RAGE inhibition blunts insulin-induced oncogenic signals in breast cancer.

The receptor for advanced glycation end products (RAGE) is implicated in diabetes and obesity complications, as well as in breast cancer (BC). Herein, we evaluated whether RAGE contributes to the oncogenic actions of Insulin, which plays a key role in BC progression particularly in obese and diabetic patients. Analysis of the publicly available METABRIC study, which collects gene expression and clinical data from a large cohort (n = 1904) of BC patients, revealed that RAGE and the Insulin Receptor (IR) are co-expressed and associated with negative prognostic parameters. In MCF-7, ZR75 and 4T1 BC cells, as well as in patient-derived Cancer-Associated Fibroblasts, the pharmacological inhibition of RAGE as well as its genetic depletion interfered with Insulin-induced activation of the oncogenic pathway IR/IRS1/AKT/CD1. Mechanistically, IR and RAGE directly interacted upon Insulin stimulation, as shown by in situ proximity ligation assays and coimmunoprecipitation studies. Of note, RAGE inhibition halted the activation of both IR and insulin like growth factor 1 receptor (IGF-1R), as demonstrated in MCF-7 cells KO for the IR and the IGF-1R gene via CRISPR-cas9 technology. An unbiased label-free proteomic analysis uncovered proteins and predicted pathways affected by RAGE inhibition in Insulin-stimulated BC cells. Biologically, RAGE inhibition reduced cell proliferation, migration, and patient-derived mammosphere formation triggered by Insulin. In vivo, the pharmacological inhibition of RAGE halted Insulin-induced tumor growth, without affecting blood glucose homeostasis. Together, our findings suggest that targeting RAGE may represent an appealing opportunity to blunt Insulin-induced oncogenic signaling in BC.

Estrogen Receptors and Chronic Venous Disease.

OBJECTIVE/BACKGROUND: Chronic venous disease (CVD) is a common and relevant problem affecting Western people. The role of estrogens and their receptors in the venous wall seems to support the major prevalence of CVD in women. The effects of the estrogens are mediated by three estrogen receptors (ERs): ERα, ERß, and G protein-coupled ER (GPER). The expression of ERs in the vessel walls of varicose veins is evaluated. METHODS: In this prospective study, patients of both sexes, with CVD and varicose veins undergoing open venous surgery procedures, were enrolled in order to obtain vein samples. To obtain control samples of healthy veins, patients of both sexes without CVD undergoing coronary artery bypass grafting with autologous saphenous vein were recruited (control group). Samples were processed in order to evaluate gene expression. RESULTS: Forty patients with CVD (10 men [25%], 30 women [75%], mean age 54.3 years [median 52 years, range 33-74 years]) were enrolled. Five patients without CVD (three men, two women [aged 61-73 years]) were enrolled as the control group. A significant increase of tissue expression of ERα, ERß and GPER in patients with CVD was recorded (p < .01), which was also related to the severity of venous disease. CONCLUSION: ERs seem to play a role in CVD; in this study, the expression of ERs correlated with the severity of the disease, and their expression was correlated with the clinical stage.

A Bodipy as a luminescent probe for detection of the G protein estrogen receptor (GPER).

We report the rational design, based on docking simulations, and synthesis of the first fluorescent and selective probe of GPER for bioimaging purposes and functional dissecting studies. It has been conceived as a Bodipy derivative and obtained by accessible and direct synthesis. Its optical properties have been measured in different solvents, showing insensitivity to their polarity. Its binding to GPER was achieved by competition assays with [3H]E2 and [5,6-3H] nicotinic acid in ER-negative and GPER-positive SkBr3 breast cancer cells. SkBr3 cells, transfected with a GPER expression vector containing a FLAG tag, were used to confirm that the fluorophore binds to GPER in a specific manner.

SIRT1 is involved in oncogenic signaling mediated by GPER in breast cancer.

A number of tumors exhibit an altered expression of sirtuins, including NAD+-dependent histone deacetylase silent information regulator 1 (SIRT1) that may act as a tumor suppressor or tumor promoter mainly depending on the tumor types. For instance, in breast cancer cells SIRT1 was shown to exert an essential role toward the oncogenic signaling mediated by the estrogen receptor-α (ERα). In accordance with these findings, the suppression of SIRT1 led to the inhibition of the transduction pathway triggered by ERα. As the regulation of SIRT1 has not been investigated in cancer cells lacking ER, in the present study we ascertained the expression and function of SIRT1 by estrogens in ER-negative breast cancer cells and cancer-associated fibroblasts obtained from breast cancer patients. Our results show that 17ß-estradiol (E2) and the selective ligand of GPER, namely G-1, induce the expression of SIRT1 through GPER and the subsequent activation of the EGFR/ERK/c-fos/AP-1 transduction pathway. Moreover, we demonstrate that SIRT1 is involved in the pro-survival effects elicited by E2 through GPER, like the prevention of cell cycle arrest and cell death induced by the DNA damaging agent etoposide. Interestingly, the aforementioned actions of estrogens were abolished silencing GPER or SIRT1, as well as using the SIRT1 inhibitor Sirtinol. In addition, we provide evidence regarding the involvement of SIRT1 in tumor growth stimulated by GPER ligands in breast cancer cells and xenograft models. Altogether, our data suggest that SIRT1 may be included in the transduction network activated by estrogens through GPER toward the breast cancer progression.

