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Background and Objective NK4, a competitive antagonist for hepatocyte growth factor (HGF) and the Met receptor, is a bifunctional molecule that acts as an HGF antagonist and an angiogenesis inhibitor.The objective of this study was to investigate the anti-tumor effects of NK4 on the cholangiocarcinoma (CCA) cell line HuCC-T1.Methods We assessed the effects of NK4 on proliferation, invasion, migration, and cell cycle progression in mock-transfected HuCC-T1 clones, empty-vector-transfected clones of HuCC-T1(Hu-Em), and NK4-transfected clones of HuCC-T 1 (Hu-NK4), with HuCC-T 1 cells serving as the control cells.Correlated with these effects on cellular functions, the mRNA levels of cyclin D1and cyclin A were monitored using RT-PCR and quantitative PCR, and the corresponding protein levels were monitored using Western blotting.In addition, Met phosphorylation and the activity of its important downstream signaling targets protein kinase B (Akt) and glycogen synthase kinase(GSK)-3 β were evaluated by Western blotting.Results Our data indicate that cell proliferation, invasion, and cell cycle progression of the three types of clones were essentially the same, while these processes were stimulated by HGF in HuCC-T1 and Hu-Em, not in Hu-NK4 cells.Moreover, when stimulated with HGF, the increases in mRNA levels of cyclin D1 and cyclin A were accompanied by corresponding increases in protein levels, and the phosphorylation of Met, Akt, and GSK-3 β were upregulated in HuCC-T1and Hu-Em cells, compared to the levels in the Hu-NK4 cells.Conclusions These findings suggest that NK4 gene therapy inhibits HGF/Met-induced growth of human CCA cells by arresting cell cycle progression.It also interferes with Met activation and the downstream phosphatidylinositol-3-kinase (PI3K)/Akt/GSK-3 β signaling pathway.