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-T1 (Hu-NK4), with HuCC-T1 cells serving as the control cells. Correlated with these effects on cellular functions, the mRNA levels of cyclin D1 and cyclin A were monitored using reverse transcription (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)-3β 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 cells, but 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-3β was upregulated in HuCC-T1 and 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/Akt/GSK-3β signaling pathway.