2-hydroxyestradiol and Breast-Neoplasms

C2- and C4-position 17beta-estradiol metabolites play an important role in breast carcinogenesis. 2-Hydroxyestradiol and 4-hydroxyestradiol are implicated in tumorigenesis via two pathways. These pathways entail increased cell proliferation and the formation of reactive oxygen species that trigger an increase in the likelihood of deoxyribonucleic acid mutations. 2-Methoxyestradiol, a 17beta-estradiol metabolite, however, causes induction of apoptosis in transformed and tumor cells; thus exhibiting an antiproliferative effect on tumor growth. The 4-hydroxyestradiol:2-methoxyestradiol and 2-hydroxyestradiol:2-methoxyestradiol ratios therefore ought to be taken into account as possible indicators of carcinogenesis.

Topics: 2-Methoxyestradiol; Animals; Breast Neoplasms; Cell Proliferation; Estradiol; Estrogens, Catechol; Humans

From these results we may conclude that estradiol 16 alpha-hydroxylation is highly correlated with tumor incidence, and that the reaction is partly regulated by MMTV and the rest by genetic influences. Elevated hydroxylation appears to be an autosomal dominant trait that is highly specific for estradiol. It is also pertinent that the product of the 16 alpha-hydroxyestrone reaction is a potent estrogen that is capable of binding covalently to amino acids and nucleotides, including the estrogen receptor molecule. The results obtained in these studies establish the usefulness of the mouse model for studying the interrelationship between enhanced 16-hydroxylation of estradiol and the incidence of mammary tumors.

Topics: Age Factors; Animals; Breast; Breast Neoplasms; Dihydrotestosterone; Estradiol; Estriol; Female; Haplorhini; Humans; Hydroxylation; Male; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Pan troglodytes; Pregnenolone; Progesterone; Rats; Risk; Species Specificity; Steroid 16-alpha-Hydroxylase; Testosterone

Germline mutations in BRCA1 predispose carriers to a high incidence of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through critical roles in DNA repair, cell-cycle arrest, and transcriptional control. A major question has been why BRCA1 loss or mutation leads to tumors mainly in estrogen-regulated tissues, given that BRCA1 has essential functions in all cell types. Here, we report that estrogen and estrogen metabolites can cause DNA double-strand breaks (DSB) in estrogen receptor-α-negative breast cells and that BRCA1 is required to repair these DSBs to prevent metabolite-induced genomic instability. We found that BRCA1 also regulates estrogen metabolism and metabolite-mediated DNA damage by repressing the transcription of estrogen-metabolizing enzymes, such as CYP1A1, in breast cells. Finally, we used a knock-in human cell model with a heterozygous BRCA1 pathogenic mutation to show how BRCA1 haploinsufficiency affects these processes. Our findings provide pivotal new insights into why BRCA1 mutation drives the formation of tumors in estrogen-regulated tissues, despite the general role of BRCA1 in DNA repair in all cell types.

Topics: BRCA1 Protein; Breast; Breast Neoplasms; Cytochrome P-450 CYP1A1; DNA Breaks, Double-Stranded; DNA Repair; Estradiol; Estrogens; Estrogens, Catechol; Female; Genomic Instability; Humans; MCF-7 Cells

The current study was undertaken to determine the involvement of cAMP/PKA and MAPK-mediated signalling pathways in the regulation of cell proliferation by hydroxylated metabolites of 17β-estradiol (E2).. MCF-7, human breast cancer cells, were cultured in phenol red-free DMEM and treated with 1 nM 2-OH-E2 or 4-OH-E2. E2 was used as a positive control. Cell proliferation was measured using the BrdU incorporation assay. Cellular levels of cAMP and PKA were determined using ELISA kits. ERK1/2 protein expression was evaluated by Western Blot analysis. To determine the involvement of different intracellular pathways in the regulation of cell proliferation appropriate activators or inhibitors were used.. Hydroxylated estrogens, as E2, exhibited no influence on cAMP accumulation and PKA activation. In concomitant treatments with forskolin, cell proliferation decreased to the amount noted under the influence of forskolin alone. A PKA inhibitor (PKI) had no statistically significant effect on proliferation stimulated by E2 and its hydroxylated metabolites. Phospho-ERK1/2 protein expression in cells stimulated with E2, 2-OH-E2 and 4-OH-E2 was not significantly different from the control. However, co-treatment with both PD98059 and E2 or its hydroxylated metabolites reversed the effect of tested compounds on cell proliferation.. We have shown that E2 hydroxylated metabolites do not activate cAMP/PKA in breast cancer cells and confirm previously published data, which showed a lack of ERK1/2 activation in a breast cancer cell line. The observed reversible action of PD98059 on cell proliferation can be explained by the fact that hydroxylated estrogens, as E2, stimulate secretion of a number of growth factors, which affect MAPK activity, suggested by Lobenhofer et al. (2000).