Multisite phosphorylation of c-Jun at threonine 91/93/95 triggers the onset of c-Jun pro-apoptotic activity in cerebellar granule neurons.

Cerebellar granule cell (CGC) apoptosis by trophic/potassium (TK) deprivation is a model of election to study the interplay of pro-apoptotic and pro-survival signaling pathways in neuronal cell death. In this model, the c-Jun N-terminal kinase (JNK) induces pro-apoptotic genes through the c-Jun/activator protein 1 (AP-1) transcription factor. On the other side, a survival pathway initiated by lithium leads to repression of pro-apoptotic c-Jun/AP-1 target genes without interfering with JNK activity. Yet, the mechanism by which lithium inhibits c-Jun activity remains to be elucidated. Here, we used this model system to study the regulation and function of site-specific c-Jun phosphorylation at the S63 and T91/T93 JNK sites in neuronal cell death. We found that TK-deprivation led to c-Jun multiphosphorylation at all three JNK sites. However, immunofluorescence analysis of c-Jun phosphorylation at single cell level revealed that the S63 site was phosphorylated in all c-Jun-expressing cells, whereas the response of T91/T93 phosphorylation was more sensitive, mirroring the switch-like apoptotic response of CGCs. Conversely, lithium prevented T91T93 phosphorylation and cell death without affecting the S63 site, suggesting that T91T93 phosphorylation triggers c-Jun pro-apoptotic activity. Accordingly, a c-Jun mutant lacking the T95 priming site for T91/93 phosphorylation protected CGCs from apoptosis, whereas it was able to induce neurite outgrowth in PC12 cells. Vice versa, a c-Jun mutant bearing aspartate substitution of T95 overwhelmed lithium-mediate protection of CGCs from TK-deprivation, validating that inhibition of T91/T93/T95 phosphorylation underlies the effect of lithium on cell death. Mass spectrometry analysis confirmed multiphosphorylation of c-Jun at T91/T93/T95 in cells. Moreover, JNK phosphorylated recombinant c-Jun at T91/T93 in a T95-dependent manner. On the basis of our results, we propose that T91/T93/T95 multiphosphorylation of c-Jun functions as a sensitivity amplifier of the JNK cascade, setting the threshold for c-Jun pro-apoptotic activity in neuronal cells.

Insulin-like growth factor-I regulates GPER expression and function in cancer cells.

Functional cross talk between insulin-like growth factor-I (IGF-I) system and estrogen signaling has been largely reported, although the underlying molecular mechanisms remain to be fully elucidated. As GPR30/GPER mediates rapid cell responses to estrogens, we evaluated the potential of IGF-I to regulate GPER expression and function in estrogen receptor (ER)α-positive breast (MCF-7) and endometrial (Ishikawa) cancer cells. We found that IGF-I transactivates the GPER promoter sequence and upregulates GPER mRNA and protein levels in both cells types. Similar data were found, at least in part, in carcinoma-associated fibroblasts. The upregulation of GPER expression by IGF-I involved the IGF-IR/PKCδ/ERK/c-fos/AP1 transduction pathway and required ERα, as ascertained by specific pharmacological inhibitors and gene-silencing. In both MCF-7 and Ishikawa cancer cells, the IGF-I-dependent cell migration required GPER and its main target gene CTGF, whereas the IGF-I-induced proliferation required both GPER and cyclin D1. Our data demonstrate that the IGF-I system regulates GPER expression and function, triggering the activation of a signaling network that leads to the migration and proliferation of cancer cells.

Recent advances in the rationale design of GPER ligands.

G-Protein Coupled Receptor (GPCR) superfamily, which comprises approximately 900 members, is the largest family of protein targets with proven therapeutic value. Although at least 500 GPCRs have been identified as therapeutically relevant, only thirteen GPCRs have been structurally characterized in apo-form or in complex with ligands. GPCRs share relatively low sequence similarity making hard the process of homology modelling, nevertheless some successful hits have been determined. Recently, the G-protein-coupled estrogen receptor 1 (GPER, formerly known as GPR30) has attracted increasing interest due to its ability in mediating estrogen signaling in different normal and cancer tissues. In this regard, the identification of selective GPER ligands has provided valuable tools in order to differentiate the specific functions elicited by this novel estrogen receptor respect to those exerted by the classical estrogen receptors (ERs). In this review, we focus on GPER examining "in silico" docking simulations and evaluating the different binding modes of diverse natural and synthetic ligands.

