Estrogen Receptor In MCF-7 Cells Research Articles

Estrogenic activity of freeze-dried silkworm extracts through the activation of estrogen receptors in MCF-7 cells

Silkworm is an insect that feeds on mulberry leaves only, and silkworm powder has been reported to have antioxidant, hypoglycemic, and anticholesterol activities. In this study, we measured the content of 1-deoxynojirimycin (1-DNJ) in 24 different extracts from silkworm cocoons and also investigated the estrogenic activities of these extracts and their effects on the activation of intracellular signaling pathways in MCF-7 breast cancer cells. Among the 24 silkworm extracts, relatively high 1-DNJ content and estrogenic activity were shown by Extract 11 (30E3H20-1), which was prepared by a single extraction of silkworm powder with 20 times the volume of 30% ethanol against each powder weight for 3 h. The estrogen receptor activity of this extract was confirmed based on its promoting effects on the phosphorylation of the estrogen receptor α (ERα) and mRNA expression of the ESR1 and ESR2 genes. In addition, treatment with Extract 11 (30E3H20-1) increased the phosphorylation of AKT, p38, and JNK, which are downstream proteins of ERα. Based on our findings, a silkworm extract could be developed as a natural estrogen supplement in the future.

Impedance-Based E-Screen Cell Biosensor for the Real-Time Screening of Xenoestrogenic Compounds

The E-screen assay is the gold standard method to screen for xenoestrogens. The method is based on the binding of estrogenic compounds to the human estrogen receptor of MCF-7 cells, resulting in increased cell proliferation. The conventional method is easy to perform but involves multiple manual steps including cell labeling and requires up to 6 days to evaluate the estrogenic activity. Here, we present an impedance-based E-screen cell biosensor in which the MCF-7 cells are cultured on electrodes. Upon estrogen treatment, the increased cell proliferation is detected as an increase in the recorded impedance with time. The estrogenic activity of test compounds is measured by comparing the increased impedance during the exponential growth phase with the impedance recorded in a hormone-free medium, which demonstrates that the impedance increase is directly proportional to the 17β-estradiol concentration. Two different compounds were tested for their estrogenic activities, confirming the known xenoestrogen, bisphenol-A, and the absence of xenoestrogenic activity of the antifouling agent, Irgarol 1051. All impedance-based results are compared to results obtained with the conventional assay. The impedance E-screen cell biosensor is label-free, easier to use, enables real-time analysis, which provides growth kinetic information upon estrogen exposure, and results in a shorter analysis time.

Ginsenoside Rg1 activates ligand-independent estrogenic effects via rapid estrogen receptor signaling pathway

BackgroundGinsenoside Rg1 was shown to exert ligand-independent activation of estrogen receptor (ER) via mitogen-activated protein kinase–mediated pathway. Our study aimed to delineate the mechanisms by which Rg1 activates the rapid ER signaling pathways. MethodsER-positive human breast cancer MCF-7 cells and ER-negative human embryonic kidney HEK293 cells were treated with Rg1 (10−12M, 10−8M), 17ß-estradiol (10−8M), or vehicle. Immunoprecipitation was conducted to investigate the interactions between signaling protein and ER in MCF-7 cells. To determine the roles of these signaling proteins in the actions of Rg1, small interfering RNA or their inhibitors were applied. ResultsRg1 rapidly induced ERα translocation to plasma membrane via caveolin-1 and the formation of signaling complex involving linker protein (Shc), insulin-like growth factor-I receptor, modulator of nongenomic activity of ER (MNAR), ERα, and cellular nonreceptor tyrosine kinase (c-Src) in MCF-7 cells. The induction of extracellular signal-regulated protein kinase and mitogen-activated protein kinase kinase (MEK) phosphorylation in MCF-7 cells by Rg1 was suppressed by cotreatment with small interfering RNA against these signaling proteins. The stimulatory effects of Rg1 on MEK phosphorylation in these cells were suppressed by both PP2 (Src kinase inhibitor) and AG1478 [epidermal growth factor receptor (EGFR) inhibitor]. In addition, Rg1-induced estrogenic activities, EGFR and MEK phosphorylation in MCF-7 cells were abolished by cotreatment with G15 (G protein-coupled estrogen receptor-1 antagonist). The increase in intracellular cyclic AMP accumulation, but not Ca mobilization, in MCF-7 cells by Rg1 could be abolished by G15. ConclusionGinsenoside Rg1 exerted estrogenic actions by rapidly inducing the formation of ER containing signalosome in MCF-7 cells. Additionally, Rg1 could activate EGFR and c-Src ER-independently and exert estrogenic effects via rapid activation of membrane-associated ER and G protein-coupled estrogen receptor.

