Estrogen-dependent Cell Proliferation Research Articles

Ganoderma lucidum inhibits proliferation of human breast cancer cells by down-regulation of estrogen receptor and NF-κB signaling

Ganoderma lucidum, an oriental medical mushroom, has been used in Asia for the prevention and treatment of a variety of diseases, including cancer. We have previously demonstrated that G. lucidum inhibits growth and induces cell cycle arrest at G0/G1 phase through the inhibition of Akt/NF-kappaB signaling in estrogen-independent human breast cancer cells. However, the molecular mechanism(s) responsible for the inhibitory effects of G. lucidum on the proliferation of estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-231) breast cancer cells remain to be elucidated. Here, we show that G. lucidum inhibited the proliferation of breast cancer MCF-7 and MDA-MB-231 cells by the modulation of the estrogen receptor (ER) and NF-kappaB signaling. Thus, G. lucidum down-regulated the expression of ERalpha in MCF-7 cells but did not effect the expression of ERbeta in MCF-7 and MDA-MB-231 cells. In addition, G. lucidum inhibited estrogen-dependent as well as constitutive transactivation activity of ER through estrogen response element (ERE) in a reporter gene assay. G. lucidum decreased TNF-alpha-induced (MCF-7) as well as constitutive (MDA-MB-231) activity of NF-kappaB. The inhibition of ER and NF-kappaB pathways resulted in the down-regulation of expression of c-myc, finally suppressing proliferation of estrogen-dependent as well as estrogen-independent cancer cells. Collectively, these results suggest that G. lucidum inhibits proliferation of human breast cancer cells and contain biologically active compounds with specificity against estrogen receptor and NF-kappaB signaling, and implicate G. lucidum as a suitable herb for chemoprevention and chemotherapy of breast cancer.

Estrogen-dependent responses of the mammary fat pad in prepubertal dairy heifers

Ovaries are absolutely required for development of the mammary parenchyma (PAR) in cattle, reflecting estrogen-dependent epithelial cell proliferation. However, the estrogen receptor (ER) that mediates the mammary estrogen effects, ERalpha, is absent in proliferating epithelial cells. In the mouse, this discrepancy is explained in part by the ability of the mammary fat pad (MFP) to synthesize epithelial cell mitogens such as IGF-I in response to estrogen. Consistent with a similar role for the bovine MFP, 30% of its fibroblasts and adipocytes were immunoreactive for ERalpha in prepubertal dairy heifers. To assess estrogen-dependent gene expression in the MFP, 16 prepubertal dairy heifers were randomly assigned to a 2x2 factorial. The first factor was ovarian status, with heifers undergoing bilateral ovariectomy or left intact at 4.6 months of age. The second factor was applied 30 days after surgery and consisted of injection of estrogen or excipient. After 3 days of injection, heifers were administered an intrajugular bolus of bromodeoxyuridine (BrdU) and slaughtered 2 h later. The estrogen injection, but not ovarian status, caused significant increases in the fraction of epithelial cells labeled with BrdU and produced tissue-specific effects on gene expression. In the PAR, estrogen injection increased IGF-I gene expression by twofold despite reductions of 50% or more in ERalpha mRNA abundance and the fraction of epithelial cells immunoreactive for ERalpha. The estrogen-dependent increase in IGF-I mRNA was greater in the MFP, presumably because estrogen failed to downregulate ERalpha expression in this mammary compartment. Finally, estrogen-responsiveness of the MFP appears unique among the bovine fat depots as estrogen injection did not induce IGF-I expression in its s.c. counterpart. Our data demonstrate that the bovine MFP is highly responsive to exogenous estrogen, consistent with a role for this tissue compartment in communicating its effects on epithelial cell proliferation.

TNF-α enhances estrogen-induced cell proliferation of estrogen-dependent breast tumor cells through a complex containing nuclear factor-kappa B

Breast tumors are usually classified according to their response to estrogens as hormone-dependent or -independent. In this work, we investigated the role of the proinflammatory cytokine TNF-alpha on the estrogen-receptor-positive T47D breast ductal tumor cells. We have found that TNF-alpha exerts a mitogenic effect, inducing cyclin D1 expression and activation of the transcription factor NF-kappaB. Importantly, activation of NF-kappaB was required for estrogen-induced proliferation and cyclin D1 expression. TNF-alpha enhanced the estrogen response by increasing the levels and availability of NF-kappaB. Chromatin immunoprecipitation analysis suggested that the action of estrogens is mediated by a protein complex that contains the activated estrogen receptor, the nuclear receptor coactivator RAC3 and a member of the NF-kappaB family. Finally, our results demonstrate that activation of this transcription factor could be one of the key signals for estrogen-mediated response.

