NK4 suppresses cholangiocarcinoma angiogenesis and invasion through targeting HIF-1α pathway

Cholangiocarcinoma (CCA) is a deadly biliary tract and hepatic malignancy with complicated genetics. This study aims to investigate the effect of hepatocyte growth factor (HGF) antagonist NK4 in inhibiting CCA cell hypoxic growth and invasion in vitro and in vivo. Human CCA cells stably transfected with NK4 (Hu-NK4) or control plasmid (Hu-Em) were treated with HGF under normoxia or hypoxia. The growth, apoptosis, and invasion of CCA cells were analyzed by MTT, flow cytometry, and transwell invasion assays, respectively. Tumor growth of human CCA in xenograft mouse model was measured by tumor size and weight. Tumor angiogenesis was assayed by cluster of differentiation 31 (CD31) positive microvessels. The expression of hypoxia-inducible factor 1-alpha (HIF-1α), vascular endothelial growth factor (vegf), and CD31 was analyzed by quantitative real-time PCR and western blot. The expression of NK4 significantly inhibited HGF-induced CCA cell growth and invasion while induced apoptosis, and hypoxia enhanced these changes. NK4 strongly inhibited tumor growth and angiogenesis of human CCA in vivo through inhibiting HIF-1α and vegf expression. These results implicated that NK4 might be an effective therapeutic reagent for CCA.