Abstract 461: Modulation of estrogen receptor (ER) and androgen receptor (AR) by a modified CRISPR-Cas9 system

Abstract Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) system is a bacterial adaptive immune defense against foreign nucleic acids from viruses, plasmids, and other mobile genetic elements. There are three types of CRISPR systems; type II uses single effector enzyme, Cas9, to cleave double-stranded DNA (dsDNA). Because of this unique feature, CRISPR/Cas9 system has been widely used as a genetic engineering tool from bacteria to mammals. In this system, Cas9 nuclease can bind and cleave genome directed by sequence-specific guide RNA (gRNA). While the wild type Cas9 can cleave double stranded DNA, the mutant Cas9 at D10A and H804A can only bind to but cannot cleave the DNA. Given this unique feature, we tried to repurpose Cas9 as a gene activation or suppression tool. To achieve this, we generated DMCas9 (double-mutant Cas9), and fused it with VP16AD (VP16 activation domain) or VP64AD (VP64 activation domain) to explore its utility for gene activation. To generate a Cas9-based gene silencing tool, we fused DMCas9 with Krab, a well-known protein domain with suppression function. Using a reporter system carrying a specific sequence that was recognized by a given gRNA and mCherry as a readout, we demonstrated that co-expression of VP16AD-DMCas9 and specific gRNAs leads to a at least three-fold induction of mCherry signal; in contrast, DMhCas9-Krab along with the specific gRNA resulted in a significant suppression of mCherry activity. Furthermore, we showed that DMCas9-Krab combined with gRNAs suppressed endogenous expression of AR and ER in MCF-7 cells, causing cell growth inhibition. Together, these results demonstrate the feasibility of Cas9-based gene activation and gene suppression. Citation Format: Nanjiang Zhou, Yin-Yuan Mo. Modulation of estrogen receptor (ER) and androgen receptor (AR) by a modified CRISPR-Cas9 system. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 461. doi:10.1158/1538-7445.AM2014-461

Internalization of a C17α-alkynylestradiol-porphyrin conjugate into estrogen receptor positive MCF-7 breast cancer cells

We hypothesized that expression of nuclear estrogen receptor (ER) in hormone-sensitive breast cancer cells could be harnessed synergistically with the tumor-accumulating effect of porphyrins to selectively deliver estrogen-porphyrin conjugates into breast tumor cells, and preferentially kill tumor cells upon exposure to visible light. In this study we synthesized a conjugate of C(17α)-alkynylestradiol and pyropheophorbide and demonstrated that this conjugate is internalized by ER-positive MCF-7 cells while pyropheophorbide did not, suggesting an ER-mediated uptake and internalization of the conjugate by incipient nuclear ER in MCF-7 cells. This study is a direct demonstration of our hypothesis about ER-mediated internalization of estrogen-porphyrin conjugates.