Expression of estrogen-responsive finger protein (Efp) is associated with advanced disease in human epithelial ovarian cancer

Objective. The estrogen-responsive ring finger protein (Efp) gene, one of estrogen receptor (ER) target genes, is considered to be essential for estrogen-dependent cell proliferation. To understand the estrogenic action on ovarian cancer, we studied the relationships between Efp and ERs expressions and the correlations of Efp expression with clinicopathological parameters in epithelial ovarian cancer. Methods. The protein expressions for Efp, ERα and ERβ were examined by immunoblotting in 12 ovarian cancer cell lines. Efp mRNA expressions were evaluated by quantitative RT-PCR in 12 ovarian cancer cell lines. A total of 100 surgical specimens diagnosed as epithelial ovarian cancer were examined immunohistochemically using antibodies for Efp, ERα and ERβ. Results. Efp protein was detected in 8 out of 12 cell lines. In Efp protein-positive cell lines, Efp mRNA was expressed higher than that in negative ( P = 0.021). All of the Efp protein-positive cell lines simultaneously expressed either ERα or ERβ protein. By immunohistochemical staining, Efp immunoreactivity was detected in 63 out of 100 ovarian cancer specimens and positive signals were in the cytoplasm of carcinoma cells. There were significant correlations between Efp and ERα, ERβ immunoreactivity (Efp and ERα, P = 0.022; Efp and ERβ, P = 0.032). Efp expression was significantly higher in a subgroup with serous adenocarcinoma ( P = 0.010) and with advanced disease ( P = 0.026). No significant relationship was detected between Efp immunoreactivity and overall survival. Conclusion. The expression of Efp was detected in human epithelial ovarian cancer and high expression of Efp was correlated with advanced disease and serous adenocarcinoma, and ERs status.

Expression of estrogen-responsive finger protein (Efp) is associated with advanced disease in human epithelial ovarian cancer

3143 Background: The estrogen-responsive ring finger protein (Efp) gene is one of the estrogen receptor (ER) target genes identified in the MCF-7 human breast cancer cell line using genomic binding-site cloning. Efp is considered to be essential for estrogen-dependent cell proliferation. In the present study, we studied correlations of Efp expression with clinicopathological parameters, Efp’s significance as a prognostic indicator, and relationships between Efp and ERs expressions in human epithelial ovarian cancer. Methods: A total of 100 epithelial ovarian cancer cases were examined immunohistochemically using antibodies for Efp, ERα, and ERβ. The correlation between Efp immunoreactivity and clinicopathological parameters was examined. The expression of Efp, ERα and ERβ and mRNA expressions of Efp, ERα, and ERβ were evaluated by immunoblotting and reverse transcription-polymerase chain reaction (RT-PCR), respectively, in 12 ovarian cancer cell lines. Results: Efp immunoreactivity was detected in the cytoplasm of carcinoma cells in 63 out of 100 cases (63%). Efp expression was significantly higher in carcinomas with serous differentiation (P=0.0102) and advanced carcinomas (P=0.0259). No significant relationship was detected between Efp immunoreactivity and overall survival. There were high correlations between Efp and ERα and β immunoreactivity (Efp and ERα, P=0.022; Efp and ERβ, P=0.0323). In immunoblotting, Efp protein was detected in 8 out of 12 cell lines; furthermore, ERα and/or ERβ protein were detected in all 8 cell lines. Significantly higher mRNA levels were detected in the 8 cancer cell lines with Efp-immunoreactive bands than in the other 4 lines without Efp-immunoreactive bands (P=0.021). Conclusion: The expression of Efp is associated with advanced disease and serous histology, and is also correlated with ER status. Our results suggest that Efp could be a prognostic indicator in human epithelial ovarian cancer. No significant financial relationships to disclose.

