.. SU5416 inhibited selleck screening library CRC cell migration To test the effect of inhibition of VEGFR-1 activation on cell migration, motility of the Bev-A cells was assessed by the scratch assay in the presence or absence of SU5416. As above, both HCT116/Bev-A and SW480/Bev-A cells migrated inwardly and covered a greater area of the defect than did control cells (HCT116/Bev-A 91% vs HCT116/control 60% SW480/Bev-A 87% vs SW480/control 50%). Treatment with SU5416 blocked cell migration (Figures 4A and D) (HCT116/Bev-A+SU5416 58% vs HCT116/Bev 91% SW480/Bev-A+SU5416 43% vs SW480/Bev-A 87%). To further confirm the result from the scratch assay. HCT116/Bev-A and SW480/Bev-A cells were pretreated with or without SU5416 (5��) for 4h, cells were then trypsinised and seeded in a modified Boyden chamber with or without SU5416 for 48h.
As above, both HCT116/Bev-A and SW480/Bev-A cell lines showed a two- to three-fold increase in migration compared with the respective control cells (P<0.0002 vs control; P<0.001 vs control, respectively). The Bev-A cells treated with SU5416 showed decreased migration compared with solvent alone (Figures 4B and E, P<0.000004 vs HCT116/Bev-A+DMSO; P<0.001 vs SW480/Bev-A+DMSO). Both HCT116/Bev-A and SW480/Bev-A cell lines exhibited growth rates similar to those of their respective controls, as determined by MTT assay (data not shown). Chronic exposure to Bev led to an increase in the level of phosphorylated VEGFR-1 in the both of HCT116/Bev-A and SW480/Bev-A cells. Treatment of HCT116/Bev-A and SW480/Bev-A cells with SU5416 led to decreased expression of phosphorylated VEGFR-1 as determined by western blotting (Figures 4C and F).
Figure 4 Effect of chronic bevacizumab exposure on CRC cell migration under SU5416 treatment. SU5416 treatment led to decreased cell migration in HCT116/Bev-A cells determined by in vitro wound healing/migration assay (A) and the modified Boyden chamber assay … Bev-A cells increased metastatic potential in vivo Because migration and invasion are theoretically associated with the metastatic phenotype, luciferase labelled HCT116/control and HCT116/Bev-A cells were injected into the tail vein of athymic nude mice. At the end of 6 weeks, all mice were killed. The mice injected with HCT116/Bev-A cells had a higher incidence of metastasis than that in mice injected with control cells (10 out of 12 Bev vs 4 out of 11 control, P<0.
05). Mice injected with HCT116/Bev-A cells showed significantly higher luciferase activity (~10-fold higher) compared with the mice injected with control cells (P<0.005, Figure 5A, upper panel). Imaging of the whole animal and harvested organs revealed that luciferase activity was higher in almost every organ harvested in mice Cilengitide injected with the HCT116/Bev-A cells (Figure 5A, lower panel). All mice underwent a detailed necropsy and all masses were removed to calculate the average number of metastases.