2-METHOXYESTRADIOL, AN ENDOGENOUS ESTROGEN METABOLITE, INDUCES APOPTOSIS IN ENDOTHELIAL-CELLS AND INHIBITS ANGIOGENESIS - POSSIBLE ROLE FORSTRESS-ACTIVATED PROTEIN-KINASE SIGNALING PATHWAY AND FAS EXPRESSION

2-methoxyestradiol (2-ME) is an endogenous metabolite of estradiol-17 beta and the oral contraceptive agent 17-ethylestradiol. 2-ME was rece ntly reported to inhibit endothelial cell proliferation. The current s tudy was undertaken to explore the mechanism of 2-ME effects on endoth elial cells, especially whether 2-ME induces apoptosis, a prime mechan ism in tissue remodeling and angiogenesis. Cultured bovine pulmonary a rtery endothelial cells (BPAEC) exposed to 2-ME showed morphological ( including ultrastructural) features characteristic of apoptosis: cell shrinkage, cytoplasmic and nuclear condensation, and cell blebbing. 2- ME-induced apoptosis in BPAEC was a time- and concentration-dependent process (EC50 = 0.45 +/- 0.09 mu M, n = 8). Nucleosomal DNA fragmentat ion in BPAEC treated with 2-ME was identified by agarose gel electroph oresis (DNA ladder) as well as in situ nick end labeling. Under the sa me experimental conditions, estradiol-17 beta and two of its other met abolites, estriol and 2-methoxyestriol (less than or equal to 10 mu M) , did not have an apoptotic effect on BPAEC. 2-ME activated stress-act ivated protein kinase (SAPK)/c-Jun amino-terminal protein kinase in BP AEC in a concentration-dependent manner. The activity of SARK was incr eased by 170 +/- 27% and 314 +/- 22% over the basal level in the prese nce of 0.4 and 2 mu M 2-ME (n = 3-6), respectively. The activation of SAPK was detected at 10 min, peaked at 20 min, and returned to basal l evels at 60 min after exposure to 2-ME. Inhibition of SAPK/c-Jun amino -terminal protein kinase activation by basic fibroblast growth factor, insulin-like growth factor, or forskolin reduced 2-ME-induced apoptos is. Immunohistochemical analysis of BPAEC indicated that 2-ME up-regul ated expression of both Fas and Bcl-2. In addition, 2-ME inhibited BPA EC migration (IC50 = 0.71 +/- 0.11 mu M, n = 4) and basic fibroblast g rowth factor-induced angiogenesis in the chick chorioallantoic membran e model. Taken together, these results suggest that promotion of endot helial cell apoptosis, thereby inhibiting endothelial cell proliferati on and migration, may be a major mechanism by which 2-ME inhibits angi ogenesis.