Published online before print June 22, 2006, doi: 10.1161/01.RES.0000233318.85181.2e
Submitted on January 26, 2006
Revised on June 7, 2006
Accepted on June 12, 2006
2-Methoxyestradiol, an Estradiol Metabolite, Inhibits Neointima Formation and Smooth Muscle Cell Growth via Double Blockade of the Cell Cycle
Federica Barchiesi ;
From the Department of Obstetrics and Gynecology (F.B., R.K.D.), Clinic for Endocrinology; Center for Integrative Human Physiology (J.F., R.K.D.); and Institute of Clinical Pathology (B.O.), University Hospital Zurich, Switzerland; Center for Clinical Pharmacology (E.K.J., D.G.G., R.K.D.) and Departments of Medicine (E.K.J., D.G.G., R.K.D.) and Pharmacology (E.K.J.), University of Pittsburgh School of Medicine, Pa; and Preclinical Pharma Research 68/209 (J.F., R.K.D.), F. Hoffmann La-Roche, Basel, Switzerland.
* To whom correspondence should be addressed. E-mail: Raghvendra.Dubey{at}usz.ch.
2-Methoxyestradiol (2-ME), an endogenous metabolite of estradiol with no affinity for estrogen receptors, is a potent anticarcinogenic agent (in phase II clinical trials) and mediates the inhibitory effects of estradiol on smooth muscle cell (SMC) growth. Here we studied the intracellular mechanisms by which 2-ME inhibits SMC growth and whether 2-ME prevents injury-induced neointima formation. 2-ME concentrations that inhibit proliferation of cycling human aortic SMCs by 
50% blocked cell-cycle progression in G0/G1 and in G2/M phase, as determined by flow cytometry. Consistent with the cell-cycle effects, at a molecular level (Western blots), 2-ME inhibited cyclin D1 and cyclin B1 expression; cyclin-dependent kinase (cdk)-1 and cdk-2 activity; and retinoblastoma protein (pRb), extracellular signal-regulated kinase (ERK) 1/2, and Akt phosphorylation. 2-ME also upregulated the Cdk inhibitor p27 and interfered with tubulin polymerization. Moreover, 2-ME augmented COX-2 expression, suggesting that it may also inhibit SMC growth via prostaglandin formation. In rats, treatment with 2-ME abrogated injury-induced neointima formation; decreased proliferating SMCs; downregulated expression of proliferating-cell nuclear antigen (PCNA), c-myc, cyclin D1, cyclin B1, phosphorylated Akt, phosphorylated ERK1/2, p21, and pRb; inhibited cdk-1 and cdk-4 activity; and upregulated expression of cyclooxygenase (COX)-2 and p27. Caspase-3 cleavage assay and fluorescence-activated cell-sorting (FACS) analysis showed no evidence of apoptosis in 2-ME-treated SMCs, and TUNEL staining in carotid segments showed no evidence of 2-ME-induced apoptosis in vivo. The antimitotic effects of 2-ME on SMCs are mediated by the inhibition of key cell-cycle regulatory proteins and effects on tubulin polymerization and COX-2 upregulation. These effects of 2-ME most likely contribute to the antivasoocclusive actions of this endogenous compound.
Key words: restenosis • stents • drugs • remodeling • signal transduction
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