We found that WT FGF1 markedly increased the number of blood vessels, whereas R50E was defective in this function (Fig. 5). Excess R50E reduced the number of blood vessels induced by WT FGF1. These findings suggest that R50E selleck chemical Enzalutamide suppresses angiogenesis induced by WT FGF1 in vivo. Figure 5 R50E suppresses angiogenesis in Matrigel plug assays in rat. R50E Suppresses Angiogenesis in Chick Embryo Chorioallantoic Membrane (CAM) Model CAM is another widely utilized in vivo system to study angiogenesis and anti-angiogenesis and it is easier to quantify angiogenesis in this assay than other assays. We placed saline- or FGF- impregnated filter disks on blood vessels in avascular sections of CAM (day 11) for 48 h to induce angiogenesis. The disks and underlying CAM tissue (day 13) were then harvested.

We scored angiogenesis by counting vessel branches present in the CAM tissue below the filter from digital images. We first determined optimum dose of wt FGF1 for angiogenesis (Fig. 6a, 6b). Five ng/ml of wt FGF1 was optimum. R50E (5 and 50 ng/ml) did not induce angiogenesis. We tested if excess R50E (50 ng/ml) suppresses angiogenesis induced by WT FGF1 (5 ng/ml). Notably, excess R50E suppressed angiogenesis induced by WT FGF1 (Fig. 6c). This suggests that R50E shows an anti-angiogenic action in this model as well. Since FGF1 binds to all known FGFRs (FGFR1-4), R50E is expected to suppress FGFR signaling induced by other members of the FGF family. We tested if R50E suppresses angiogenesis induced by FGF2. We found that this is the case: excess R50E suppressed angiogenesis induced by WT FGF2 (Fig.

6c). The data suggest that R50E suppresses FGF1- and FGF2-induced angiogenesis in the CAM model. Figure 6 R50E suppresses FGF1- and FGF2-induced angiogenesis (branching formation) in CAM models. Taken together, R50E was defective in inducing angiogenesis, and effectively suppressed angiogenesis in different in vitro and in vivo angiogenesis models. It is likely that R50E may indirectly suppress tumorigenesis in vivo through suppressing angiogenesis. Discussion R50E is an Anti-angiogenic Agent In the present study, we establish that R50E suppressed tumor growth in vivo while WT FGF1 enhanced it using cancer cells that stably express WT FGF1 or R50E. Since R50E showed little or no effect on proliferation of cancer cells in vitro, we hypothesized that R50E indirectly suppressed tumorigenesis through suppressing angiogenesis.

Excess R50E suppressed migration and tube formation of HUVEC, and suppressed angiogenesis in aorta ring assays and matrigel plug assays, suppressed angiogenesis in chick embryo chorioallantoic membrane (CAM) assays, which is induced by WT FGF1. Taken together, our results suggest that R50E suppresses angiogenesis induced by FGF1 and thereby may indirectly suppress tumorigenesis, in addition Cilengitide to its possible direct effect on tumor cell proliferation in vivo.