Effects of analogs of indole-3-carbinol cyclic trimerization product in human breast cancer cells

5,6,11,12,17,18-Hexahydrocyclonona[1,2- b:4,5- b*:7,8- b**]triindole (CTr) is a major digestive product of indole-3-carbinol (I3C) from Brassica vegetables and exhibits strong estrogenic activities. CTr increases proliferation of estrogen-dependent breast tumor cells, binds with strong affinity for the estrogen receptor-alpha (ERα), and activates expression of estrogen (E 2)-dependent genes. To begin to examine the structural features that determine the biological activity of CTr, we prepared and studied the effects of two analogs, 9,18-dihydro-12 H-[1,2,5]trithionino[3,4- b:6,7- b*:9,8- b**]triindole (S 3CTr) and 5,6,11,12,17,18-hexahydro-5,11,17-trimethylcyclonona[1,2- b:4,5- b*:7,8- b**]triindole (Me 3CTr). N-Methylation of CTr completely ablated the estrogenic activities of CTr. In the dose range in which CTr was clearly estrogenic, Me 3CTr exhibited no detectable effect on cell growth, ERα binding to E 2, or ERα-responsive gene expression. S 3CTr showed mixed ERα agonist activities. It bound to the ERα and activated receptor binding with DNA, weakly activated expression of transfected E 2-responsive reporter gene constructs, and strongly inhibited the E 2-induced activation of these reporter constructs. S 3CTr activated aryl hydrocarbon receptor (AhR)-mediated pathways, consistent with the moderately strong binding affinity of S 3CTr for the AhR. Comparisons of the conformational characteristics among CTr and its two analogs indicated that the estrogenic effects of CTr are highly sensitive to apparently minor structural modifications, and further supported the hypothesis for a central role of hydrogen bonding around the nitrogen atom in CTr binding to the ligand binding site of ERα.

Daucane Sesquiterpenes from Ferula hermonis

The dried roots of Ferula hermonis yielded three new daucanes, (1R,4R)-4-hydroxydauca-7-ene-6-one (1), (1R,4R)-4-hydroxydauca-7-ene-6,9-dione (2), and (1R,3S,8S)-3-ethoxy-8-angeloyloxydauca-4-en-9-one (3), together with the three known sesquiterpenes, ferutinin, teferidin, and (+)-alpha-bisabolol. The structures of compounds 1-3 were elucidated on the basis of spectroscopic evidence. The effect of these compounds on the proliferation of estrogen-dependent MCF-7 cells was evaluated, and it was found that compounds 1 and 3 exhibited proliferative activity, whereas 2 showed an antiproliferative effect.

Cdk4 Is Indispensable for Postnatal Proliferation of the Anterior Pituitary

For proper development and tissue homeostasis, cell cycle progression is controlled by multilayered mechanisms. Recent studies using knock-out mice have shown that animals can develop relatively normally with deficiency for each of the G1/S-regulatory proteins, D-type and E-type cyclins, cyclin-dependent kinase 4 (Cdk4), and Cdk2. Although Cdk4-null mice show no embryonic lethality, they exhibit specific endocrine phenotypes, i.e. dwarfism, infertility, and diabetes. Here we have demonstrated that Cdk4 plays an essential non-redundant role in postnatal proliferation of the anterior pituitary. Pituitaries from wild-type and Cdk4-null embryos at embryonic day 17.5 are morphologically indistinguishable with similar numbers of cells expressing a proliferating marker, Ki67, and cells expressing a differentiation marker, growth hormone. In contrast, anterior pituitaries of Cdk4-null mice at postnatal 8 weeks are extremely hypoplastic with markedly decreased numbers of Ki67+ cells, suggesting impaired cell proliferation. Pituitary hyperplasia induced by transgenic expression of human growth hormone-releasing hormone (GHRH) is significantly diminished in the Cdk4+/- genetic background and completely abrogated in the Cdk4-/- background. Small interfering RNA (siRNA)-mediated knockdown of Cdk4 inhibits GHRH-induced proliferation of GH3 somato/lactotroph cells with restored expression of GHRH receptors. Cdk4 siRNA also inhibits estrogen-dependent cell proliferation in GH3 cells and closely related GH4 cells. In contrast, Cdk6 siRNA does not diminish proliferation of these cells. Furthermore, Cdk4 siRNA does not affect GHRH-induced proliferation of mouse embryonic fibroblasts or estrogen-dependent proliferation of mammary carcinoma MCF-7 cells. Taken together, Cdk4 is dispensable for prenatal development of the pituitary or proliferation of other non-endocrine tissues but indispensable specifically for postnatal proliferation of somato/lactotrophs.

