association with AMPK/mTORC1 pathway

背景与目的:柚皮素(NAR)是天然的黄酮类化合物,能够抑制宫颈癌、胃癌、舌鳞癌及肝癌细胞的生长,但其对甲状腺癌细胞的作用尚不明确。本实验探讨NAR对甲状腺细胞的影响,并初步研究其作用机制,以期为甲状腺癌的药物研发提供理论基础。方法:不同浓度的NAR处理甲状腺癌ACT-1细胞不同时间后,用MTT法检测细胞存活率,以观察NAR对ACT-1细胞的时间、浓度效应并计算IC_(50)值。在以上实验基础上,选择适合的浓度的NAR与适合的时间处理ACT-1细胞,然后分别采用Annexin V-FITC/PI法检测细胞凋亡;绿色荧光蛋白(GFP)标记质粒转染检测自噬小体变化;Western blot法检测自噬相关蛋白(LC3 I、LC3 II、p62)以及AMPK/mTORC1通路相关蛋白表达的变化,以及AMPK抑制剂对NAR作用的影响。结果:NAR能明显抑制ACT-1细胞的存活,呈时间与浓度依赖性(均P<0.05),其12、24和48 h的IC_(50)值分别为85.65、50.12、38.94μg/mL。选用25、50、100μg/mL的NAR处理ACT-1细胞24 h后,细胞凋亡率、自噬小体数量、LC3 II/LC3 I和p-AMPK/AMPK蛋白表达明显升高,p62和p-mTORC1/mTORC1蛋白表达明显降低,均呈浓度依赖性(均P<0.05)。用100μg/mL NAR同时加入25μmol/L AMPK抑制剂后处理ACT-1细胞后,NAR的以上作用以及升高凋亡效应蛋白caspase-3的作用被明显抑制(均P<0.05)。结论:NAR能够抑制甲状腺癌ACT-1细胞增殖,诱导凋亡,这可能与其调控AMPK/mTORC1信号通路,增强自噬相关。

Background and Aims:Naringin(NAR)is a natural flavonoid compound,which can inhibit the growth of cells of cervical cancer,gastric cancer,tongue squamous cell carcinoma and liver cancer,but its effect on thyroid cancer cells is not clear.Therefore,this study was conducted to investigate the effects of NAR on thyroid cells,and to preliminarily analyze its mechanism,so as to provide a theoretical basis for research and development of drugs against thyroid cancer.Methods:Thyroid cancer ACT-1 cells were cultured with different concentrations of NAR for different time periods,and then,the cell viability rates were determined by MTT assay to observe the time and concentration effects of NAR on ACT-1 cells,and calculate the IC_(50) values.Based on above experiment,the proper concentrations of NAR and proper time span were chosen to treat the ACT-1 cells.After that,the cell apoptosis was detected by Annexin V-FITC/PI staining,change in autophagic bodies was observed by green fluorescent protein(GFP)-labeled plasmid transfection,and the changes in expressions of the autophagy-associated proteins(LC3 I,LC3 II,p62)and proteins in the AMPK/mTORC1 pathway,as well as the influences of AMPK inhibitor on NAR actions were examined by Western blot analysis.Results:The survival of ACT-1 cells was significantly suppressed in a time-and concentration dependent manner by NAR treatment(all P<0.05),and the IC_(50) values for 12,24 and 48 h were 85.65,50.12 and 38.94μg/mL,respectively.In ACT-1 cells after treatment with the selected concentrations of 25,50 and 100μg/mL NAR,the apoptosis rates,numbers of autophagic bodies and the protein expression levels of LC3 II/LC3 I and p-AMPK/AMPK were significantly increased,and the protein expression levels of p62 and p-mTORC1/mTORC1 were significantly decreased,with a concentration-dependent manner(all P<0.05).In ACT-1 cells after treatment with 100μg/mL NAR and simultaneous 25μmol/L AMPK inhibitor,the above effects as well as the apoptosis-functional protein caspase-3 increasing effect of NAR on ACT-1 cells were all significantly suppressed(all P<0.05).Conclusion:NAR can inhibit the proliferation and induce the apoptosis in thyroid cancer ACT-1 cells,which may be related to its regulating AMPK/mTORC1 signaling pathway and the enhancing autophagy.