Topics: Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Drug Evaluation, Preclinical; Enzyme Activation; Estradiol; Estrogens; Estrogens, Catechol; Female; Humans; Hydroxylation; MAP Kinase Signaling System; Signal Transduction

Sex Hormone-Binding Globulin (SHBG) - specific carrier for sex steroids - regulates hormone bioavailable fraction and estrogen signaling system in breast cancer cells. This study was conducted to elucidate the effects of hydroxylated estrogen (E2) metabolites (2-OH-E2 and 4-OH-E2) on sex hormone binding globulin (SHBG) mRNA and protein expression as well as on intracellular and extracellular SHBG levels.. MCF-7 human breast cancer cells were cultured with 2-OH-E2 or 4-OH-E2 in concentration of 1, 10 and 100 nM for 24 h, 17β-estradiol being used as a positive control. SHBG levels were measured in medium and cells by ELISA, SHBG mRNA expression was evaluated by real-time-PCR and protein expression by Western blot analysis.. 4-OH-E2 in high doses and 2-OH-E2 in the highest dose, while 17β-estradiol in all doses used increased intracellular but not extracellular SHBG levels. Both metabolites increased SHBG mRNA expression, the rank order of potency being E2 > 4-OH-E2 > 2-OH-E2. Both E2 and its metabolites increased SHBG protein expression.. Although the metabolites showed a lower potency than 17β-estradiol, further studies are needed to clarify whether hydroxylated metabolites of E2 are potent ligands for SHBG.

Topics: Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Estradiol; Estrogens, Catechol; Female; Gene Expression; Humans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sex Hormone-Binding Globulin

A 17beta-estradiol (E(2)) is hydrolyzed to 2-hydroxy-E(2) (2-OHE(2)) and 4-hydroxy-E(2) (4-OHE(2)) via cytochrome P450 (CYP) 1A1 and 1B1, respectively. In estrogen target tissues including the mammary gland, ovaries, and uterus, CYP1B1 is highly expressed, and 4-OHE(2) is predominantly formed in cancerous tissues. In this study, we investigated the inhibitory effects of chrysoeriol (luteorin-3'-methoxy ether), which is a natural methoxyflavonoid, against activity of CYP1A1 and 1B1 using in vitro and cultured cell techniques. Chrysoeriol selectively inhibited human recombinant CYP1B1-mediated 7-ethoxyresorufin-O-deethylation (EROD) activity 5-fold more than that of CYP1A1-mediated activity in a competitive manner. Additionally, chrysoeriol inhibited E(2) hydroxylation was catalyzed by CYP1B1, but not by CYP1A1. Methylation of 4-OHE(2), which is thought to be a detoxification process, was not affected by the presence of chrysoeriol. In human breast cancer MCF-7 cells, chrysoeriol did not affect the gene expression of CYP1A1 and 1B1, but significantly inhibited the formation of 4-methoxy E(2) without any effects on the formation of 2-methoxy E(2). In conclusion, we present the first report to show that chrysoeriol is a chemopreventive natural ingredient that can selectively inhibit CYP1B1 activity and prevent the formation of carcinogenic 4-OHE(2) from E(2.).

