Raloxifene (Rlx) Has Antiestrogenic Effect on Vascular Endothelial Growth Factor (VEGF) mRNA and Protein While Down-Regulating Progesterone Receptors in a Human Endometrial Cell Line In Vitro

Abstract

To determine if the effects of Rlx on VEGF are mediated through the estrogen receptor (ER) or the progesterone receptor (PR). Angiogenesis is regulated by a balance between pro-angiogenic (VEGF) and antiangiogenic (TSP-1) proteins. In human endometrial cells, mRNA and protein for VEGF are upregulated by E, blocked by specific E-antagonists and only minimally increased by P; whereas for TSP-1 mRNA is stimulated by P with the effect being blocked by anti-P mifepristone (RU 486). Estradiol does not stimulate TSP-1 expression in these cells. Raloxifene is a selective estrogen receptor modulator (SERM) that acts as an E-agonist on bone and lipid metabolism but as an E-antagonist in breast and uterus. The effect of Rlx on endometrial angiogenesis is currently unknown. If Rlx were to behave as an estrogen agonist, one would expect it to increase VEGF and to have no effect on TSP-1. In contrast, our preliminary studies have shown that Rlx has E-antagonistic effects: no VEGF up-regulation and increase of TSP-1 mRNA (progestational activity) in Ishikawa cellsin vitro. prospective basic research, experimental in vitro cell culture Ishikawa cells (human epithelial adenocarcinoma, ER+, PR+) were cultured for 24 hrs with control, E, P, Rlx, Rlx and E plus an antiestrogen ICI 182.780 and Rlx and P plus the antiprogestin RU486. Treatment effect on VEGF, ER and PR proteins was assessed by immunocytochemistry of the Ishikawa cell. VEGF mRNA gene expression was estimated by semiquantitative RT-real time PCR (cyclophilin used as housekeeping gene). The percentage of stained cells was compared by χ2 and the relative band intensity of the PCR products was compared by Wilcoxon Signed Ranks Test. Raloxifene significantly increased VEGF mRNA (P<0.03125), though to a lesser extent than estrogen. The Rlx effect was partially inhibited by both ICI and RU 486. Rlx did not increase VEGF protein staining by immunocytochemistry. Estrogen is a more potent stimulator of VEGF mRNA than progesterone and Rlx. Progesterone did not alter VEGF expression either at the mRNA or protein level. Rlx decreased PR immunostaining (p<0.025), which was not affected by either ICI or RU 486 and Rlx did not alter ER protein staining. Tabled 1 Raloxifene clinically does not stimulate endometrial growth or results in endometrial bleeding in postmenopausal women. Rlx may inhibit endometrial growth through reduced induction of the angiogenic factor VEGF. Our data show that Rlx moderately increased VEGF mRNA that was only partially inhibited by ICI, and antagonized E-dependent target gene induction based on the expression level of PR. Future studies are needed further to delineate Rlx’s mechanism of action on the endometrium