ascorbic-acid and 4-hydroxyestradiol

Estradiol 17β (E2β) and ascorbic acid (AA) have been implicated in cancer progression. However, little is known about the actions of biologically active metabolites of E2β, 2-hydroxyestradiol (2OHE2), 4-hydroxyestradiol (4OHE2), 2-methoxyestradiol (2ME2), and 4-methoxyestradiol (4ME2) synthesized sequentially by cytochrome P450, family 1, subfamily A (CYP1A1) and B (CYP1B1), polypeptide 1, and catechol-O-methyltransferase (COMT) on ovarian cancer. Herein, we examined the expression of CYP1A1, CYP1B1, COMT, and estrogen receptor α (ERα) and β (ERβ) in human ovarian surface epithelial (IOSE-385) and cancer cell lines (OVCAR-3, SKOV-3, and OVCA-432). We also investigated the roles of E2β, 2OHE2, 4OHE2, 2ME2, and 4ME2 in cell proliferation, and their interactive effects with AA on ovarian cells. We found the expression of CYP1A1, CYP1B1, COMT, ERα, and ERβ in most cell lines tested. Treating cells with physiological concentrations of E2β and its metabolites promoted (13%-42% of the control) IOSE-385 and OVCAR-3 proliferation. The ER blockade inhibited IOSE-385 (∼76%) and OVCAR-3 (∼87%) proliferative response to E2β but not to its metabolites. The ERα blockade inhibited (∼85%) E2β-stimulated OVCAR-3 proliferation, whereas ERβ blockade attenuated (∼83%) E2β-stimulated IOSE-385 proliferation. The AA at ≥250 μmol/L completely inhibited serum-stimulated cell proliferation in all cell lines tested; however, such inhibition in IOSE-385, OVCAR-3, and OVCA-432 was partially (∼10%-20%) countered by E2β and its metabolites. Thus, our findings indicate that E2β and its metabolites promote cell proliferation and antagonize the AA-suppressed cell proliferation in a subset of ovarian cancer cells, suggesting that blocking the actions of E2β and its metabolites may enhance AA's antiovarian cancer activity.

Topics: 2-Methoxyestradiol; Aryl Hydrocarbon Hydroxylases; Ascorbic Acid; Catechol O-Methyltransferase; Cell Line, Tumor; Cell Proliferation; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Dose-Response Relationship, Drug; Drug Interactions; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens, Catechol; Female; Humans; Ovarian Neoplasms; Time Factors

The mechanisms underlying the pathogenesis of estrogen-induced breast carcinogenesis remain unclear. The present study investigated the roles of estrogen metabolism and oxidative stress in estrogen-mediated mammary carcinogenesis in vivo. Female August Copenhagen Irish (ACI) rats were treated with 17beta-estradiol (E(2)), the antioxidant vitamin C, the estrogen metabolic inhibitor alpha-naphthoflavone (ANF), or cotreated with E(2) + vitamin C or E(2) + ANF for up to 8 months. E(2) (3 mg) was administered as an subcutaneous implant, ANF was given via diet (0.2%) and vitamin C (1%) was added to drinking water. At necropsy, breast tumor incidence in the E(2), E(2) + vitamin C and E(2) + ANF groups was 82, 29 and 0%, respectively. Vitamin C and ANF attenuated E(2)-induced alterations in oxidative stress markers in breast tissue, including 8-iso-prostane F(2alpha) formation and changes in the activities of antioxidant enzymes superoxide dismutase and glutathione peroxidase. Quantification of 2-hydroxyestradiol (2-OHE(2)) and 4-hydroxyestradiol (4-OHE(2)) formation in breast tissue confirmed that ANF inhibited 4-hydroxylation of E(2) and decreased formation of the highly carcinogenic 4-OHE(2). These results demonstrate that antioxidant vitamin C reduces the incidence of estrogen-induced mammary tumors, increases tumor latency and decreases oxidative stress in vivo. Further, our data indicate that ANF completely abrogates breast cancer development in ACI rats. The present study is the first to demonstrate the inhibition of breast carcinogenesis by antioxidant vitamin C or the estrogen metabolic inhibitor ANF in an animal model of estrogen-induced mammary carcinogenesis. Taken together, these results suggest that E(2) metabolism and oxidant stress are critically involved in estrogen-induced breast carcinogenesis.

