金属镉促进甲状腺未分化癌FRO细胞增殖的作用及机制

Role of cadmium in promoting proliferation in human anaplastic thyroid carcinoma FRO cells

目的探讨金属镉对甲状腺未分化癌FRO细胞增殖的影响及其作用机制。方法Western blot法检测FRO、MCF-7和MAD-MB-231细胞中G蛋白偶联受体1(G protein-coupled estrogen receptor,GPER1)表达水平。不同浓度(0、0.25、0.50、0.75、1.00 mmol/L)镉(Cd Cl2)处理FRO、MCF-7及MAD-MB-231细胞48 h后,MTT法检测细胞增殖率。0.5 mmol/L镉分别处理FRO细胞0、5、10、15、30 min,Western blot法检测ERK1/2和Akt的磷酸化水平。GPER1抑制剂G15处理FRO细胞后,设计合成针对GPER1的小干扰RNA(GPER-siRNA)并转染FRO细胞,采用Western blot再次检测ERK1/2和Akt磷酸化水平。分别用GPER1抑制剂G15、ERK1/2抑制剂(PD98059)和PI3K-Akt抑制剂(LY294002)、GPER-siRNA处理FRO细胞,MTT法检测细胞增殖率。结果 GPER1在MAD-MB-231细胞中表达水平明显低于MCF-7、FRO细胞。不同浓度Cd Cl2处理FRO、MCF-7及MAD-MB-231细胞48 h后,低浓度Cd Cl2促进FRO、MCF-7细胞增殖,对MAD-MB-231细胞增殖无显著影响;高浓度Cd Cl2对细胞均具有抑制作用。0.5 mmol/L Cd Cl2处理FRO细胞不同时间后,ERK1/2与Akt的磷酸化水平在15 min达最大值。GPER1抑制剂G15处理FRO细胞,ERK1/2与Akt的磷酸化水平显著降低(P<0.05)。GPER1的小干扰RNA干扰后,ERK1/2与Akt的磷酸化水平明显降低(P<0.05)。G15、PD、LY和GPER-siRNA处理FRO细胞,细胞增殖率均显著下降。结论金属镉通过GPER1-ERK/Akt信号通路促进FRO细胞的增殖。

Objective To determine the effect of cadmium on the proliferation of anaplastic thyroid carcinoma FRO cells,and investigate the underlying mechanisms. Methods Western blotting was used to detect the expression of G protein-coupled estrogen receptor 1（ GPER1） in FRO cells,MCF-7 cells and MAD-231 cells respectively. After the above cells were separately treated with different concentrations of Cd Cl2（ 0,0. 25,0. 50,0. 75 and 1. 00 nmol/L） for 48 h,the cell viability was evaluated by MTT assay. The FRO cells were treated by 0. 5 mmol / L Cd Cl2 for 0,5,10,15 or 30 min,the levels of phosphorylated ERK1 /2and Akt were analyzed by Western blot analysis. FRO cells were treated with G15 or transfected with the designed and synthesized RNA interference against GPER（ GPER-RNAi）,the levels of phosphorylated ERK1 /2 and Akt were analyzed by Western blotting respectively. FRO cells were treated with GPER1 inhibitor G15,ERK1 /2 inhibitor PD98059（ PD） and PI3K-Akt inhibitor LY294002（ LY）,alone or in combination,and MTT assay was used to determine the cell proliferation. Results The expression level ofGPER1 was significantly lower in the MAD-MB-231 cells than in the MCF-7 and FRO cells. Low-dosed Cd Cl2 treatment for 48 h promoted the proliferation of the FRO cells and MCF-7 cells,but had no effect on that of the MAD-MB-231 cells. However,Cd Cl2 at high concentrations suppressed the cell proliferation in the 3 kinds of cells. After the cells were treated by 0. 5 mmol / L Cd Cl2 for different periods,the levels of phosphorylated ERK1 /2 and Akt reached peak at 15 min in FRO cells,and the levels of phosphorylated ERK1 /2 and Akt were decreased significantly in FRO cells（ P〈0. 05） when the cells were treated with GPER1 inhibitor G15,transfected with GPER-siRNA,ERK1 /2 inhibitor PD or Akt inhibitor LY. Conclusion Cadmium promotes the proliferation of anaplastic thyroid carcinoma FRO cells through GPER1-ERK / AKT pathway.