N-methyl-N-nitro-N-nitrosoguanidine-mediated ING4 downregulation contributed to the angiogenesis of transformed human gastric epithelial cells

Angiogenesis is associated with the progression and mortality of gastric cancer. Epidemiological evidences indicate that long-term N-nitroso compounds (NOCs) exposure predominantly contributes to the mortality of gastric cancer. Therefore, further reduced mortality of gastric cancer demands to explore the exact mechanisms of NOCs induced angiogenesis. As a tumor suppressor gene, inhibitor of growth protein 4 (ING4) plays an important role in pathological angiogenesis. In this study, we will investigate ING4 expression level in human gastric epithelial cells after the long-term low dose exposure of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and the pathological impact of MNNG-reduced ING4 on angiogenesis of transformed cells. The soft agar colony formation assay, Western blotting, immunofluorescence and wound healing assay were used to evaluate the characteristics of transformed cells. HUVEC growth and tube formation assays were performed to test the angiogenic abilities. EMSA, luciferase reporter gene assay, real-time PCR and Western blotting were used to explore the exact mechanism. By establishing transformed human gastric epithelial cells via chronic low dose treatment, a gradually ING4 downregulation was observed in the later-stage of MNNG-induced cell transformation. Moreover, we demonstrated that MNNG exposure-reduced ING4 expression significantly resulted into aggravating angiogenesis through increasing the phosphorylation level of NF-κB p65 and subsequently DAN binding activity and regulating the expressions of NF-κB p65 downstream pro-angiogenic genes, MMP-2 and MMP-9. Our findings provided a significant mechanistic insight into angiogenesis of MNNG-transformed human gastric epithelial cell and supported the concept that ING4 may be a relevant therapeutic target for gastric cancer.