二甲双胍通过巨噬细胞抑制食管癌TE-1细胞的侵袭和迁移

Metformin inhibits the invasion and migration of esophageal cancer TE-1 cells through macrophages

目的:探究二甲双胍是否通过巨噬细胞抑制食管癌TE-1细胞的侵袭迁移及其可能的分子机制。方法:100 ng/mL PMA诱导THP-1巨噬细胞。取对数生长期TE-1细胞和巨噬细胞,分别用最终浓度为0.5 mmol/L、1 mmol/L和2 mmol/L二甲双胍处理,Western blot检测MMP9、VEGF蛋白表达水平,ELISA检测细胞TNF-α和IL-8浓度,筛选二甲双胍最佳作用浓度(2 mmol/L)。分别用100 ng/mL LPS、2 mmol/L二甲双胍、100 ng/mL LPS+2 mmol/L二甲双胍处理巨噬细胞制备条件培养基。将TE-1细胞分为4组:对照组、LPS-条件培养基组(LPS组)、二甲双胍-条件培养基组(Met组)和LPS+二甲双胍-条件培养基组(LPS+Met组)。划痕实验观察各组细胞迁移能力。Transwell小室检测细胞侵袭能力。Western blot检测各组细胞VEGF、E-cad、Vimentin和NEDD9的表达。ELISA检测各组细胞TNF-α和IL-8浓度。结果:与对照组相比,二甲双胍可显著降低TE-1细胞和巨噬细胞中MMP9、VEGF、TNF-α和IL-8水平(P<0.05),且呈剂量依赖性。二甲双胍可显著抑制TE-1细胞迁移和侵袭(P<0.05),显著抑制VEGF、Vimentin和NEDD9蛋白表达(P<0.05),促进E-cad蛋白表达(P<0.05);显著降低TE-1细胞中TNF-α和IL-8水平(P<0.05)。结论:二甲双胍可通过巨噬细胞降低食管癌TE-1细胞NEDD9的表达,抑制食管癌细胞侵袭迁移。

Objective:To investigate whether metformin inhibits the invasion and metastasis of esophageal cancer TE-1 cells through macrophages and its possible molecular mechanism.Methods:THP-1 macrophages were induced by 100 ng/mL PMA.TE-1 cells and macrophages at logarithmic growth stage were treated with metformin at final concentrations of 0.5 mmol/L,1 mmol/L and 2 mmol/L,respectively.Western blot was used to detect the protein expression levels of MMP9 and VEGF,and ELISA was used to detect the concentrations of TNF-αand IL-8.The optimal concentration of metformin(2 mmol/L)was selected.Macrophages were treated with 100 ng/mL LPS,2 mmol/L metformin and 100 ng/mL LPS+2 mmol/L metformin to prepare conditioned medium.TE-1 cells were divided into 4 groups:Control group,LPS conditioned medium group(LPS group),metformin conditioned medium group(Met group)and LPS+metformin conditioned medium group(LPS+Met group).The migration ability of each group was observed by scratch test.Transwell chamber was used to detect cell invasion.Western blot was used to detect the expression of VEGF,Vimentin,E-cad and NEDD9.The concentrations of TNF-αand IL-8 were detected by ELISA.Results:Metformin significantly reduced the levels of MMP9,VEGF,TNF-αand IL-8 in TE-1 cells and macrophages in a dose-dependent manner compared with the control group(P<0.05).Metformin significantly inhibited the migration and invasion of TE-1 cells(P<0.05),significantly inhibit the expression of VEGF,Vimentin and NEDD9(P<0.05),and promote the expression of E-cad protein(P<0.05).TNF-αand IL-8 levels in TE-1 cells were significantly decreased(P<0.05).Conclusion:Metformin can reduce the expression of NEDD9 in esophageal carcinoma cells through macrophages,and inhibit the invasion and migration of esophageal carcinoma TE-1 cells.