ER regulates an evolutionarily conserved apoptosis pathway

Estrogen receptor (ER) is regarded as a major causal factor in breast cancer and FoxA1, a winged-helix transcription factor belonging to the forkhead family, has been found to function as a pioneer factor in the recruitment of ER to several cis-regulatory elements in the genome. High throughput chromatin immunoprecipitation analyzed by hybridization to microarrays (ChIP-chip) can reveal ER and FoxA1 binding sites occupied by transcription factors. However, these results are blind to lineage-specific cis-regulatory elements. In this study, we identified ER and FoxA1 binding sites conserved in evolution by using Detection of LinEage-Specific Selection (DLESS) method. We also analyzed target genes close to conserved cis-regulatory elements by combining gene-expression data. A total of 7877 ER binding sites and 18,135 FoxA1 binding sites were identified in MCF-7 cells by performing an unbiased genome-wide ChIP-chip with False Discovery Rate (FDR) of 5%. Using DLESS method, we found target candidate genes closest to fully conserved cis-regulatory elements related to apoptosis according to gene ontology analysis. Furthermore, network analysis of apoptosis-related genes within 10 kb of fully conserved cis-regulatory elements was constructed using Ingenuity Pathway Analysis (IPA). Apoptosis genes in the network showed over- and under-expression in MCF-7 cell-line. And these apoptosis-related genes closest to fully conserved cis-regulatory elements in network showed strong correlation with ER in MCF-7 cells. These results elucidate that ER regulates an evolutionarily conserved apoptosis pathway. This opens up new perspectives in the apoptosis of human breast cancer from the evolution of cis-regulatory elements.

FKBPL Regulates Estrogen Receptor Signalling and Determines Response to Endocrine Therapy.

Abstract Approximately 40% of patients with ER+ breast cancers do not respond to endocrine therapies; furthermore, most tumours eventually become resistant. We have identified a novel Hsp90 co-chaperone and immunophilin, FKBPL, that binds to Hsp90 and affects the stability and signalling of the estrogen receptor (ER) with implications for breast cancer growth and sensitivity to endocrine therapies.Using co-immunoprecipitations, we have demonstrated that FKBPL interacts within Hsp90 complexes associated with the estrogen receptor in MCF-7 cells. Cells stably overexpressing FKBPL become dependent on estrogen for their growth and even in its presence, FKBPL over-expression slows the rate of proliferation of these cells. More importantly, this dependence on estrogen, rendered FKBPL over-expressing cells dramatically more sensitive (up to 90%) to the anti-estrogens, tamoxifen and fulvestrant. Furthermore, knock-down of FKBPL using a targeted siRNA approach, dramatically increased the resistance of these cells to tamoxifen, supporting a role for FKBPL as a determinant of response to endocrine therapies.We have also identified putative estrogen responsive elements within FKBPL's promoter and show that FKBPL is upregulated in response to estrogen suggesting that FKBPL itself is an estrogen responsive gene. As FKBPL levels increased in response to estrogen, ER levels fell; implicating FKBPL in the stabilisation of ER. This is supported by data demonstrating that FKBPL over-expressing cells exhibit decreased levels of ER and cathepsin D, an estrogen responsive gene, critical to breast cancer growth, survival and invasion. Furthermore, knockdown of FKBPL using an siRNA approach increased ER and cathepsin D levels. The regulation of this ER-responsive gene supports a functional role for FKBPL in physiological ER-mediated signalling. FKBPL is also important for the stabilisation of the cyclin dependent kinase inhibitor, p21 (Jascur et al., 2005). We have seen a dramatic fall in p21 levels when FKBPL is knocked down with a targeted siRNA. Loss of p21 has been associated with a tamoxifen growth inducing phenotype and hyperphosphorylation of ER at S118, and with subsequent increased expression of ER-regulated genes. Here we show that ER phosphorylation is increased in FKBPL knockdown cells and decreased in FKBPL over-expressing cells. Together, these data support a model in which high levels of FKBPL would stabilise p21, reducing ER phosphorylation, abrogating tamoxifen-induced agonist potency and so increase sensitivity to the drug.Finally, having established that FKBPL expression may result in p21-mediated growth arrest and sensitisation to endocrine therapies, we hypothesised that FKBPL may have prognostic value that might impact upon tumour proliferative capacity and improve outcome independent of ER status. This was verified in two publically available breast cancer patient cohorts where we demonstrate that high FKBPL expression was correlated with increased overall survival and distant metastasis-free survival. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5126.

Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to the estrogen receptor of MCF-7 cells in cytosolic extracts and in nuclei of intact cells.

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.

Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells

Select changes in microRNA (miRNA) expression correlate with estrogen receptor α (ERα) expression in breast tumors. miR-21 is higher in ERα positive than negative tumors, but no one has examined how estradiol (E2) regulates miR-21 in breast cancer cells. Here we report that E2 inhibits miR-21 expression in MCF-7 human breast cancer cells. The E2-induced reduction in miR-21 was inhibited by 4-hydroxytamoxifen (4-OHT), ICI 182 780 (Faslodex), and siRNA ERα indicating that the suppression is ERα-mediated. ERα and ERβ agonists PPT and DPN inhibited and 4-OHT increased miR-21 expression. E2 increased luciferase activity from reporters containing the miR-21 recognition elements from the 3′-UTRs of miR-21 target genes, corroborating that E2 represses miR-21 expression resulting in a loss of target gene suppression. The E2-mediated decrease in miR-21 correlated with increased protein expression of endogenous miR-21-targets Pdcd4, PTEN and Bcl-2. siRNA knockdown of ERα blocked the E2-induced increase in Pdcd4, PTEN and Bcl-2. Transfection of MCF-7 cells with antisense (AS) to miR-21 mimicked the E2-induced increase in Pdcd4, PTEN and Bcl-2. These results are the first to demonstrate that E2 represses the expression of an oncogenic miRNA, miR-21, by activating estrogen receptor in MCF-7 cells.

E6AP and Calmodulin Reciprocally Regulate Estrogen Receptor Stability

Estrogen promotes the proliferation of human breast epithelial cells by interacting with the estrogen receptor (ER). Physiological responses of cells to estrogen are regulated in part by degradation of the ER. Previous studies revealed that calmodulin binds directly to the ER, thereby enhancing its stability. Consistent with these findings, cell-permeable calmodulin antagonists dramatically reduced the number of ER in MCF-7 human breast epithelial cells. Here we investigated the molecular mechanism by which calmodulin attenuates ER degradation. MG132 and lactacystin, inhibitors of the ubiquitin-proteasome pathway, prevented the calmodulin antagonist CGS9343B from reducing the amount of ER in MCF-7 cells. In contrast, protease inhibitors afforded no protection. Moreover, CGS9343B enhanced ER ubiquitination. A point mutant ER construct that is unable to bind calmodulin, termed ERDeltaCaM, is ubiquitinated to a greater extent than wild type ER. The ubiquitin-protein isopeptide ligase E6-associated protein (E6AP) associated with and promoted the degradation of ER. The possible convergence of calmodulin and E6AP on ER degradation was examined. ERDeltaCaM bound E6AP with higher affinity than that of wild type ER. Moreover, calmodulin attenuated the in vitro interaction between ER and E6AP in a Ca(2+)-dependent manner. Collectively, our data reveal that E6AP is a component of ER degradation via the ubiquitin-proteasome pathway and that Ca(2+)/calmodulin modulates this degradation mechanism. These results have potential implications for the development of selectively targeted therapeutic agents for breast cancer.

Novel Interactions of Vitamin E and Estrogen in Breast Cancer

The prevention of breast cancer through dietary modification is an active area of clinical and epidemiological research. It has been proposed that dietary supplementation of vitamin E may reduce a woman's risk of developing breast cancer. However, the exact mechanism remains unknown. α-Tocopherol is the most biologically active form of vitamin E. We investigated the effect of vitamin E (α-tocopherol) on breast cancer cell growth. A dose-dependent inhibition of cell proliferation was found in estrogen receptor (ER)-positive cells showing a potent suppression of growth at 100 μM vitamin E in MCF-7 (53%) and T47D (75%) cells. Vitamin E reduced significantly the response of both cell lines to estrogen (10 nM), and cell proliferation was decreased in MCF-7 and T47D cells by 69% and 84%, respectively. No growth inhibition was observed when cells were grown in the absence of estrogen. Vitamin E altered and decreased the growth inhibition induced by tamoxifen (10 μM) in MCF-7 (33%) and T47D (54%) cells. In addition, the immunostaining of ER of MCF-7 cells was reduced by 30% in the presence of vitamin E, suggesting an effect of vitamin E on the expression of ER. This provides evidence that vitamin E may inhibit ER-positive cell growth by altering the cellular response to estrogen.

Selenium Disrupts Estrogen Signaling by Altering Estrogen Receptor Expression and Ligand Binding in Human Breast Cancer Cells

Cancer prevention studies suggest that selenium is effective in reducing the incidence of cancers including prostate, colon, and lung cancers. Previous reports showed that selenium inhibits premalignant human breast MCF-10AT1 and MCF10AT3B cell growth in vitro and reduces mammary tumor incidence after exposure to carcinogens in tumor models. Because estrogen is critical to the development and differentiation of estrogen target tissues, including the breast, the present study was designed to examine the effect of selenium on estrogen receptor (ER) expression and activation using methylseleninic acid (MSA), an active form of selenium in vitro. Selenium decreased the levels of expression of ERalpha mRNA and protein and reduced the binding of labeled estradiol to estrogen receptor in MCF-7 cells. Selenium inhibited the trans-activating activity of estrogen receptor in MCF-7 cells expressing functional estrogen receptor using a luciferase reporter construct linked to estrogen responsive element. Selenium decreased the binding of estrogen receptor to the estrogen responsive element site using an electrophoretic mobility gel shift assay. Selenium suppressed estrogen induction of the endogenous target gene c-myc. In contrast to the effect on ERalpha in MCF-7 cells, selenium increased ERbeta mRNA expression in MDA-MB231 human breast cancer cells. Thus, differential regulation of ERalpha and ERbeta in breast cancer cells may represent a novel mechanism of selenium action and provide a rationale for selenium breast cancer prevention trial.

2-Methoxyestradiol Is an Estrogen Receptor Agonist That Supports Tumor Growth in Murine Xenograft Models of Breast Cancer

2-Methoxyestradiol (2MEO) is being developed as a novel antitumor agent based on its antiangiogenic activity, tumor cell cytotoxicity, and apparent lack of toxicity. However, pharmacologic concentrations of 2MEO bind to estrogen receptors (ER). We have therefore examined the ER activity of 2MEO. Estrogenic actions of 2MEO were evaluated by changes in gene expression of the ER-positive (MCF7) breast tumor cell line and, in vivo, estrogenicity was assessed in breast tumor xenograft models and by measuring endocrine responses in uterus and liver. In the ER-positive breast tumor cell line (MCF7), microarray experiments revealed that 269 of 279 changes in gene expression common to 2MEO and estradiol were prevented by the ER antagonist, ICI 182,780. Changes in the expression of selected genes and their sensitivity to inhibition by ICI 182,780 were confirmed by quantitative reverse transcription-PCR measurement. Activation of ER in MCF7 cells by 2MEO was further confirmed by stimulation of an estrogen response element-dependent reporter gene that was blocked by ICI 182,780 (1 micromol/L). Doses of 2MEO (15-150 mg/kg) that had no antitumor efficacy in either nu/nu BALB/c or severe combined immunodeficient mice bearing ER-negative MDA-MB-435 tumors had uterotropic and hepatic estrogen-like actions. In female nu/nu BALB/c mice inoculated with the estrogen-dependent MCF7 tumor cells, 2MEO (50 mg/kg/d) supported tumor growth. Tumor growth enhancement by 2MEO at doses generating serum levels (100-500 nmol/L) that have estrogenic activity suggests that a conservative approach to the further clinical evaluation of this agent should be adopted and that its evaluation in breast cancer is inappropriate.

Ginsenoside-Rb1 from Panax ginseng C.A. Meyer Activates Estrogen Receptor-α and -β, Independent of Ligand Binding

We studied the estrogenic activity of a component of Panax ginseng, ginsenoside-Rb1. The activity of ginsenoside-Rb1 was characterized in a transient transfection system, using estrogen receptor isoforms and estrogen-responsive luciferase plasmids, in COS monkey kidney cells. Ginsenoside-Rb1 activated both alpha and beta estrogen receptors in a dose-dependent manner with maximal activity observed at 100 microm, the highest concentration examined. Activation was inhibited by the estrogen receptor antagonist ICI 182,780, indicating that the effects were mediated through the estrogen receptor. Treatment with 17beta-estradiol or ginsenoside-Rb1 increased expression of the progesterone receptor, pS2, and estrogen receptor in MCF-7 cells and of AP-1-driven luciferase genes in COS cells. Although these data suggest that it is functionally very similar to 17beta-estradiol, ginsenoside-Rb1 failed to displace specific binding of [(3)H]17beta-estradiol from estrogen receptors in MCF-7 whole-cell ligand binding assays. Our results indicate that the estrogen-like activity of ginsenoside-Rb1 is independent of direct estrogen receptor association.

Waste Crankcase Oil: An Environmental Contaminant with Potential to Modulate Estrogenic Responses

Used engine, or crankcase, oil is frequently discarded into the environment resulting in significant pollution of both aquatic and terrestrial ecosystems. The chemical composition of crankcase oils changes with use, and in general, used, or waste, crankcase oil is considered more toxic than the original oil. Polycyclic aromatic hydrocarbons (PAHs) are major constituents of crankcase oil and may exhibit both estrogenic and antiestrogenic activity. In the present study, the estrogenic activity and antiestrogenic activity of both new (unused) and waste crankcase oils were examined in a human breast cancer cell culture assay. Concentrations of 5, 10, 15, 20, or 25 ppm of new oil or waste oil did not alter either the preconfluent or postconfluent cell growth when tested in control medium. In contrast, waste crankcase oil significantly reduced the postconfluent growth of cells grown in medium containing 0.1 nM17β-estradiol. Results from mechanistic assays using [3H]-17β-estradiol demonstrated that waste crankcase oil both increased the metabolism of 17β-estradiol, and displaced 17β-estradiol from the estrogen receptor in MCF-7 cells. The observed antiestrogenic activity of the waste crankcase oil suggests that this pollutant has the potential to alter estrogenic responses, and therefore its presence in the environment may be of concern for reproductive health.

Two antiestrogens affect differently chromatin remodeling of trefoil factor 1 (pS2) gene and the fate of estrogen receptor in MCF7 cells

We show here that the two antagonists ICI 182 780, a pure estrogen antagonist, and 4-hydroxy-tamoxifen, a selective estrogen receptor modulator (SERM) have distinct effects on TFF1 (formerly pS2) gene chromatin structure and transcription. Indeed, ICI 182 780 decreased both the intensity of the hormone-dependent DNase I hypersensitive site pS2 HS-1 and transcription of the pS2 gene whereas 4-hydroxy-tamoxifen (OH-Tam) increased the intensity of pS2-HS1 and had no effect on pS2 gene transcription. Interestingly, these differential effects are associated with different fates of ERα following the two treatments: The ERα–OH-Tam complex was retained in the nucleus more efficiently than the ERα–estradiol complex. In contrast, ICI 182 780 provoked a rapid relocation of ERα complex to an insoluble nuclear fraction, followed by its degradation. Taken together, these data suggest that regulating the amount of ERα in the nucleus is a major way of action of estrogen antagonists with respect to chromatin remodeling and transcriptional control.

Estrogenic Effects of Cimicifuga racemosa (Black Cohosh) in Mice and on Estrogen Receptors in MCF-7 Cells.

