Vascular Endothelial Growth Factor Mediates Intracrine Survival in Human Breast Carcinoma Cells through Internally Expressed VEGFR1/FLT1
Figure 1
Expression of VEGF Receptors in Breast Cancer Cells and Apoptosis of MDA-MB-231 Cells by Down-Regulation of VEGF
(A) VEGF receptor expression was examined by using immunoprecipitation (IP) and Western blot (WB) analyses in several breast cancer cell lines.
(B) VEGFR2 expression in MDA-MB-231 cells was analyzed by confocal microscopy. Cells were stained with anti-VEGFR2 antibody or IgG (inset). The asterisk indicates cells transduced with adenovirus encoding soluble human VEGFR2, which served as a positive control.
(C) MDA-MB-231 cells were transfected with antisense VEGF vector and selected in the presence of Zeocin (1 mg/ml). VEGF expression in the MDA-MB-231 transfectants (pZeoSV, AS-C1, and AS-C2) was assessed by Western blotting (WB) using a polyclonal anti-VEGF antibody. Total protein extracts were analyzed by Western blotting using anti-Csk antibody as an internal control.
(D and E) MDA-MB-231 cells were stably transfected with antisense VEGF vector and the survival of these cells was measured by using cell cycle analysis (D) and TUNEL assay (E).
(F) MDA-MB-231 cells were transiently transfected with siCTL or siVEGF oligonucleotides, and VEGF was quantitated in the supernatants (left graph) using an ELISA kit as directed by the manufacturer. The apoptosis of these cells was measured by using cell cycle analysis. The data are representative of three individual studies (right graphs).