NF‐κB promotes iNOS and VEGF expression in salivary gland adenoid cystic carcinoma cells and enhances endothelial cell motility in vitro

Abstract

Tumour cell-derived angiogenic-related factors and endothelial cell mobility are essential for neovascularization and haematogenous metastasis of adenoid cystic carcinoma (ACC) of the human salivary glands. Our previous study demonstrated that vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and nuclear factor kappaB (NF-kappaB) staining correlated with ACC microvessel density. However, there still remains a lack of direct evidence to clarify the function of liposaccharide-induced NF-kappaB activity in ACC angiogenesis. This study aimed to demonstrate the function of liposaccharide-induced NF-kappaB signalling pathway (which would be relevant to angiogenesis) of ACC cell lines. A PCMV-IkappaBalphaM vector transfection assay was performed to inhibit NF-kappaB activity. Constitutive and liposaccharide-induced NF-kappaB activity, along with VEGF and iNOS expression, was detected by electrophoretic mobility shift assays, reverse transcription-polymerase chain reaction and Western blot assay. Tumour cell-derived NO(2) (-) was evaluated by a nitrite determination assay, and endothelial cell mobility was investigated by endothelial cell proliferation, migration and tube formation assays. We demonstrated that regulation of VEGF and iNOS expression utilized the NF-kappaB signalling pathway in ACC cell lines. In the constitutive and liposaccharide-induced condition, NF-kappaB activity was blocked by phosphorylation-defective IkappaBalpha. Accordingly, mRNA and protein expression of VEGF and iNOS were significantly decreased, with a great drop in endogenous NO(2) (-). Furthermore, supernatant from ACC-2/IkBaM and ACC-M/IkBaM cells resulted in much lower endothelial cell mobility than that from ACC-2 and ACC-M cells. Our results suggest that NF-kappaB promotes iNOS and VEGF expression in salivary gland ACC cells and enhances endothelial cell motility in vitro.