The estrogenic effects of Cimicifuga racemosa or Actacea racemosa (black cohosh, CR) extracts were tested in mice, and their effects on estrogen receptor (ER) levels in human breast cancer MCF-7 cells were also investigated. Four groups of weanling female Kunming mice were given 0 (control), 75, 150, or 300 mg/kg body weight CR extracts orally for 14 days. The estrus cycle and the weights of the uterus and ovary of mice, as well as serum estradiol (E(2)) were measured. The proliferation patterns of MCF-7 cells exposed to CR extracts or 17beta-estradiol were studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Subsequently, growth of MCF-7 cells in 0 (control) or 4.75 &mgr;g/L of CR extracts or 0.3 nmol/L of 17beta-estradiol groups were observed for 5 days. ER levels in MCF-7 cells were analyzed by indirect immunofluorescence assay using flow cytometry. The uterine weights of mice increased with the increase in dosage of CR extracts, and the estrus duration was significantly prolonged in the group receiving 300 mg/kg body weight (P <.05). However, CR extracts did not increase the serum E(2) concentration significantly. In the in vitro study, a dose-response relationship was demonstrated when cells were treated with low doses of CR extracts, and the optimal enhancement concentration of CR extracts was 4.75 &mgr;g/L on MCF-7 cells. The doubling times (T(D)) of cell growth in the CR extracts group and the 17beta-estradiol group were 32.1 and 31.7 hours, respectively, both shorter than that of the negative control group (T(D) = 35.3 hours). Additionally, 4.75 &mgr;g/L of CR extracts resulted in significantly increased ER levels compared with the control group (P <.01). In conclusion, CR extracts produced an estrogenic action. The effect of increasing ER levels by CR extracts may be one of the potential mechanisms of its phytotherapeutic effects for postmenopausal symptoms.

2-Methoxymethylestradiol: a new 2-methoxy estrogen analog that exhibits antiproliferative activity and alters tubulin dynamics

An estradiol metabolite, 2-methoxyestradiol (2-MeOE 2), has shown antiproliferative effects in both hormone-dependent and hormone-independent breast cancer cells. Previously, a series of 2-hydroxyalkyl estradiol analogs had been synthesized in our laboratories as potential probes for comparison of estrogen receptor (ER)-mediated versus non-ER-mediated effects in breast cancer cells. A methoxy derivative of 2-hydroxymethyl estradiol was prepared for biological evaluation and comparison with 2-MeOE 2. Estrogenic activity of the synthetic analogs was evaluated in two ways, one by examining affinity of the analogs for the estrogen receptor in MCF-7 cells and the other by examining the ability of the analogs to induce estrogen-responsive gene expression. The analog, 2-methoxymethyl estradiol (2-MeOMeE 2), demonstrated weak affinity for the estrogen receptor (0.9% of estradiol) and weak ability to stimulate estrogen-induced expression of the pS2 gene (0.02% of estradiol). Antitumor activity was evaluated both in vitro and in vivo. The steroidal nucleus seems to be an attractive target for developing novel tubulin polymerization inhibitors. Additionally, such steroidal compounds may have low toxicity compared to the natural products known to interact with tubulin. Interestingly, 2-MeOMeE 2 inhibited tubulin polymerization in vitro at concentrations of 1 and 3 μM and was more effective than 2-MeOE 2. In cells, 2-MeOMeE 2 was effective in suppressing growth and inducing cytotoxicity in MCF-7 and MDA-MB-231 breast cancer cells. The cytotoxic effects of 2-MeOMeE 2 are associated with alterations in tubulin dynamics, with the frequent appearance of misaligned chromosomes, a significant mitotic delay, and the formation of multinucleated cells. In comparison, 2-MeOE 2 was more effective than 2-MeOMeE 2 in producing cytotoxicity and altering tubulin dynamics in intact cells. Assessment of in vivo antitumor activity was performed in athymic mice containing human breast tumor xenografts. Nude mice bearing MDA-MB-435 tumor xenografts were treated i.p. with 50 mg/kg per day of 2-MeOMeE 2 or vehicle control for 45 days. Treatment with 2-MeOMeE 2 resulted in an approximate 50% reduction in mean tumor volume at treatment day 45 when compared to control animals and had no effect on animal weight. Thus, 2-MeOMeE 2 is an estrogen analog with minimal estrogenic properties that demonstrates antiproliferative effects both in vitro and in the human xenograft animal model of human breast cancer.