Topics: Animals; Antioxidants; Ascorbic Acid; Benzoflavones; Cell Transformation, Neoplastic; Dinoprost; Estradiol; Estrogens, Catechol; Female; Mammary Neoplasms, Experimental; Neoplasms, Hormone-Dependent; Oxidative Stress; Rats; Rats, Inbred ACI

Catechol estrogens are considered critical intermediates in estrogen-induced carcinogenesis. We demonstrated previously that 17beta-estradiol (E(2)), estrone (E(1)) and four of their catechol estrogens, 2- and 4-hydroxyestradiols (2- and 4-OHE(2)), and 2- and 4-hydroxyestrones (2- and 4-OHE(1)) induce morphological transformation in Syrian hamster embryo (SHE) fibroblasts, and the transforming abilities vary as follows: 4-OHE(1) > 2-OHE(1) > 4-OHE(2) > 2-OHE(2) vertical line E(2), E(1). To examine the involvement of catechol estrogens in the initiation of hormonal carcinogenesis, we studied the ability of E(2), E(1) and their catechol estrogens to induce DNA adducts in SHE cells by using a (32)P-post-labeling assay. DNA adducts were detected in cells treated with each of all the catechol estrogens at concentrations of 10 microg/ml for 1 h and more. 2- or 4-OHE(2) formed a single DNA adduct, which was chromatographically distinct from each other. In contrast, 2- or 4-OHE(1) produced one major and one minor adduct, and the two adducts formed by each catechol estrogen exhibited identical mobilities on the chromatograms. Neither E(2) nor E(1) at concentrations up to 30 microg/ml induced DNA adducts. The abilities of the estrogens to induce DNA adducts were ranked as follows: 4-OHE(1) > 2-OHE(1) > 4-OHE(2) > 2-OHE(2) > > E(2), E(1), which corresponds well to the transforming and carcinogenic abilities of the estrogens. In addition, the level of DNA adducts induced by the catechol estrogens was markedly decreased by co-treatment of cells with the antioxidant L-ascorbic acid. The results indicate the possible involvement of oxidative metabolites of catechol estrogens of E(2) and E(1) in the initiation of endogenous estrogen-induced carcinogenesis.

Topics: Animals; Antioxidants; Ascorbic Acid; Cell Survival; Cell Transformation, Neoplastic; Cricetinae; DNA Adducts; Estradiol; Estrogens, Catechol; Fibroblasts; Hydroxyestrones; Mesocricetus

Formation of the catechol estrogens 2- and 4-hydroxyestradiol (2-OHE2 and 4-OHE2) from estradiol by pig blastocysts was studied using a direct product isolation assay for estrogen-2/4-hydroxylase (E-2/4-H). Blastocyst E-2/4-H activity was characterized biochemically using homogenates of blastocysts obtained on day 12 of pregnancy. This information was used to establish appropriate incubation conditions for the assay of E-2/4-H activity in blastocysts during the preimplantation period. Catechol estrogen formation was linear with time for up to 30 min and with blastocyst protein concentrations of up to 100 micrograms in a reaction volume of 150 microliters. The E-2/4-H activity of pig blastocysts was maximal at pH 7.9 and was not affected by the nonionic detergent Tween-80. The E-2/4-H activity was dependent on nicotinamide cofactor, with NADPH preferred over NADH for 2-OHE2 formation. The predominant catechol estrogen formed was 2-OHE2: maximum velocities (Vmax) for the formation of 2- and 4-OHE2 were 1570 and 174 pmol/mg protein . 30 min, respectively. The apparent Km values with respect to estradiol for 2- and 4-OHE2 were similar, 4.39 and 4.27 microM, respectively. Blastocyst E-2/4-H activity was detectable in one of two samples of blastocysts from day 10 of pregnancy (4.4 pmol 2-OHE2/mg protein . 30 min), increased to a maximum on days 12 and 13 (628 +/- 153 and 516 +/- 227 pmol 2-OHE2/mg protein . 30 min, respectively), and declined by day 14 (63.2 +/- 32.9 pmol 2-OHE2/mg protein . 30 min). The activity of E-2/4-H was positively correlated with aromatase activity assayed in the same tissue samples from days 10-14 of pregnancy. The surge in E-2/4-H activity coincides with several of the critical events that occur near the time of implantation. Our findings are consistent with the hypothesis that catechol estrogens mediate some of the actions of estrogens in early pregnancy in the pig.

Topics: Animals; Aromatase; Ascorbic Acid; Blastocyst; Cytochrome P-450 CYP1A1; Embryonic Development; Estradiol; Estrogens, Catechol; Female; Hydrogen-Ion Concentration; Kinetics; NAD; Polysorbates; Pregnancy; Proteins; Steroid Hydroxylases; Swine