Inhibition of Metastatic Potential in Breast Carcinoma In Vivo and In Vitro through Targeting VEGFRs and FGFRs
Figure 3
Significant antimetastatic and antilymphangiogenic effect of 194-A in the 4T1 orthotopic graft model. (a) 4T1-Luc+ (5 × 105 cells/mouse) were orthotopically implanted into the fat pad of male BALB/c mice. Mice were p.o. administered with 50 mg/kg/day of 194-A or vehicle control for 30 days. At the end of the study, lung metastasis was measured by in vivo bioluminescent signals and ex vivo visualization. Arrow indicates metastatic tumor nodules. (b) Histological analyses of lung, liver, and lymph node metastasis from vehicle or 194-A-treated group. T, tumor part; NT, nontumor part. (c) The incidence of tumor metastasis was evaluated by histological analyses of the lung, liver, and lymph node. Mean values of two replicates; bars, SE. (d) Cryostat sections of 4T1 tumors, from mice treated with 194-A or vehicle control, were stained against LYVE-1 for the quantification of the mean lymphatic vessel length (LVL). Vessel length per 200x field was assessed from 3 to 4 fields per tumor section. Columns, mean (); bars, SE. ** as compared to the vehicle control group. Scale bar, 20 μm. (e) and (f) Relative proliferation or migration of LECs grown in serum-free media supplemented with 500 ng/mL of VEGF-C as indicated. Proliferation (e) or migration (f) 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. (g) 194-A inhibited VEGF-C-induced activation of VEGFR-3 and its common downstream signaling molecules. Serum-starved LECs were pretreated with 194-A for 30 min and then stimulated with 100 ng/mL VEGF-C for 10 min. Lysates were subjected to immunoprecipitation (IP) with VEGFR-3-specific antibody and resolved in SDS-PAGE, or resolved in SDS-PAGE directly and probed with specific antibodies against p-Tyr, VEGFR-3, p-ERK1/2, ERK1/2 p-Akt, and Akt.