Topics: 2-Methoxyestradiol; Aryl Hydrocarbon Hydroxylases; Biocatalysis; Breast Neoplasms; Carcinogens; Catechol O-Methyltransferase; Cell Line, Tumor; Chemoprevention; Culture Media, Conditioned; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Cytosol; Enzyme Inhibitors; Estradiol; Estrogens, Catechol; Female; Flavones; Flavonoids; Gene Expression; Glucuronidase; Humans; Hydroxylation; Kinetics; Methylation; Oxazines; Recombinant Proteins; Sulfatases

We examined associations between polymorphisms in genes related to estrogen metabolism (CYP1B1 codon 432G --> C rs#1056836, CYP1B1 codon 453A --> G rs#1800440, COMT codon 158G --> A rs#4680) and biosynthesis (CYP17 T --> C promoter rs#743572, CYP19 exon 4 TTTA repeat) and urinary estrogen metabolites (2-hydroxyestrogens (2-OHE), 16alpha-hydroxyestrone (16alpha-OHE1), and their ratio) in a pilot study of 64 pre- and post-menopausal women with a family history of breast cancer. Women were participants in the Metropolitan New York Registry of Breast Cancer Families, one of six international sites of the National Cancer Institute's Breast Cancer Family Registry. We used linear regression to examine the effects of genetic variants on log-transformed urinary estrogen metabolites. After adjusting for menopausal status, BMI, and age, carriers of the CYP1B1 codon 453G variant allele had 31.0% lower levels of 2-OHE (P-value = 0.05) and 40.2% lower levels of 16alpha-OHE1 (P = 0.01). Results were similar after restricting the analyses to pre-menopausal women (n = 41). Consistent with other studies, among pre-menopausal women, carriers of the COMT codon 158A variant allele had increased 2-OHE levels (P = 0.03) and an increased 2-OHE/16alpha-OHE1 ratio (P = 0.04); carriers of the CYP17 C promoter variant allele had increased 2-OHE levels (P = 0.08). To our knowledge this is the first report showing associations between the CYP1B1 codon 453G variant allele and urinary 2-OHE and 16alpha-OHE1 metabolites. Further larger studies should be conducted to confirm these results. Future identification of individuals with genetic polymorphisms that affect estrogen metabolism and biosynthesis may help characterize women at higher breast cancer risk and could guide breast cancer prevention strategies for those individuals.

Topics: Adult; Aged; Aryl Hydrocarbon Hydroxylases; Breast Neoplasms; Catechol O-Methyltransferase; Codon; Cytochrome P-450 CYP1B1; Estradiol; Estrogens; Female; Humans; Hydroxyestrones; Middle Aged; Polymorphism, Genetic; Steroid 17-alpha-Hydroxylase

Certain estradiol metabolites may play a pivotal role in breast carcinogenesis. Of special interest are the metabolites 2-hydroxyestradiol (2-OHE2), which can react anti-carcinogenically, and 4-hydroxyestradiol (4-OHE1) and 16a-hydroxyestrone (16-OHE1), which may have procarcinogenic potential. In the present study, we have compared for the first time the effect of these metabolites and their parent substance 17beta-estradiol (E2) on proliferation, apoptosis, apoptosis markers and markers of metastatic property of human breast cancer cells.. MCF-7 cells (human estrogen-receptor positive metastatic breast cancer cell line) were incubated with the estrogens at concentrations of 0.1-100 nM. Cell proliferation rate was measured by the ATP-assay. Apoptosis was measured by cell death assay and the apoptosis markers cytochrome C, Bcl-2, Fasl and p53 were determined in cell lysates by ELISAs. The markers of metastatic property of the cell line, VEGF and MCP-1 were measured in the cell supernatant by ELISAs.. The estrogens E2, 4-OHE2 and 16-OHE1 display a proliferative effect on MCF-7 cells which is accompanied by a down-regulation of apoptosis. Various markers of apoptosis such as Bcl-2, cytochrome C and p53 appear to be involved. No significant effect was found for the metabolite 2-OHE2. VEGF and MCP-1 were up-regulated by E2 and 16-OHE1, whereas 2-OHE2 and 4-OHE2 did not show any effect.. The most potent estrogen regarding proliferation, apoptosis and metastasis of breast cancer cells seems to be estradiol. However, the estradiol metabolites 4-OHE2 and 16-OHE1 elicit similar properties on cell proliferation, apoptosis and metastasis as compared to estradiol but only at higher concentrations. In contrast 2-OHE2 did not show any significant effect on these parameters. Thus, intracellular estradiol metabolism may determine an individual's risk for breast carcinogenesis.

