Figure 2.

The antiangiogenesis efficacy of 194-A in the 4T1 orthotopic graft model. (a) Tumor vascularity was monitored after 10 days of 194-A (50 mg/kg) p.o. treatment, by using non-contrast-enhanced flow-sensitive ultrasound (Vevo 770 micro-ultrasound system). (b) Immunohistochemical staining of 4T1 tumor sections with CD31 antibody, counterstained with hematoxylin. Vessel density per 200x field was assessed from 3 to 4 fields per tumor section. Columns, mean (n = 6); bars, SE. **P < 0.01 as compared to the vehicle control group. Scale bar, 20 μm. (c) Relative proliferation of HUVECs grown in serum-free media supplemented with 100 ng/mL of VEGF-A as indicated. Proliferation was reduced in a dose-dependent manner in response to 194-A treatment. Mean values of three replicates, normalized to the untreated controls; bars, SE. (d) Treatment with increasing concentrations of 194-A reduced VEGF-A-induced migration in HUVECs. The number of migrating cells was normalized to DMSO control and values are displayed as mean values from three independent experiments. (e) 194-A inhibited VEGF-A-induced activation of VEGFR-2 and its common downstream signaling molecules. Serum-starved HUVECs were pretreated with 194-A for 30 min and then stimulated with 100 ng/mL VEGF-A for 10 min. Lysates were resolved in SDS-PAGE and probed with specific antibodies against p-Tyr1054 VEGFR-2, VEGFR-2, p-ERK1/2, ERK1/2 p-Akt, and Akt.