title: 2-Methoxyestradiol, an endogenous estrogen metabolite, induces apoptosis in endothelial cells and inhibits angiogenesis: possible role for stress-activated protein kinase signaling pathway and Fas expression.
abstract: 2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol-17beta and the oral contraceptive agent 17-ethylestradiol. 2-ME was recently reported to inhibit endothelial cell proliferation. The current study was undertaken to explore the mechanism of 2-ME effects on endothelial cells, especially whether 2-ME induces apoptosis, a prime mechanism in tissue remodeling and angiogenesis. Cultured bovine pulmonary artery 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 microM, n = 8). Nucleosomal DNA fragmentation in BPAEC treated with 2-ME was identified by agarose gel electrophoresis (DNA ladder) as well as in situ nick end labeling. Under the same experimental conditions, estradiol-17beta and two of its other metabolites, estriol and 2-methoxyestriol (< or =10 microM), did not have an apoptotic effect on BPAEC. 2-ME activated stress-activated protein kinase (SAPK)/c-Jun amino-terminal protein kinase in BPAEC in a concentration-dependent manner. The activity of SAPK was increased by 170 +/- 27% and 314 +/- 22% over the basal level in the presence of 0.4 and 2 microM 2-ME (n = 3-6), respectively. The activation of SAPK was detected at 10 min, peaked at 20 min, and returned to basal levels 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 apoptosis. Immunohistochemical analysis of BPAEC indicated that 2-ME up-regulated expression of both Fas and Bcl-2. In addition, 2-ME inhibited BPAEC migration (IC50 = 0.71 +/- 0.11 microM, n = 4) and basic fibroblast growth factor-induced angiogenesis in the chick chorioallantoic membrane model. Taken together, these results suggest that promotion of endothelial cell apoptosis, thereby inhibiting endothelial cell proliferation and migration, may be a major mechanism by which 2-ME inhibits angiogenesis.
authors.affiliation: Department of Cardiovascular Phamacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA. tian-li_yue@sbphrd.com
authors.forename: T L
authors.initials: TL
authors.lastname: Yue
authors.forename: X
authors.initials: X
authors.lastname: Wang
authors.forename: C S
authors.initials: CS
authors.lastname: Louden
authors.forename: S
authors.initials: S
authors.lastname: Gupta
authors.forename: K
authors.initials: K
authors.lastname: Pillarisetti
authors.forename: J L
authors.initials: JL
authors.lastname: Gu
authors.forename: T K
authors.initials: TK
authors.lastname: Hart
authors.forename: P G
authors.initials: PG
authors.lastname: Lysko
authors.forename: G Z
authors.initials: GZ
authors.lastname: Feuerstein
mesh_terms: D000077584:2-Methoxyestradiol; D000482:Allantois / Q000098:blood supply; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D002417:Cattle; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D002642:Chick Embryo; D002823:Chorion / Q000098:blood supply; D005576:Colforsin / Q000494:pharmacology; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004347:Drug Interactions; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000037:antagonists & inhibitors / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D018919:Neovascularization, Physiologic / Q000187:drug effects* / Q000502:physiology; D009707:Nucleosomes / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013002:Somatomedins / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects; D019096:Vitronectin / Q000494:pharmacology; D019014:fas Receptor / Q000096:biosynthesis*
chemical_list: D009707:Nucleosomes; D019253:Proto-Oncogene Proteins c-bcl-2; D013002:Somatomedins; D019096:Vitronectin; D019014:fas Receptor; D016222:Fibroblast Growth Factor 2; D005576:Colforsin; D004958:Estradiol; D000077584:2-Methoxyestradiol; D004247:DNA; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases