XAF1 inhibits angiogenesis of mouse endothelial cells

Abstract

BACKGROUND/OBJECTIVES XIAP associated factor 1 (XAF1) is a tumor suppressor gene and is expressed at low or undetectable levels in many cancers. In cancer cells, it is also densely hyper-methylated. Up-regulation of XAF1 leads to a sensitization of cancer cells to anticancer drug-induced apoptosis and growth inhibition. Angiogenesis is regarded as an important mechanism of tumor growth and development, and thus become a promising focus for cancer therapy. In this study, we tried to investigate whether XAF1 has any anti-angiogenesis effect. MATERIAL AND METHODS MS1 cells (a mouse pancreatic islet endothelial cell line, ATCC) are cultured in 6-well plates in DMEM supplemented with 10% fetal bovine serum (FBS). When cells were 60-70% confluent, they were transiently transfected with pCDNA3.1-XAF1-sense (XAF1-S) or an adenoviral vector ZD55-ZAF1-S. Controls were transfected with empty vector and ZD55-EGFP, respectively. The migratory ability of transfected cells were detected by wound healing assay, and the new blood vessel forming ability of the cells was evaluated by tube formation assay using the In Vitro Angiogenesis Assay Kit (Chemicon). Western blot analysis was used to determine the expression of angiogenesis associated genes. Cell proliferation was detected by WST-1 assay, and apoptosis was detected by APOPercentageTM Assay. To prepare the conditioned medium (CM) from colon cancer HCT116 cells, cells were cultured in McCoy's 5A medium supplemented with 10% FCS, infected with ZD55-XAF1 or ZD55-EGFP for 48 h, and medium from each treatment condition was collected. This medium was then used to treat the mouse endothelial cells, and the effect of such treatment on the proliferation of mouse endothelial cells was detected by WST-1 assay. RESULTS Forty eight hours after transfection, both XAF1-S and ZD55-XAF1 induced a significant up-regulation of XAF1 at mRNA and protein levels in MEC cells. Cells transfected with XAF1-S and ZD55-XAF1 showed a decreased migration and a suppressed tube formation. Over-expression of XAF1 in MS1 caused a significant decrease in the expression of Tie-1, Ang-1, and c-myc. The expression of VEGF was not significantly affected. Enhanced expression of XAF1 also reduced the proliferation of MS1 and induced apoptosis in MS1 cells. CONCLUSIONS Our preliminary data suggest that XAF1 possess a potential anti-angiogenesis effect, possibly through inhibition of the angiogeneic factor Tie-1 and Ang-1. Suppression of cell growth and induction of apoptosis may also play a role. More studies are needed to unveil the potential anti-angiogenesis role of XAF1 and the underlying molecular mechanisms.