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. 2015 Mar 12;6(12):10016–10029. doi: 10.18632/oncotarget.3540

Figure 6. WP1066 inhibits the invasion and migration ability of bEnd.3 cells.

Figure 6

(A) Cytotoxicity of WP1066 in bEnd.3 cells by MTT assay. bEnd.3 cells were treated with various concentrations of WP1066 or with DMSO for 72 hours. Values are means ± SD for triplicate experiments. *, P<0.1; #, P<0.001. (B) bEnd.3 cells were treated with vehicle or WP1066 1 μM in CM of MDA-MB-231BR cells. Protein expression of VEGFR-2, pVEGFR-2, and β-actin was analyzed by immunoblotting. (C) bEnd.3 cells were cultured with serum-free medium or CM from MDA-MB-231BR cells treated with vehicle or WP1066 1 μM. Then migration ability was determined using the migration assay at 12 hours. Left panels, representative photos of the results. Right panel, quantification of results. Each column indicates the mean ± SD from results of three different experiments. **, P<0.01; ***, P<0.001. (D) bEnd.3 cells were cultured medium as described in (C). Then invasiveness was determined using the invasion assay at 16 hours. Left panels, representative photos of the results. Right panel, quantification of results. Each column indicates the mean ± SD from results of three different experiments. **, P<0.01; ***, P<0.001. (E) Stat3 regulates breast cancer cell brain metastasis by affecting interaction between tumor cells and endothelial cells and tumor cell invasive. Activated Stat3 in tumor cells upregulates the expression of VEGF that induces the phosphorylation of VEGFR2 in endothelial cells. The phosphorylated VEGFR2 leads to phosphorylation hence activation of Stat3 in endothelial cells. Then the activated Stat3 directly upregulates VEGFR2 expression in endothelial cells, which leads to further activation of VEGFR2 in the cells, increasing their infiltration and angiogenesis. Therefore, Stat3 activation orchestrates the interaction between endothelial and tumor cells. Stat3 also directly regulates MMP-9 expression hence breast cancer cell and endothelial invasion. WP1066 can effectively inhibit tumor angiogenesis and invasion thus brain metastasis mediated by Stat3.