Deviation from Additivity with Estrogenic Mixtures Containing 4-Nonylphenol and 4-tert-Octylphenol Detected in the E-SCREEN Assay

An intriguing deviation from expected additivity is reported with mixtures containing 17beta-estradiol, 17alpha-ethinylestradiol, genistein, bisphenol A, 4-nonylphenol, and 4-tert-octylphenol. The effect of these chemicals on the proliferation of estrogen-dependent MCF-7 human breast cancer cells (the E-SCREEN) was measured. Data variance-component analyses, carried out to optimize the assay for mixture studies, showed that between-experiment variability was the dominant source of data variation. Adoption of a data-normalization procedure reduced the impact of this variability and allowed the pooling of historical E-SCREEN data. Concentration-response relationships for all six chemicals were recorded and utilized to calculate predictions of their joint effects by employing the model of concentration addition. Surprisingly, the observed combination effects of the mixture fell short of the additivity expectations, indicating weak antagonism. Experimental or prediction errors were ruled out as possible explanations for this deviation, which suggested that it might be the result of interactions between mixture components. With the aim of identifying the responsible components, mixtures were designed by excluding one or more of the chemicals from the original six-component mixture, and the resulting combination effects were assessed. These permutation studies allowed us to conclude thatthe presence of 4-nonylphenol and 4-tert-octylphenol is associated with the antagonisms observed with the six-component mixture and thus negatively affected the predictability of mixture effects. Future mixture studies utilizing the E-SCREEN with endocrine disrupters that also exhibit toxicity or growth-inhibitory effects will have to take account of the possibility that such interactions might compromise the predictability of estrogenic combination effects.

Application of a newly developed ELISA for BCAR1 protein for prediction of clinical benefit of tamoxifen therapy in patients with advanced breast cancer.

Advanced breast cancer is frequently treated with the antiestrogen tamoxifen. Response is observed in approximately one half of estrogen receptor (ER)-positive patients but ultimately fails because the cancer becomes resistant. The mechanisms for resistance of breast cancer to tamoxifen treatment are not yet understood (1). The breast cancer anti-estrogen resistance protein 1 ( BCAR1 ) gene was identified in a search for genes that cause proliferation of estrogen-dependent breast cancer cells in the presence of an antiestrogen drug (2). To assess the role of the BCAR1 protein in breast cancer progression, we developed a specific and sensitive ELISA. Here we report the use of this BCAR1 ELISA to measure BCAR1 protein concentrations in human breast cancer cytosols to predict success of tamoxifen treatment for advanced disease. Primary invasive breast tumors were collected between 1978 and June 1995. Our study design was approved by the medical ethics committee of the Erasmus MC Rotterdam. Selection of samples for analysis of response to tamoxifen in recurrent breast cancer was based on the availability of stored cytosol extracts (in liquid nitrogen), which remained after routine ER and progesterone receptor analyses (3). Tissue specimens that were sampled after neoadjuvant treatment or obtained from a biopsy were not included. In addition, the samples selected were only from ER-positive (>10 fmol/mg of protein) tumors of patients who developed recurrent disease and were treated with tamoxifen as first-line therapy. Of these 592 patients, 133 had received adjuvant chemotherapy (89 receiving cyclophosphamide–methotrexate–5-fluorouracil and 44 receiving 5-fluorouracil–epirubicin–cyclophosphamide), and none had received adjuvant hormonal therapy. The median age at the start of tamoxifen treatment for recurrent disease was 61 years (range, 28–91 years); 144 patients were premenopausal and 448 were postmenopausal. During the follow-up period, 527 of 592 patients (89%) showed tumor progression, and 433 patients (73%) died. …

Mechanism of carcinogenesis induced by a veterinary antimicrobial drug, nitrofurazone, via oxidative DNA damage and cell proliferation

Nitrofurazone, a veterinary antimicrobial drug, causes mammary and ovarian tumors in animals. We investigated the mechanisms of carcinogenesis by nitrofurazone. Nitrofurazone significantly stimulated the proliferation of estrogen-dependent MCF-7 cells. Nitrofurazone caused Cu(II)-mediated damage to 32P-5′-end-labeled DNA fragments obtained from human genes only when cytochrome P450 reductase was added. DNA damage was inhibited by catalase and bathocuproine. DNA damage was preferably induced at the 5′-A CG-3′ sequence, a hotspot of the p53 gene. These findings suggest that nitrofurazone metabolites are involved in tumor initiation through oxidative DNA damage and nitrofurazone itself enhances cell proliferation, leading to promotion and/or progression in carcinogenesis.