Topics: Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Estradiol; Estriol; Estrogens; Estrogens, Catechol; Fas Ligand Protein; Female; Humans; Membrane Glycoproteins; Proto-Oncogene Proteins c-bcl-2; Tumor Necrosis Factors; Tumor Suppressor Protein p53; Up-Regulation

An elevated incidence of breast cancer in women has been associated with prolonged exposure to high levels of estrogens. Our laboratory demonstrated that treatment of the immortalized human breast epithelial cells MCF-10F with 17beta-estradiol (E2), 4-hydroxyestradiol (4-OHE2) or 2-hydroxyestradiol (2-OHE2) induces phenotypical changes indicative of neoplastic transformation. MCF-10F cells treated with E2, 4-OHE2 or 2-OHE2 formed colonies in agar methocel and lost their ductulogenic capacity in collagen, expressing phenotypes similar to those induced by the carcinogen benzo[a]pyrene. To investigate whether the transformation phenotypes were associated with genomic changes, cells treated with E2, 4-OHE2 or 2-OHE2 at different doses were analyzed using microsatellite markers. Since microsatellite instability (MSI) and loss of heterozygosity (LOH) in chromosomes 13 and 17 have been reported in human breast carcinomas, we tested these parameters in MCF-10F cells treated with E2, 2-OHE2, or 4-OHE2 alone or in combination with the antiestrogen ICI182780. MCF-10F cells treated with E2 or 4-OHE2, either alone or in combination with ICI182780, exhibited LOH in the region 13q12.3 with the marker D13S893 located at approximately 0.8 cM telomeric to BRCA2. Cells treated with E2 or 4-OHE2 at doses of 0.007 and 70 nM and 2-OHE2 only at a higher dose (3.6 microM) showed a complete loss of 1 allele with D13S893. For chromosome 17, differences were found using the marker TP53-Dint located in exon 4 of p53. Cells treated with E2 or 4-OHE2 at doses of 0.007 nM and 70 nM and 2-OHE2 only at a higher dose (3.6 microM) exhibited a 5 bp deletion in p53 exon 4. Our results show that E2 and its catechol estrogen metabolites are mutagenic in human breast epithelial cells. ICI182780 did not prevent these mutations, indicating that the carcinogenic effect of E2 is mainly through its reactive metabolites 4-OHE2 and 2-OHE2, with 4-OHE2 and E2 being mutagenic at lower doses than 2-OHE2.

Topics: Base Sequence; Breast; Breast Neoplasms; Cell Culture Techniques; Cell Transformation, Neoplastic; Epithelial Cells; Estradiol; Estrogens, Catechol; Female; Humans; Loss of Heterozygosity; Molecular Sequence Data; Mutagenesis; Phenotype

Catechol estrogens, the hydroxylated metabolites of 17beta-estradiol (E2), have been considered to be implicated in estrogen-induced carcinogenesis. 2-Hydroxyestradiol (2-OHE2), a major oxidized metabolite of E2 formed preferentially by cytochrome P-450 1A1, reacts with DNA to form stable adducts and exerts genotoxicity. 2-OHE2 can be oxidized to quinone, which is accompanied by generation of reactive oxygen species (ROS). In the present study, 2-OHE2 induced strand scission in phiX174 phage DNA and oxidative base modifications in calf thymus DNA in the presence of cupric ion. In cultured human mammary epithelial (MCF-10A) cells, 2-OHE2 treatment produced ROS accumulation, 8-oxo-7,8-dihydroxy-2'-deoxyguanosine formation, cytotoxicity, and disruption of mitochondrial transmembrane potential, all of which were prevented by N-acetylcysteine. These findings, taken together, suggest that 2-OHE2-induced oxidative DNA damage and apoptosis in MCF-10A cells might be mediated by ROS generated via the redox cycling of this catechol estrogen.

Topics: Animals; Apoptosis; Breast; Breast Neoplasms; Cattle; Cell Culture Techniques; DNA Damage; Epithelial Cells; Estradiol; Humans; Oxidative Stress; Reactive Oxygen Species; Thymus Gland

