Curcumin协同ABT-737对肝癌细胞上皮间质转化的抑制作用及相关机制初步研究

The combined inhibiting effect and related mechanism of Curcumin and ABT-737 on epithelial-mesenchymal transition of human liver cancer cell

目的探讨Curcumin协同ABT-737对肝癌7404细胞上皮间质转化(epithelial-mesenchymal transition,EMT)的抑制作用及机制研究。方法2μmol/L Curcumin、5μmol/L ABT-737或2μmol/L Curcumin+5μmol/L ABT-737作用肝癌7404细胞后,观察细胞形态的变化,细胞划痕实验检测7404细胞的迁移能力,蛋白质印迹法检测7404细胞中E-cadherin、Vimentin、N-cadherin、ZEB1蛋白的表达和p-beta-catenin、p-JNK、Snail、Twist蛋白的表达。构建肝癌动物模型(Alb-Cre;P53f/f;Ras),在苏木精伊红染色下观察肝转移瘤情况,并对在肝脏形成的转移灶进行数量统计。结果相比2μmol/L Curcumin、5μmol/L ABT-737,2μmol/L Curcumin+5μmol/L ABT-737作用后,7404细胞梭形化明显减少(P<0.05),细胞迁移能力显著降低(P<0.05),Vimentin、N-cadherin、ZEB1蛋白的表达水平明显抑制(P<0.05),E-cadherin蛋白的表达水平明显上调(P<0.05),beta-catenin和JNK的磷酸化水平明显上调(P<0.05),beta-catenin下游靶基因Snail、Twist的表达水平明显抑制(P<0.05)。相比对照组,Curcumin+ABT-737作用后,肝癌动物模型(Alb-Cre;P53f/f;Ras)中肝转移瘤数量明显减少(P<0.05)。结论Curcumin协同ABT-737能抑制肝癌细胞上皮间质转化,JNK-beta-catenin可能参与EMT转化的抑制。

Objective To explore the combined inhibiting effect and related mechanism of Curcumin and ABT-737 on epithelial-mesenchymal transition(EMT)of human liver cancer cell.Methods After treatment with 2μmol/L Curcumin,5μmol/L ABT-737 alone or 2μmol/L Curcumin in combination with 5μmol/L ABT-737,the morphological change of 7404 cells was observed under an inverted microscope,the cell migration was analyzed by Wound Healing,the expression levels of Vimentin,N-cadherin,ZEB1 and E-cadherin proteins in 7404 cells were detected by Western blotting.The expression levels of p-beta-catenin,p-JNK,Snail and Twist proteins in 7404 cells were also detected by Western blotting.The animal models of liver cancer(Alb-Cre;P53f/f;Ras)was established.The numbers of metastatic tumor in liver was observed and calculated under hematoxylin eosin stain.Results As compared with the 7404 cells with treatment of 2μmol/L Curcumin or 5μmol/L ABT-737,the numbers of 7404 cells that changed shape to spindle-like after treatment with 2μmol/L Curcumin+5μmol/L ABT-737 were obviously reduced(P<0.05),with significantly decline of cell migration ability.Compared with the 2μmol/L Curcumin or 5μmol/L ABT-737 group,the expression levels of E-cadherin protein were significantly increased while the expression levels of Vimentin,N-cadherin and ZEB1 protein were significantly decreased in 7404 cells after treatment with 2μmol/L Curcumin and 5μmol/L ABT-737(P<0.05).What’s more,as compared with the 7404 cells treated with the 2μmol/L Curcumin or 5μmol/L ABT-737 group,the expression levels of p-beta-catenin and p-JNK protein were significantly increased while the expression levels of Snail and Twist protein were decreased significantly in the 7404 cells after treatment with 2μmol/L Curcumin+5μmol/L ABT-737(P<0.05).The numbers of metastatic tumor in liver were decreased significantly in the animal models of liver cancer(Alb-Cre;P53f/f;Ras)after Curcumin and ABT-737 treatment than those in the animal models without any treatment(as a control)(P<0.05).Conclusion The combination treatment of curcumin and ABT-737 may inhibit the epithelial-mesenchymal transition of human liver cancer cell,and JNK-beta-catenin signaling pathway may be involved in EMT.