Figure 4
TAM- and CXB- inhibit overexpressed VEGFR2 induced angiogenesis in MCF7 cells (left) and MDA-MB-231 cells (right). (A) Cells were treated with TAM, CXB, or both and incubated in serum-free conditioned medium for 24 h. VEGF levels were determined by ELISA. (B) Cells (5 × 105/ml) were transfected with VEGFR-luciferase plasmid, incubated for 24 h, and treated with TAM, CXB, or both for 4 h. Whole-cell extracts were then prepared and analyzed for luciferase activity. Absolute values are normalized to untreated cells without VEGFR2. Data are means ± SD of three independent experiments. (C) Western blot analysis for VEGFR2 and phosphorylated VEGFR2. (D) Densitometric analysis of phosphorylated VEGFR2 protein levels. Data are means ± SD of three independent experiments. P < 0.05 (t-test). (E) The level of VEGFR2 mRNA in MCF7 and MDA-MB-231 cells examined by RT-PCR analysis following TAM, CXB and T + C treatment for 24 h. Data are means ± SD of three independent experiments using different cell preparations. *P < 0.05 vs. untreated cells. EGF, epidermal growth factor; T + C, TAM plus CXB; UT, untreated cells.