A novel mechanistic hypothesis is proposed which suggests that hyperhomocysteinemia is a risk factor for the development of estrogen-induced hormonal cancer in humans. Mechanistically, hyperhomocysteinemia may exert its pathogenic effects largely through metabolic accumulation of intracellular S-adenosyl-L-homocysteine, a strong non-competitive inhibitor of the catechol-O-methyltransferase-mediated methylation metabolism of endogenous and exogenous catechol estrogens (mainly 2-hydroxyestradiol and 4-hydroxyestradiol). While a strong inhibition of the methylation metabolism of 2-hydroxyestradiol would decrease the formation of 2-methoxyestradiol (an antitumorigenic endogenous metabolite of 17beta-estradiol), an inhibition of the methylation of 4-hydroxyestradiol would lead to accumulation of this hormonally-active and strongly procarcinogenic catechol estrogen metabolite. Both of these effects resulting from inhibition of the methylation metabolism of catechol estrogens would facilitate the development of estrogen-induced hormonal cancer in the target organs. This hypothesis also predicts that adequate dietary intake of folate, vitamin B6, and vitamin B12 may reduce hyperhomocysteinemia-associated risk for hormonal cancer. Experimental studies are warranted to determine the relations of hyperhomocysteinemia with the altered circulating or tissue levels of 4-hydroxyestradiol and 2-methoxyestradiol and also with the altered risk for estrogen-induced hormonal cancer.

Topics: Anticarcinogenic Agents; Breast Neoplasms; Catechol O-Methyltransferase; Catechol O-Methyltransferase Inhibitors; Estradiol; Estrogens; Estrogens, Catechol; Female; Folic Acid; Humans; Hyperhomocysteinemia; Kinetics; Methylation; Models, Biological; Mutagenicity Tests; Neoplasms, Experimental; Neoplasms, Hormone-Dependent; Risk Factors; S-Adenosylhomocysteine; Uterine Neoplasms; Vitamin B 12; Vitamin B 6

The main goal of our study was to assess estrogen contents of breast tumor tissues, having different estrogen receptor status, in relation to long-term follow-up of patients.. Twenty-one breast cancer cases, all collected from January 1986 to January 1988 at the M. Ascoli Cancer Hospital Centre in Palermo, were included in the study and compared with 6 healthy women as a control group. Average follow-up time of patients was 144 +/- 10 months. The estrogen receptor status of tissues was determined by both ligand binding and immunohistochemical assays. A high performance liquid chromatography-based approach, jointly with gas chromatography/mass spectrometry, was used to identify and measure main estrogens, various hydroxyestrogens, and their methoxy derivatives in both normal and tumor tissues.. Although variable concentrations of hydroxylated estrogens were detected, they consistently accounted for >80% of all of the estrogens. Significantly greater amounts of both 2- and 4-hydroxyestradiol, along with a marked increase of 16 alpha-hydroxyestrone (OHE(1)), were observed in cancer with respect to normal breast tissues. A significant positive association was observed with elevated 16 alpha OHE(1) (P = 0.015) in patients alive, leading to significantly lower (P = 0.043) 2OHE(1):16 alpha OHE(1) ratio values. Conversely, ratio values of 4:2 hydroxy+methoxy estrogens was significantly lower (P = 0.006) in deceased patients. Using cutoff values of 1.2 for 4:2 hydroxy+methoxy ratio and 150 fmol/mg tissue for 16 alpha OHE(1) we achieved a clear-cut separation of patients, with over-cutoff patients having 147 months and under cutoff patients showing only 47 months median survival time (P = 0.00008).. Our data imply that individual hydroxyestrogens may have a distinct role in the onset and the clinical progression of breast cancer, with greater 16 alpha OHE(1) levels being in turn associated to cancer with respect to normal tissues and to a prolonged survival of breast cancer patients.

Topics: Adult; Aged; Binding Sites; Breast Neoplasms; Chromatography, High Pressure Liquid; Estradiol; Estrogens; Estrogens, Catechol; Female; Follow-Up Studies; Humans; Hydroxyestrones; Immunoenzyme Techniques; Middle Aged; Receptors, Estrogen; Survival Rate