Two novel GPER agonists induce gene expression changes and growth effects in cancer cells.

Although the action of estrogens has been traditionally explained by the binding to and transactivation of the nuclear estrogen receptor (ER)α and ERß, recently the G protein-coupled receptor GPR30/GPER has been involved in the rapid estrogen signaling. We investigated the ability of two original molecules, which were named GPER-L1 and GPERL2, to bind to and activate the GPER transduction pathway in cancer cells. Competition assays, docking simulations, transfection experiments, real-time PCR, immunoblotting, gene silencing technology and growth assays were performed to ascertain the selective action of GPER-L1 and GPER-L2 in activating the GPER-mediated signaling. Both compounds, which did not show any ability to bind to and activate the classical ERs, were able to bind to GPER and to trigger the rapid activation of the GPER/EGFR/ERK transduction pathway which led to the up-regulation of GPER-target genes. Notably, GPER-L1 and GPER-L2 induced the proliferation of SkBr3 breast and Ishikawa endometrial cancer cells at nM concentrations through GPER, hence providing further evidence on their capability to elicit relevant biological responses mediated by GPER. The identification and characterization of these novel compounds as selective GPER agonists represent a valuable tool to further dissect the pharmacology of this novel estrogen receptor and to better differentiate the specific functions elicited by each estrogen receptor subtype in cancer cells.

Structure-based approach for the discovery of novel selective estrogen receptor modulators.

In the last twenty years the efforts to design and optimize new drugs have been based on the three dimensional structure of the selected target proteins. In this regard, useful information has been achieved mainly by protein crystallography, which has recently turned from a low into a high-throughput process thanks to the improvement in robot technologies, automation procedure and the use of synchrotron radiation facilities [1-3]. This review examines the impact of Structure Based Drug Design (SBDD) on the discovery of ligands as the selective estrogen receptor modulators (SERMs) of the Estrogen Receptor (ER)α, which is involved in the regulation of several physiological and pathological processes.

Gper and ESRs are expressed in rat round spermatids and mediate oestrogen-dependent rapid pathways modulating expression of cyclin B1 and Bax.

Spermatogenesis is a precisely controlled and timed process, comprising mitotic divisions of spermatogonia, meiotic divisions of spermatocytes, maturation and differentiation of haploid spermatids giving rise to spermatozoa. It is well known that the maintenance of spermatogenesis is controlled by gonadotrophins and testosterone, the effects of which are modulated by a complex network of locally produced factors, including oestrogens. However, it remains uncertain whether oestrogens are able to activate rapid signalling pathways directly in male germ cells. Classically, oestrogens act by binding to oestrogen receptors (ESRs) 1 and 2. Recently, it has been demonstrated that rapid oestrogen action can also be mediated by the G-protein-coupled oestrogen receptor 1 (Gper). The aim of the present study was to investigate ESRs and Gper expression in primary cultures of adult rat round spermatids (RS) and define if oestradiol (E2) is able to activate, through these receptors, pathways involved in the regulation of genes controlling rat RS apoptosis and/or maturation. In this study, we demonstrated that rat RS express ESR1, ESR2 and Gper. Short-time treatment of RS with E2, the selective Gper agonist G1 and the selective ESR1 and ERß agonists, 4,4',4"-(4-propyl-[1H]pyrazole-1,3,5-triyl) trisphenol (PPT) and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), respectively, determined activation of Extra-cellular signal-regulated kinase (ERK1/2) through the involvement of epidermal growth factor receptor transactivation. In addition, we investigated the effects of ESRs and Gper pathway activation on factors involved in RS maturation. Expression of cyclin B1 mRNA was downregulated by E2, G1 and PPT, but not by DPN. A concomitant and inverse regulation of the pro-apoptotic factor Bax mRNA expression was observed in the same conditions, with DPN being the only one determining an increase in this factor expression. Collectively, these data demonstrate that E2 activates, through ESRs and Gper, pathways involved in the regulation of genes controlling rat RS apoptosis and differentiation such as cyclin B1 and Bax.

c-Jun activation is required for 4-hydroxytamoxifen-induced cell death in breast cancer cells.