Calmodulin Enhances the Stability of the Estrogen Receptor

The estrogen receptor mediates breast cell proliferation and is the principal target for chemotherapy of breast carcinoma. Previous studies have demonstrated that the estrogen receptor binds to calmodulin-Sepharose in vitro. However, the association of endogenous calmodulin with endogenous estrogen receptors in intact cells has not been reported, and the function of the interaction is obscure. Here we demonstrate by co-immunoprecipitation from MCF-7 human breast epithelial cells that endogenous estrogen receptors bind to endogenous calmodulin. Estradiol treatment of the cells had no significant effect on the interaction. However, incubation of the cells with tamoxifen enhanced by 5-10-fold the association of calmodulin with the estrogen receptor and increased the total cellular content of estrogen receptors by 1.5-2-fold. In contrast, the structurally distinct calmodulin antagonists trifluoperazine and CGS9343B attenuated the interaction between calmodulin and the estrogen receptor and dramatically reduced the number of estrogen receptors in the cell. Neither of these agents altered the amount of estrogen receptor mRNA, suggesting that calmodulin stabilizes the protein. This hypothesis is supported by the observation that, in the presence of Ca2+, calmodulin protected estrogen receptors from in vitro proteolysis by trypsin. Furthermore, overexpression of wild type calmodulin, but not a mutant calmodulin incapable of binding Ca2+, increased the concentration of estrogen receptors in MCF-7 cells, whereas transient expression of a calmodulin inhibitor peptide reduced the estrogen receptor concentration. These data demonstrate that calmodulin binds to the estrogen receptor in intact cells in a Ca2+-dependent, but estradiol-independent, manner, thereby modulating the stability and the steady state level of estrogen receptors.

Ligand-independent activation of estrogen receptor function by 3,3′-diindolylmethane in human breast cancer cells

3,3′-Diindolylmethane (DIM), a major in vivo product of acid-catalyzed oligomerization of indole-3-carbinol (I3C), is a promising anticancer agent present in vegetables of the Brassica genus. We investigated the effects of DIM on estrogen-regulated events in human breast cancer cells and found that DIM was a promoter-specific activator of estrogen receptor (ER) function in the absence of 17β-estradiol (E 2). DIM weakly inhibited the E 2-induced proliferation of ER-containing MCF-7 cells and induced proliferation of these cells in the absence of steroid, by approximately 60% of the E 2 response. DIM had little effect on proliferation of ER-deficient MDA-MB-231 cells, suggesting that it is not generally toxic at these concentrations. Although DIM did not bind to the ER in this concentration range, as shown by a competitive ER binding assay, it activated the ER to a DNA-binding species. DIM increased the level of transcripts for the endogenous pS2 gene and activated the estrogen-responsive pERE-vit-CAT and pS2-tk-CAT reporter plasmids in transiently transfected MCF-7 cells. In contrast, DIM failed to activate transcription of the simple E 2- and diethylstilbesterol-responsive reporter construct pATC2. The estrogen antagonist ICI 182780 (7α-[9-[(4,4,5,5,5-pentafluoropentyl)sulfonyl]nonyl]-estra-1,3,5(10)-triene-3,17β-diol) was effective against DIM-induced transcriptional activity of the pERE-vit-CAT reporter, which further supports the hypothesis that DIM is acting through the ER. We demonstrated that ligand-independent activation of the ER in MCF-7 cells could be produced following treatment with the D1 dopamine receptor agonist SKF-82958 [(±)6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepinehydrobromide]. We also demonstrated that the agonist effects of SKF-82958 and DIM, but not of E 2, could be blocked by co-treatment with the protein kinase A (PKA) inhibitor H-89 ( N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide). These results have uncovered a promoter-specific, ligand-independent activation of ER signaling for DIM that may require activation by PKA, and suggest that this major I3C product may be a selective activator of ER function.