Certain estrogen metabolites are involved in carcinogenesis and the development of resistance to methotrexate (MTX). In this study, we determined whether these well-established biological effects correlate with the relative efficiency of several estrogen metabolites to induce DNA strand breaks in the presence of copper, and investigated the potential enhancing effect of reduced nicotinamide adenine dinucleotide (NADH). DNA strand breaks induced by estradiol metabolites were measured by the conversion of supercoiled phage phiX-174 RF1 DNA to open circular and linear forms. The most active catecholestrogens were the 4-hydroxy derivatives, which produced about 2.5 times more DNA double strand breaks than the 2-hydroxy derivatives, while estradiol and 16alpha-hydroxyestrone were inactive. In addition, our results show that 4-hydroxyestradiol (4-OHE2) at physiological concentrations was capable of exhibiting DNA cleaving activity. The formation of these catecholestrogen-induced DNA strand breaks was associated with the utilization of oxygen and the generation of H2O2, because catalase inhibited the DNA cleaving activity of 4-OHE2. Interestingly, we also observed that NADH enhanced the induction of DNA strands breaks by 4-OHE2/Cu(II), probably by perpetuating the redox cycle between the quinone and the semiquinone forms of the catecholestrogen. In conclusion, this study demonstrated that the relative efficiency of 2-, and 4-hydroxyestrogen in carcinogenesis and for the enhancement of MTX resistance correlates with their relative capability to induce DNA strand breaks. In order to inhibit these estrogen-mediated biological effects, it may be important to develop different strategies to block the production of reactive oxygen species by the catecholestrogen-redox cycle.

Topics: Bacteriophage phi X 174; Breast Neoplasms; Copper; DNA Damage; DNA, Superhelical; DNA, Viral; Electrophoresis, Agar Gel; Estradiol; Estrogens, Catechol; Humans; Hydrogen Peroxide; NAD; Oxidation-Reduction; Oxygen Consumption; Reactive Oxygen Species; Superoxides

The comparative mitogenic activities of 17beta-estradiol (E2) and four metabolites, 2-hydroxyestradiol (2-OHE2), 2-hydroxyestrone (2-OHE1), 16alpha-hydroxyestradiol (16alpha-OHE2) and 16alpha-hydroxyestrone (16alpha-OHE1) were determined in estrogen receptor (ER)-positive MCF-7 and T47D human breast cancer cells. E2 (1 nM) induced a 7- to 13-fold increase in cell number in both cell lines compared to untreated cells and the mitogenic potencies of 16alpha-OHE1 or 16alpha-OHE2 were comparable to or greater than E2. In contrast, 2-OHE1 and 2-OHE2 were weak mitogens in both cell lines and in cells cotreated with 1 nM E2 and 100 or 1000 nM 2-OHE1 or 2-OHE2, there was a significant inhibition of hormone-induced cell proliferation. The comparative ER agonist/antagonist activities of E2 and the metabolites on transactivation were determined in T47D cells transiently transfected with constructs containing promoter inserts from the cathepsin D (pCD) and creatine kinase B (pCKB) genes. E2, 16alpha-OHE2 and 16alpha-OHE1 induced reporter gene activity in both MCF-7 or T47D cells transfected with pCKB or pCD. In contrast, 2-OHE1 and 2-OHE2 did not exhibit ER agonist activity for these transactivation assays, but in cells cotreated with E2 plus 2-OHE1 or 2-OHE2, there was a significant decrease in the hormone-induced response. These results demonstrate that 16alpha-OHE1/16alpha-OHE2 exhibit estrogenic activities similar to that observed for E2, whereas the 2-catecholestrogens are weak ER agonists (cell proliferation) or antagonists (cell proliferation and transactivation).

Topics: Breast Neoplasms; Cathepsin D; Cell Division; Creatine Kinase; Estradiol; Estriol; Female; Humans; Hydroxyestrones; Isoenzymes; Promoter Regions, Genetic; Receptors, Estrogen; Recombinant Proteins; Transfection; Tumor Cells, Cultured

Synthetic estrogens possessing hydroxyalkyl side chains at the C-2 position of the A-ring were designed in order to further elucidate the structural and electronic requirements of the estrogen receptor to A-ring modifications. Furthermore, these compounds were envisaged as being stable analogs of the estradiol metabolite 2-hydroxyestradiol. The homologous series of 2-(hydroxyalkyl)estradiols 1-3 has been prepared by chain extension of 2-formylestradiol 6, which, in turn, was prepared via ortholithiation of estradiol. The substituted estradiols 1-3 were assayed for their abilities to bind to the estrogen receptor in MCF-7 cells and induce estrogen-responsive gene expression. The estradiol homologs exhibited significantly weaker affinity than estradiol for the MCF-7 cell estrogen receptor, with relative binding affinities (estradiol = 100) ranging from 1.11 for 2-(hydroxymethyl)estradiol (1) to 0.073 for 2-(hydroxypropyl)estradiol (3). The relative activities for mRNA induction of the pS2 gene by the estradiol homologs closely parallel the relative binding affinities for the estrogen receptor in MCF-7 cells. 2-(Hydroxymethyl)-estradiol exhibited similar estrogen receptor affinity and pS2 gene induction to the catechol estrogen 2-hydroxyestradiol and may prove useful in examination of the further biological effects of 2-hydroxyestrogen homologs.

