Objective: To explore the effect of aspirin combined with metformin on the apoptosis of thyroid cancer TPC-1 cells and its mechanism. Methods: The proliferation and apoptosis of TPC-1 cells treated with different concentrations of aspirin and metformin were detected using cell count kit-8 (CCK-8) assay and flow cytometry, respectively. Western blot was used to detect the expressions of microtubule-associated protein light chain 3 (LC3), p62 and cysteinyl aspartate specific proteinase 3 (caspase-3) after treatment with aspirin, metformin and 3-Methyladenine (3-MA). Results: The relative cell viability of TPC-1 cells treated with 0.5, 1.0, 2.0, 4.0 mmol/L aspirin for 24 and 48 hours were (85.6±9.1)%, (79.9±8.6)%, (57.0±5.3)%, (55.7±5.4)%; (76.7±2.8)%, (75.4±6.1)%, (46.1±4.1)%, (36.3±3.2)%, respectively. The value of half maximal inhibitory concentration (IC(50)) for 24 and 48 hours were 4.297 mmol/L, 2.133 mmol/L, respectively. The apoptotic rate in the 1 mmol/L aspirin treatment group and negative control group were (29.2±8.5)%, (4.2±2.9)%, respectively (P<0.05). Moreover, treatment with metformin increased the protein expression of LC3Ⅱ/Ⅰ ratio, and decreased the expression of p62, while treatment with aspirin decreased the expression of LC3Ⅱ/Ⅰ ratio and increased the expression of p62. The relative cell viability of TPC-1 cells treated with metformin, 3-MA, an autophagy inhibitor, and 3-MA combined with metformin were (73.2±9.2)%, (95.8±3.3)%, (59.9±9.2)%, respectively. The apoptotic rates in these groups were (35.5±1.5)%, (12.3±1.4)%, (49.9±5.4)%, respectively. Compared with the metformin group, the relative cell viability in metformin combined with 3-MA group was significantly lower while the apoptotic rate was higher (P<0.05), which indicated that treatment with 3-MA enhanced the metformin-induced apoptosis of TPC-1 cells. The relative cell viability of TPC-1 cells in metformin group, aspirin group, metformin combined with aspirin group were (87.3±11.8)%, (85.7±9.6)%, (72.4±8.8)%, respectively. The apoptotic rates in these groups were (29.7±4.0)%, (30.5±6.5)%, (52.5±4.6)%, respectively. Compared with the metformin or aspirin group, the relative cell viability in metformin combined with aspirin group was significantly lower, while the apoptotic rate was higher (P<0.05), which indicated that aspirin enhanced the metformin-induced apoptosis of TPC-1 cells. Conclusions: Our findings indicate that metformin-mediated autophagy plays a protective role in metformin-induced apoptosis and proliferation inhibition. Aspirin enhances the metformin-induced apoptosis of thyroid cancer TPC-1 cells through inhibition of autophagy.
目的： 探讨阿司匹林和二甲双胍对甲状腺癌细胞TPC－1凋亡的影响和作用机制。 方法： 采用CCK－8法和流式细胞术检测阿司匹林和二甲双胍对甲状腺癌TPC－1细胞增殖和凋亡的影响，采用Western blot检测阿司匹林、二甲双胍和3－MA对自噬相关蛋白微管相关蛋白轻链3(LC3)、p62和caspase－3表达水平的影响。 结果： CCK－8结果显示，0.5、1.0、2.0、4.0 mmol/L阿司匹林作用24 h后，TPC－1细胞的存活率分别为(85.6±9.1)%、(79.9±8.6)%、(57.0±5.3)%和(55.7±5.4)%，半数抑制浓度(IC(50))为4.297 mmol/L；0.5、1.0、2.0、4.0 mmol/L阿司匹林作用48 h后，TPC－1细胞的相对存活率分别为(76.7±2.8)%、(75.4±6.1)%、(46.1±4.1)%和(36.3±3.2)%，IC(50)为2.133 mmol/L。流式细胞术显示，1 mmol/L阿司匹林作用TPC－1细胞24 h后，TPC－1细胞的凋亡率为(29.2±8.5)%，与阴性对照组[(4.2±2.9)%]比较，差异有统计学意义(P<0.05)。Western blot检测结果显示，阿司匹林处理后，与对照组比较，LC3Ⅱ/LC3Ⅰ比值下降，而p62的表达水平升高；二甲双胍处理后，与对照组比较，LC3Ⅱ/LC3Ⅰ比值升高，而p62的表达水平下降。阿司匹林抑制细胞的自噬，而二甲双胍促进细胞的自噬。自噬抑制剂3－MA预处理TPC－1细胞后，二甲双胍组、3－MA组和二甲双胍联合3－MA组TPC－1细胞的相对存活率分别为(73.2±9.2)%、(95.8±3.3)%和(59.9±9.2)%，凋亡率分别为(35.5±1.5)%、(12.3±1.4)%和(49.9±5.4)%；与二甲双胍组比较，二甲双胍联合3－MA组TPC－1细胞的相对生长抑制率和凋亡率上升，差异均有统计学意义(均P<0.05)。二甲双胍联合阿司匹林处理后，二甲双胍组、阿司匹林组和二甲双胍联合阿司匹林组TPC－1细胞的相对存活率分别为(87.3±11.8)%、(85.7±9.6)%和(72.4±8.8)%，凋亡率分别为(29.7±4.0)%、(30.5±6.5)%和(52.5±4.6)%；与二甲双胍组和阿司匹林组比较，二甲双胍联合阿司匹林组TPC－1细胞的相对抑制率和凋亡率升高，差异均有统计学意义(均P<0.05)。 结论： 二甲双胍活化的自噬降低其上调的生长抑制率和细胞凋亡水平，而阿司匹林可通过抑制细胞的自噬，协同二甲双胍促进甲状腺癌TPC－1细胞的凋亡。.
Keywords: Apoptosis; Aspirin; Autophagy; Metformin; Thyroid neoplasms.