胃肠安对人胃癌细胞侵袭转移及RUFY3、MAPKs通路的影响

Effects of Weichang'an(胃肠安) on Invasion and Metastasis of Human Gastric Cancer Cells and Pathway of RUFY3 and MAPKs

目的探讨胃肠安抑制胃癌侵袭转移的作用及可能作用机制。方法采取细胞增殖实验筛选胃肠安进行实验的药物浓度。实验分为空白组,胃肠安高、低剂量组,5-氟尿嘧啶(5-FU)组。细胞培养24 h后胃肠安高、低剂量组用拟筛选浓度的胃肠安100μl进行干预,5-FU组加入32 mg/L 5-FU注射液100μl,空白组给予无血清培养基100μl。采用CCK8法检测细胞增殖的变化,划痕实验和Transwell侵袭实验检测胃癌细胞迁移和侵袭能力;采用免疫印迹法检测RUN和FYVE结构域蛋白3 (RUFY3)、磷酸化细胞外信号调节激酶(p-ERK)、促分裂原活化蛋白激酶(MEK)、磷酸化促分裂原活化蛋白激酶(p-MEK)、Twist1、E-钙粘蛋白、波形蛋白、N-钙粘蛋白、血管内皮生长因子(VEGF)、基质金属蛋白酶2 (MMP2)、基质金属蛋白酶9 (MMP9)蛋白表达水平。结果筛选出500 mg/L和1000 mg/L浓度的胃肠安为胃肠安低、高剂量组给药量。给药48 h时胃肠安高、低剂量组和5-FU组细胞迁移率均明显低于空白组(P <0. 01)。胃肠安高剂量组、5-FU组穿膜细胞数显著低于空白组和胃肠安低剂量组(P <0. 01)。胃肠安高、低剂量组侵袭抑制率分别为54. 11%、7. 65%,5-FU组侵袭抑制率为47. 88%。与空白组比较,胃肠安低剂量组p-ERK、Twist1蛋白表达降低,E-钙粘蛋白表达增加(P <0. 05);胃肠安高剂量组RUFY3、p-ERK、p-MEK、Twist1、N-钙粘蛋白及波形蛋白表达降低,E-钙粘蛋白表达增加(P <0. 05)。与胃肠安低剂量组比较,胃肠安高剂量组RUFY3、p-MEK、N-钙粘蛋白表达降低更明显(P <0. 05)。结论胃肠安可抑制胃癌的侵袭转移,并且以1000 mg/L浓度效果最好,其机制可能是通过调控RUFY3、MAPKs通路来调节侵袭转移相关蛋白和抑制胃癌细胞上皮-间充质转化。

Objective To investigate the effects of Weichang’an(胃肠安)on the invasion and metastasis of gastric cancer and its possible mechanism.Methods The cell proliferation assay was used to screen the drug concentration of Weichang’an.Samples were divided into blank group,Weichang’an high dose group,Weichang’an low dose group and 5-FU group.After cell culture for 24 h,the Weichang’an high and low dose groups were treated with the screening concentration of Weichang’an 100μl for intervention.The 5-FU group was added with 32 mg/L 5-FU injection 100μl.The blank group was treated with serum-free culture base 100μl.The cell proliferation was detected by cell counting kit-8(CCK8)method,and the migration and invasion ability of gastric cancer cells were detected by scratch test and Transwell invasion assay.RUN and FYVE domain protein 3(RUFY3),phosphorylated extracellular signal-regulated kinase(p-ERK),mitogen-activated protein kinase(MEK),phosphorylated mitogen-activated protein kinase(p-MEK),Twist1,E-cadherin,vimentin,N-cadherin,vascular endothelial growth factor(VEGF),matrix metalloproteinase 2(MMP2),matrix metalloproteinase 9(MMP9)protein expression levels were detected by Western blot.Results The concentrations of 500 mg/L and 1000 mg/L Weichang’an were screened and set as Weichang’an low and high treated dose.After drug administration for 48 h,the cell migration rate of Weichang’an high,low dose group and 5-FU group was significantly lower than that of the blank group(P<0.01).The number of transmembrane cells in the Weichang’an high dose group and 5-FU group was significantly lower than that in the blank group and Weichang’an low dose group(P<0.01).The invasive inhibition rates of Weichang’an high and low dose groups were 54.11%and 7.65%,respectively,and the invasion inhibition rate of 5-FU group was 47.88%.Compared with the blank group,the expression of p-ERK and Twist1 protein was decreased in Weichang’an low dose group,and the expression of E-cadherin protein was increased(P<0.05).The expression of RUFY3,p-ERK,pMEK,Twist1.N-cadherin and Vimentin protein in the Weichang’an high dose group was decreased,and the expression of E-cadherin protein was increased(P<0.05).Compared with Weichang’an low dose group,the expression of RUFY3,p-MEK and N-cadherin protein in Weichang’an high dose group was more significantly decreased(P<0.05).Conclusion Weichang’an can inhibit the invasion and metastasis of gastric cancer,and the effect is best at the concentration of 1000 mg/L.The mechanism may be related to the regulation of invasion and metastasis-related proteins and inhibition of epithelial mesenchymal transition in gastric cancer cells by regulating RUFY3 and MAPKs pathways.