Topics: Base Sequence; Breast Neoplasms; Cell Line; DNA Primers; Estradiol; Humans; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Mass Spectrometry; Molecular Sequence Data; Receptors, Estrogen; Structure-Activity Relationship; Tumor Cells, Cultured

Estrogen is a known risk factor in human breast cancer. In rodent models, estradiol has been shown to induce tumors in those tissues in which this hormone is predominantly converted to the catechol metabolite 4-hydroxyestradiol by a specific 4-hydroxylase enzyme, whereas tumors fail to develop in organs in which 2-hydroxylation predominates. We have now found that microsomes prepared from human mammary adenocarcinoma and fibroadenoma predominantly catalyze the metabolic 4-hydroxylation of estradiol (ratios of 4-hydroxyestradiol/2-hydroxyestradiol formation in adenocarcinoma and fibroadenoma, 3.8 and 3.7, respectively). In contrast, microsomes from normal tissue obtained either from breast cancer patients or from reduction mammoplasty operations expressed comparable estradiol 2- and 4-hydroxylase activities (corresponding ratios, 1.3 and 0.7, respectively). An elevated ratio of 4-/2-hydroxyestradiol formation in neoplastic mammary tissue may therefore provide a useful marker of benign or malignant breast tumors and may indicate a mechanistic role of 4-hydroxyestradiol in tumor development.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; Breast; Breast Neoplasms; Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme System; Estradiol; Estrogens; Estrogens, Catechol; Female; Fibroadenoma; Humans; Hydroxylation; In Vitro Techniques; Microsomes; Steroid Hydroxylases

The catechol estrogens (CE), 2-hydroxyestradiol (2-OH-E2) and 4-hydroxyestradiol (4-OH-E2) were analyzed for their binding affinity to the estrogen receptor of MCF-7 cells. Applying a competitive binding assay to cytosols prepared from MCF-7 breast cancer cells, we measured a relative binding affinity of 23% (2-OH-E2) and 26% (4-OH-E2) compared to E2. Nuclear binding assays with the same cell line demonstrated a high specific binding with Kd's of 0.31 nM (2-OH-E2) and 0.21 nM (4-OH-E2). The relative binding affinity measured was 25% and 42% for 2-OH-E2 and 4-OH-E2, respectively. Based on this nuclear binding it can be concluded that the estrogen receptor occupied by CE is bound within the nucleus and might therefore be transcriptionally active.

Topics: Binding, Competitive; Breast Neoplasms; Cell Nucleus; Cytosol; Estradiol; Estrogens, Catechol; Humans; Prolactin; Receptors, Estrogen; Receptors, Progesterone; Tumor Cells, Cultured

Catecholestrogens are important metabolites of estradiol and estrone in the human. Considerable interest has focused on the catecholestrogens 2-hydroxy- and 4-hydroxyestradiol since they bind to the estrogen receptor with an affinity in the range of estradiol. Using the MCF-7 cell line, we analysed the capacity of purified catecholestrogens to transform the estrogen receptor into its high affinity nuclear binding form and to affect receptor-dependent processes such as proliferation and expression of the progesterone receptor (PR). Incubations with 2-hydroxy- and 4-hydroxyestradiol at 10(-8) M for 1 h resulted in tight nuclear binding of the estrogen receptor. During treatment of the cells with catecholestrogens we obtained a marked increase in proliferation rate of 36 and 76% for 2-hydroxy- and 4-hydroxyestradiol, respectively, relative to the inductive effect of estradiol (100%). The PR level, was slightly increased by treatment with 2-hydroxyestradiol (10%), whereas treatment with 4-hydroxyestradiol increased the PR level at 28%, compared to estradiol (100%). From these results we conclude that the 2- and 4-hydroxylated derivatives of estradiol are active hormones and are able to initiate estrogen receptor mediated processes in MCF-7 cells.