Systemic distribution of estrogen-responsive finger protein (Efp) in human tissues

Estrogen-responsive finger protein (Efp), a target gene product of estrogen receptor (ER), is considered essential for estrogen-dependent cell proliferation. The biological significance of Efp remains unclear in human tissues, and therefore, we examined systemic distribution of Efp in human adult and fetal tissues using RT-PCR and immunohistochemistry. Efp mRNA expression was marked in the placenta and uterus, high in the thyroid gland, aorta, and spleen in adult, and relatively low in other human adult and fetal tissues examined in this study. Efp immunoreactivity was detected in epithelium of various adult tissues, and was also detected in cytotrophoblasts of the placenta and splenic macrophages. Efp immunolocalization in human fetus was generally similar as that in adult. These Efp-positive cells were previously reported to be associated with ERα and/or ERβ expression. Therefore, these results indicate that Efp is widely expressed and may play important roles in various human tissues possibly through ERs.

Hormonal regulation and localization of estrogen, progestin and androgen receptors in the endometrium of nonhuman primates: effects of progesterone receptor antagonists

This article reviews the effects of estradiol (E(2)), progesterone (P) and P receptor antagonists (PA) on the rhesus macaque endometrium. Ovariectomized macaques can be treated with implants of estradiol (E(2)) and P to induce precisely controlled, artificial menstrual cycles. During these cycles, treatment with E(2) alone induces an artificial proliferative phase marked by extensive endometrial epithelial cell proliferation and increased expression of stromal and epithelial estrogen receptor (ER) and P receptor (PR). Androgen receptor (AR) is also upregulated by E(2) but is expressed only by the endometrial stroma. Progesterone acts on the E(2) primed endometrium to induce secretory differentiation and causes suppression of epithelial and stromal ER, epithelial PR, and stromal AR in the functionalis zone. However, epithelial ER and PR are retained in the basalis zone during the secretory phase. When potent P antagonists (PA) are administered acutely at the end of an E(2) + P induced cycle, menses typically ensues similar to P withdrawal at the end of the menstrual cycle. When PAs are administered chronically there is significant blockage of all P- dependent effects including upregulation of ER, PR and AR and suppression of glandular secretory function. However, chronic PA administration also inhibits estrogen-dependent endometrial cell proliferation and growth. This endometrial antiproliferative effect is the basis of the clinical use of PA to control various diseases such as endometriosis.

Transit of rat uterine stromal cells through G1 phase of the cell cycle requires temporal and cell-specific hormone-dependent changes on cell cycle regulators.

Progesterone pretreatment increases the number of synchronously proliferating stromal cells in the ovariectomized rat uterus, but estrogen is necessary to stimulate reentry into the cell cycle. To investigate the mechanisms underlying differential hormone actions, sexually mature ovariectomized rats were injected with progesterone (2 mg) for three consecutive days. Estradiol 17-beta (0.6 microg) was administered to initiate cell proliferation. Uterine samples were collected at timed intervals. Cell entry into DNA replication was monitored by injecting 5-bromo-2'-deoxyuridine (1 mg/100 g body weight) 2 h before necropsy. Demicolchicine (400 microg) was injected 30 min before necropsy to assess transit into M phase. Temporal progress through G1 was determined by spatial changes in cyclin D1/D3 proteins. Total cyclin D1/D3 protein and mRNA was measured by Western and Northern blotting. Estrogen increased the number of 5-bromo-2'-deoxyuridine-positive stromal cells (P < 0.05), compared with the number in rats treated with progesterone alone. An increase (P < 0.05) in the number of M-phase cells occurred at 12 h post estrogen. There was no evidence for epithelial cell proliferation in response to steroid treatments. Cyclin D1/D3 mRNA was expressed in the uteri of ovariectomized and hormone treated rats. The D-type cyclin proteins, however, were not evident in stromal cells without estrogen treatment. Progesterone pretreatment inhibited estrogen-dependent epithelial cell proliferation while redirecting D-type cyclin expression to the uterine stroma. Stromal cell transit through G1 required nongenomic steroid-dependent action on signal transduction pathways that control the nuclear localization and cell type-specific expression of the D-type cyclin proteins.

