二甲双胍对甲状腺乳头状癌TPC-1细胞凋亡的影响及其机制研究

Effect and mechanism of metformin on apoptosis of papillary thyroid carcinoma TPC-1 cells

目的探讨二甲双胍对甲状腺乳头状癌TPC-1细胞凋亡的影响及其机制。方法用不同浓度(0mmol/L、1mmol/L、5mmol/L、10mmol/L、20mmol/L、40mmol/L)二甲双胍分别处理TPC-1细胞24h。采用四甲基偶氮唑蓝(MTT)法检测细胞增殖能力;采用AnnexinⅤ-FITC/PI流式细胞术检测不同浓度二甲双胍对TPC-1细胞凋亡的影响,同时在40mmol/L组加入腺苷酸活化蛋白激酶(AMPK)抑制剂Compound C,观察其对TPC-1细胞凋亡的影响;采用Western blot法检测磷酸化AMPK(p-AMPK)及Caspase-9蛋白的表达。结果与0mmol/L组比较,1mmol/L及5mmol/L组的TPC-1细胞增殖能力无明显改变(P>0.05);而10mmol/L、20mmol/L、40mmol/L二甲双胍均可明显抑制TPC-1细胞的增殖能力,且呈剂量依赖性(P<0.05)。与0mmol/L组比较,不同浓度二甲双胍均可明显增加TPC-1细胞凋亡率(P<0.001);而加入Compound C后,40mmol/L组TPC-1细胞凋亡率较前明显下降(P<0.001)。与0mmol/L组比较,1mmol/L、5mmol/L、10mmol/L、20mmol/L、40mmol/L组p-AMPK、Caspase-9表达量增加。结论二甲双胍可诱导甲状腺乳头状癌TPC-1细胞凋亡,且二甲双胍诱导TPC-1细胞凋亡的过程可能与激活AMPK通路有关,可能由Caspase-9介导。

Objective To investigate the effect and mechanism of metformin on the apoptosis of papillary thyroid carcinoma TPC-1 cells.Methods TPC-1 cells were treated with different doses(0mmol/L,1mmol/L,5mmol/L,10mmol/L,20mmol/L and 40mmol/L)of metformin for 24h.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay was used to measure the proliferation viability of cell lines.AnnexinⅤ-FITC/PI flow cytometry was used to detect the rate of apoptosis of TPC-1 cells treated by different concentrations of metformin.At the same time,the adenosine 5’-monophosphate-activated protein kinase(AMPK)inhibitor Compound C was added to the 40mmol/L group,and its effect on the apoptosis of TPC-1 cells was observed.Western blotting was performed to determine the protein expressions of p-AMPK and Caspase-9.Results Compared with the 0mmol/L group,the proliferation viability of TPC-1 cells in 1mmol/L and 5mmol/L groups had no significant changes(P>0.05).But 10mmol/L,20mmol/L and 40mmol/L metformin all significantly inhibited the proliferation activity of TPC-1 cells in a dosedependent manner(P<0.05).Compared with the 0 mmol/L group,the different concentrations of metformin could significantly increase the apoptosis rate of TPC-1 cells(P<0.001).However,after the addition of Compound C,the apoptosis rate of TPC-1 cells in 40mmol/L group was significantly lower than that before(P<0.001).Compared with the 0mmol/L group,the different concentrations of metformin could increase the protein expression of p-AMPK and Caspase-9.Conclusion Metformin can induce apoptosis of TPC-1 cells.The process of apoptosis induced by metformin is probably related to the activation of AMPK signaling pathway,which may be mediated by Caspase-9.