Topics: Breast Neoplasms; Cell Division; Cell Nucleus; Estradiol; Estrogens, Catechol; Female; Humans; Kinetics; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen; Time Factors; Tumor Cells, Cultured

Different cell growth effects were observed in MCF-7 cells after six daily exposures to either 17 beta-estradiol (E2), 2-hydroxyestradiol (2-OHE2), or 2-methoxyestradiol (2-MeOE2) at 10 nM levels. 2-OHE2 enhanced cell growth significantly (P < 0.05) more than did the parent compound, whereas 2-MeOE2 inhibited cell growth. To identify the estrogen-affected cellular processes involved in cell cycle progression, hydroxy urea-synchronized MCF-7 cells were studied. No effects on DNA synthesis in mid-S-phase or on mitotic indices were observed after E2 or 2-OHE2 treatment. 2-MeOE2, however, significantly (P < 0.05) inhibited DNA synthesis and mitosis. Synchronized cells were exposed for 1 h to E2, 2-OHE2, or 2-MeOE2 before cAMP levels were determined in early S-phase and mid-S-phase, as well as during mitosis. E2 and 2-OHE2 had no effect, but 2-MeOE2 caused a significant (P < 0.05) increase in cAMP concentration in early S-phase and a decrease during mitosis. Phosphorylation of S-phase proteins was also studied. [32P]Pi incorporation was significantly (P < 0.05) enhanced in many proteins in 2-MeOE2-exposed cells. Small proteins (M(r) < 25,000), as well as large proteins (M(r) > 220,000), were most prominently affected. In comparison, E2 and 2-OHE2 had little effect. We suggest that the enhanced 2-MeOE2-induced protein phosphorylation during S-phase may affect S-phase events, which subsequently causes inhibition of mitosis. Protein synthesis during G2/M transition was unexpectedly enhanced by 2-OHE2 and was not enhanced by E2. [35S]Methionine incorporation into proteins in the order of M(r) 32,000-46,000, 47,000-50,000, 58,000-67,000, and 83,000-89,000 was significantly (P < 0.05) increased. 2-MeOE2 had no effect. The results of this study indicate that 2-OHE2 may be the more potent mitogen, whereas 2-MeOE2 acts as a cytostatin.

Topics: 2-Methoxyestradiol; Breast Neoplasms; Cell Cycle; Cyclic AMP; DNA, Neoplasm; Estradiol; G2 Phase; Humans; Methionine; Mitosis; Molecular Weight; Neoplasm Proteins; Phosphorylation; S Phase; Time Factors; Tumor Cells, Cultured

The binding of catechol estrogens, epoxyenones and methoxyestrogens was evaluated using estrogen receptors in cytosol prepared from human breast cancers. The relative affinity of 2-hydroxyestradiol, a metabolite formed in vitro from estradiol-17 beta by breast cancer cells, was indistinguishable from that of estradiol-17 beta. 4-Hydroxyestradiol, which is also a metabolite of estradiol-17 beta, associated with the estrogen receptor with a relative affinity approximately 1.5-fold greater than that of estradiol-17 beta. Epoxyenones and methoxyestrogens were weak competitors compared to the binding of estradiol-17 beta, exhibiting relative affinities 3% or less than the affinity of estradiol-17 beta. Sucrose density gradient centrifugation revealed that both 2- and 4-hydroxyestradiol inhibited the binding of estradiol-17 beta to both the 4S and 8S isoforms of the estrogen receptor in a competitive manner, with a Ki = 0.94 nM for 2-hydroxyestradiol and a Ki = 0.48 nM for 4-hydroxyestradiol. It can be concluded that these data demonstrate a specific receptor-mediated estrogenic action for both of these catechol estrogens.

Topics: Binding, Competitive; Breast Neoplasms; Centrifugation, Density Gradient; Cytosol; Estradiol; Estrogens, Catechol; Female; Humans; Kinetics; Receptors, Estrogen; Tumor Cells, Cultured

Topics: Animals; Breast Neoplasms; Equilenin; Equilin; Estradiol; Estrogens; Estrogens, Catechol; Ethinyl Estradiol; Female; Humans; Hydroxyestrones; Hydroxylation; Microsomes, Liver; Papio