Biocompatibility of Hydroxylated Metabolites of BISGMA and BFDGE

Unpolymerized dental monomers can leach out into the oral biophase and are bioavailable for metabolism. We hypothesize that metabolites would be less toxic than parent monomers. We first identified the formation of metabolites from bisphenol F diglycidyl ether (BFDGE) and Bisphenol A glycidyl methacrylate (BISGMA) after their exposure to liver S9 fractions. Then, the metabolites and parent compounds were subjected to in vitro cytotoxicity, mutagenicity, and estrogenicity studies. Bisphenol A bis(2,3-dihydroxypropyl) ether and bisphenol F bis(2,3-dihydroxypropyl) ether were the hydroxylated metabolites of BISGMA and BFDGE, respectively. Cytotoxicity against L929 cells showed that the metabolites were significantly (p < 0.05) less cytotoxic than the parent monomers. Only BFDGE was mutagenic in the Ames assay with strain TA100 of Salmonella typhimurium. Parent and metabolite compounds did not stimulate estrogen-dependent MCF-7 cell proliferation above solvent controls. These results indicated that the hydroxylated metabolites were non-mutagenic, non-estrogenic, and less cytotoxic than their parent monomers.

Oxytocin modulates estrogen receptor α expression and function in MCF7 human breast cancer cells

Oxytocin (OT) inhibits the proliferation of MCF7 estrogen-dependent human breast cancer cells, via specific OT receptors (OTR). Besides this effect, we report that OT modulates the expression of estrogen receptor alpha (ERalpha) in MCF7 cells, both at mRNA and protein level. Since the first 24 h of OT treatment the ERalpha mRNA levels are down-regulated; in contrast, ERalpha protein expression decreases at a later time. The reduced number of ERalpha goes in parallel to a temporary increase in the binding affinity of these receptors as well as to a significant increase in their estradiol (E2)-induced transcription activity. The increase in both binding affinity and transcriptional activity of ERalpha likely balances the reduction in the number of ERalpha binding sites, ruling out the hypothesis that part of the OT contrasting effect on E2-induced cell proliferation could depend on the reduced E2 binding to MCF7 cells and supporting the hypothesis of an exclusively direct OT-antimitogenic effect. This is the first evidence that OT modulates the expression of ERalpha receptors in human neoplastic cells.

In Vitro Inhibition of Proliferation of Estrogen-Dependent and Estrogen-Independent Human Breast Cancer Cells Treated with Carotenoids or Retinoids

Both estrogen-receptor (ER) positive MCF-7 and ER-negative Hs578T and MDA-MB-231 human breast cancer cells were treated with carotenoids (beta-carotene, canthaxanthin and lycopene) and retinoids (all-trans-, 9-cis- and 13-cis-retinoic acid and all-trans-retinol). Among carotenoids, beta-carotene significantly reduced the growth of MCF-7 and Hs578T cells, and lycopene inhibited the growth of MCF-7 and MDA-MB-231 cells. Canthaxanthin did not affect the proliferation of any of the three cell lines. All-trans- and 9-cis-retinoic acid significantly reduced the growth of both MCF-7 and Hs578T cells, whereas 13-cis-retinoic acid and all-trans-retinol had a significant effect only on MCF-7 cells. MCF-7 and Hs578T cells treated with all-trans-retinoic acid (all-t-RA) were further studied for the mechanism behind growth inhibition. Retinoic acid receptors alpha and gamma (RARalpha, gamma) in MCF-7 cells and RARalpha, beta and gamma in Hs578T cells were not induced by all-t-RA treatment at either the protein or mRNA level. Hs578T cells treated with all-t-RA had significantly more cells in the G0/G1 stage of the cell cycle, but the same was not observed for MCF-7 cells. All-t-RA induced a dose-dependent cell death in MCF-7 cells, which may be a necrotic phenomenon. These results demonstrate that ER status is an important, although not essential factor for breast cancer cell response to carotenoid and retinoid treatments, and the mode of action of all-t-RA in MCF-7 and Hs578T cells is not through the induction of RAR. Other mechanistic pathways that are either followed by or concomitant with growth inhibition are possible.

Catechol estrogens induce oxidative DNA damage and estradiol enhances cell proliferation.

