Cariporide对胆囊癌细胞株GBC—SD转移及基质金属蛋白酶表达的影响

Effects of Cariporide on metastasis and MMPs expression of GBC-SD cells

目的研究钠氢交换蛋白1（NHl）特异性抑制剂Cariporide对人胆囊癌细胞株GBC—SD体外转移能力的影响，初步探讨其作用机制。方法采用双羧乙基碳氧荧光素四乙酰氧甲酯（BCECF-AM）荧光染料和酚红检测细胞内外氢离子浓度（pH）值。采用细胞-基质黏附实验、细胞划痕实验和细胞迁移侵袭（Trans well）实验分别检测Cariporide对GBC—SD细胞的黏附、迁移和侵袭能力的影响；实时PCR、蛋白印迹法分别检测Cariporide对GBC—SD细胞基质金属蛋白酶（MMPs）mRNA及蛋白表达的影响。结果Cariporide处理后GBC—SD细胞的细胞内pH和NHEl活性明显下降，胞外pH明显上升。细胞-基质黏附实验结果显示，Cariporide处理后的GBC-SD细胞体外黏附率降低至对照组的80％和6l％，差异有统计学意义（P〈0．05）。细胞划痕实验和Transwell侵袭实验结果显示细胞损伤愈合的速度和穿过滤膜的细胞数量均少于对照组。实时PCR及蛋白印迹结果表明Cariporide可显著降低MMP-2和MMP-9mRNA和蛋白水平的表达量（P均〈0．05）。结论Cariporide可有效抑制人胆囊癌细胞株GBC—SD的体外转移能力，其作用机制可能与细胞内外pH值改变降低MMPs的表达有关。

Objective To study the effects of sodium hydrogen exchange protein 1 （Na~/H ~ ex- change 1 ; NHE1 ） -specific inhibitor Cariporide on the metastasis of GBC-SD cells in vitro and to preliminarily explore the potential anti-metastatic mechanism of Cariporide. Methods The regulatory effect of Cariporide on intracellular pH and extracellular pH of GBC-SD cells were analyzed by BCECF and phenol red. The effect of Cariporide on the adhesion, migration, and invasion of GBC-SD cells were determined using cell-matrix adhesion assay, wound healing assay, and cell invasion assay. Real-time PCR （RT-PCR） and Western blot assay were also used to detect the effects of Cariporide on the levels of MMP-2 and MMP-9 mRNA and on the protein expression in the GBC-SD cells, respectively. Results Cariporide can significantly decrease the intracellular pH and the NHE1 activity, but increase the extracellular pH in a dose-dependent manner. The cell-matrix adhesion assay showed that the adhesive ratio of the GBC-SD cells treated with Cariporide was reduced to 80% and 61% compared with the controls. The wound healing and cell invasion assays showed that after the Cariporide treatment, the healing speed of GBC-SD cells injury and the amount of the cells passing through the filter membrane were less in the treatment group than that in the control group. RT-PCR and Western blot results showed that Cariporide produced a distinguished inhibition of MMP-2 and MMP-9 expression in GBC-SD cells, both on the mRNA and on the protein levels. Conclusions Cariporide can inhibit the in vitro metastasis of human GBC-SD cells effectively. The mechanism may be related to the downregulated expression of MMPs.