Estrogen and tamoxifen metabolites protect smooth muscle cell membrane phospholipids against peroxidation and inhibit cell growth

Abstract

The goal of this study was to test the hypothesis that antioxidant estrogens, by a mechanism independent of the estrogen receptor, protect phospholipids residing in the plasma membrane of vascular smooth muscle cells from peroxidation and peroxidation-induced cell growth and migration. Peroxidation of membrane phospholipids was assessed by HPLC analysis of phospholipids extracted from rat aortic vascular smooth muscle cells prelabeled with cis-parinaric acid (a fatty acid that is susceptible to peroxidation, which quenches its fluorescent-properties). Incubation of cells for 2 hours with the peroxyl radical donor 2,2'-azobis-2,4- dimethylvaleronitrile (AMVN) caused peroxidation of all measured membrane phospholipids. This effect was attenuated by pretreating cells for 15 minutes with 50 to 5000 ng/mL of 2-hydroxyestradiol (strong antioxidant but weak estrogen-receptor ligand) or 4-hydroxytamoxifen (strong antioxidant and potent estrogen-receptor ligand), but not by estrone or droloxifene (both weak antioxidants but potent estrogen-receptor ligands). Moreover, pretreatment of cells for 20 hours with physiological concentrations (0.3 ng/mL) of 2-hydroxyestradiol or pharmacologically relevant concentrations of 4-hydroxytamoxifen (40 ng/mL) also decreased AMVN-induced phospholipid peroxidation. Both 2-hydroxyestradiol and 4-hydroxytamoxifen were as effective as 2,2,5,7,8-pentamethyl-6-hydrochromane (an antioxidant homolog of vitamin E) in attenuating AMVN-induced peroxidation of membrane phospholipids. Also, physiological concentrations of 2-hydroxyestradiol, but not estrone, and pharmacologically relevant concentrations of 4- hydroxytamoxifen attenuated AMVM-induced DNA synthesis, cell proliferation, and cell migration. These studies demonstrate in vascular smooth muscle cells that antioxidant estrogens via a non-estrogen receptor-dependent mechanism attenuate peroxidation of membrane phospholipids and peroxidation-induced cell growth and migration.