Estrogen-induced carcinogenesis involves enhanced cell proliferation (promotion) and genotoxic effects (initiation). To investigate the contribution of estrogens and their metabolites to tumor initiation, we examined DNA damage induced by estradiol and its metabolites, the catechol estrogens 2-hydroxyestradiol (2-OHE(2)) and 4-hydroxyestradiol (4-OHE(2)). In the presence of Cu(II), catechol estrogens formed piperidine-labile sites at thymine and cytosine residues in (32)P 5'-end-labeled DNA fragments and induced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine. NADH markedly enhanced Cu(II)-dependent DNA damage mediated by nanomolar concentrations of catechol estrogens. Catalase and bathocuproine inhibited the DNA damage, suggesting the involvement of H(2)O(2) and Cu(I). These results suggest that H(2)O(2), generated during Cu(II)-catalyzed autoxidation of catechol estrogens, reacts with Cu(I) to form the Cu(I)-peroxide complex, leading to oxidative DNA damage, and that NADH enhanced DNA damage through the formation of redox cycle. To investigate the role of estrogens and their metabolites in tumor promotion, we examined their effects on proliferation of estrogen-dependent MCF-7 cells. Estradiol enhanced the proliferation of MCF-7 cells at much lower concentrations than catechol estrogens. These findings indicate that catechol estrogens play a role in tumor initiation through oxidative DNA damage, whereas estrogens themselves induce tumor promotion and/or progression by enhancing cell proliferation in estrogen-induced carcinogenesis.

Steroid analysis and xenosteroid potentials in two small streams in southwest Germany

The presence of estrogenic substances in thewater of the small streams Korsch (Ko)and Krahenbach (Kr), Southwest Germany, wasdetermined by chemical and biological analysis.Because a large proportion of the Ko waternear its mouth consists of sewage treatmentplant (STPs) effluents, the impact of STPs onlevels of estrogens in surface water is anenvironmental issue of concern. In July 1996,water samples were taken from Kr and Ko(four sites) and tested in the E-Screen assaywith human MCF-7 breast cancer cells. AllKo samples induced estrogen-dependent cellproliferation resulting in 17β-estradiolequivalent concentrations (EEQ) between 3.3 and9.7 ng/L while the Kr water showed no effect.In 1998/99 eight samples from Ko (near itsmouth) and nine samples from Kr were collectedand tested in the E-Screen after solid phaseextraction. Some estrogenicity was detectablein three Kr samples but Ko samples had amedian EEQ of 3.1 ng/L (range: 1.2–42 ng/L).GC/MS analysis revealed differences in thelevels of 17β-estradiol and estronebetween the two streams. 17β-estradiolwas detectable in five Ko samples only (0.7–1.8 ng/L). Estrone was found in the Kofrom 2.5 to 38 ng/L (median: 7.6 ng/L) and inthe Kr between 0.8 and 22 ng/L (median: 1.7 ng/L). Analysis for nine phenolic xenoestrogensrevealed rather low levels for five compoundswhich occurred more frequently and in higherconcentrations in the Ko. After asemi-field exposure of adult male rainbow troutfor 4 weeks in Ko water, plasmavitellogenin levels were significantly highercompared to those levels detected in the sameanimals before exposure.

Stereochemistry of cis- and trans-hinokiresinol and their estrogen-like activity.

Naturally occurring phenylpropanoids, hinokiresinol (trans-hinokiresinol) and nyasol (cis-hinokiresinol) were found to possess appreciable estrogen receptor binding activity. Strong differences in activity were observed between the geometrical isomers and enantiomers. Among these, (3S)-cis-hinokiresinol displayed the highest activity, one order of magnitude greater than the activity of genistein. Furthermore, cis- and trans-hinokiresinol stimulated the proliferation of estrogen-dependent T47D breast cancer cells, and their stimulatory effects were blocked by an estrogen antagonist, indicating that the compounds are estrogen agonists. In addition, the absolute configuration of C-3 in (+)-cis-hinokiresinol has been assigned as S by comparison with the circular dichroism spectra of the hydrogenated products prepared from cis and trans ((3S)-trans-hinokiresinol: previously assigned) isomers. These results incidentally provide us with an unambiguous answer to contradictory reports regarding the assignment of the full stereochemisry of cis- and trans-hinokiresinol that have existed in the literature for more than two decades.