The c-Jun N-terminal kinase (JNK) has been shown to mediate tamoxifen-induced apoptosis in breast cancer cells. However, the downstream mediators of the JNK pathway linking tamoxifen to effectors of apoptosis have yet to be identified. In this study, we analysed whether c-Jun, the major nuclear target of JNK, has a role in tamoxifen-induced apoptosis of SkBr3 breast cancer cells. We show that before DNA fragmentation and caspase 3/7 activation, cytotoxic concentrations of 4-hydroxytamoxifen (OHT) induced JNK-dependent phosphorylation of c-Jun at JNK sites earlier shown to regulate c-Jun-mediated apoptosis. In addition, OHT induced ERK-dependent expression of c-Fos and transactivation of an AP-1-responsive promoter. In particular, the ectopic expression of dominant-negative constructs blocking either AP-1 activity or c-Jun N-terminal phosphorylation prevented DNA fragmentation after OHT treatment. Furthermore, both c-Fos expression and c-Jun N-terminal phosphorylation preceded OHT-dependent activation of caspase 3-7 in different types of tamoxifen-sensitive cancer cells, but not in OHT-resistant LNCaP prostate cancer cells. Taken together, our results indicate that the c-Jun/c-Fos AP-1 complex has a pro-apoptotic role in OHT-treated cancer cells and suggest that pharmacological boosts of c-Jun activation may be useful in a combination therapy setting to sensitize cancer cells to tamoxifen-mediated cell death.

Evidence that bergapten, independently of its photoactivation, enhances p53 gene expression and induces apoptosis in human breast cancer cells.

Psoralens (5-MOP and 8-MOP), a class of naturally occurring compounds, in combination with ultraviolect light are potent modulators of epidermal cell growth and differentiation. For a long time, photo-chemotherapy has been used in the treatment of psoriasis where it can reduce the number of cycling keratinocytes and decrease the IGF-1 receptors. However, the molecular mechanism of PUVA therapy remains unclear. In this study, we have evaluated, for the first time, in MCF-7 and SKBR-3 breast cancer cells the effects of 5-MOP (Bergapten), independently of its photoactivation, on the signalling pathways involved in cell cycle arrest and in apoptosis. Drug treatment induced a block in the G0/G1 phase and increased mRNA and protein levels of p53 and p21waf. These data correlate with a functional activation of caspase 8/caspase 9 together with DAPI staining and DNA ladder. Bergapten can transactivate p53 gene promoter in these cells and site-direct mutagenesis studies showed that the binding sequence of the nuclear factor NF-Y on p53 promoter is required for 5-MOP responsiveness. Besides, Bergapten increases NF-Y nuclear translocation through p38 MAPK activation. The same treatment impairs the PI3Kinase/AKT survival signal, in hormone-dependent MCF-7 cells even in the presence of IGF-I/E2 mitogenic factors. Here, we demonstrated that Bergapten, independently on the exposure to UV, generates membrane signalling pathways able to address apoptotic responses in breast cancer cells and to counteract the stimulatory effect of IGF-I/E2 on estrogen-receptor positive MCF-7 cell growth and progression.

A new membrane G protein-coupled receptor (GPR30) is involved in the cardiac effects of 17beta-estradiol in the male rat.

In the present study, we evaluated the transduction pathways involved in the cardiac effects elicited by 17beta-estradiol (E2) on the isolated, Langendorff perfused male Wistar rat heart. E2 and selective agonists for ERalpha and ERbeta induced a dose-dependent reduction of contractility which was blocked by the ER inhibitor ICI 182,780. Moreover, the potential involvement of the novel membrane estrogen receptor GPR30 in mediating estrogen activity was determined using the selective GPR30 ligand G-1. Notably, specific inhibitors of ERK, PI3K, PKA, and eNOS transduction pathways abolished the cardiac responses to E(2). Taken together, our data suggest that ERalpha and ERbeta along with several signaling cascades are involved in the action of E(2) on the male rat heart. Our results also point to a potential role of GPR30, however further evaluation is required in order to fully understand the contribution of the different estrogen receptors in mediating estrogen activity on cardiac performance.

Antiestrogens upregulate estrogen receptor beta expression and inhibit adrenocortical H295R cell proliferation.

The molecular mechanisms involved in adrenocortical tumorigenesis are still not completely understood. In this study, using the H295R cell line as a model system, we investigated the role of estrogens and estrogen receptor (ER) alpha and ER beta in the growth regulation of adrenocortical tumors. We demonstrated that H295R cells are able to convert androgens to estrogens by a constitutive expression of active cytochrome P450 aromatase protein and express ER beta to a greater extent than ER alpha. Moreover, physiological concentrations of 17beta-estradiol (E2) determined an increase of thymidine incorporation, suggesting the presence of an autocrine mechanism in maintaining H295R cell proliferation. Evaluating the response to ER antagonists like 4-hydroxytamoxifen (OHT) and ICI 182 780 (ICI), we observed an up-regulation of ER beta and a dose-dependent inhibition of H295R cell proliferation. Whereas ICI determined the growth arrest of H295R cells, OHT induced morphological changes that were characteristic of apoptosis. According to the above-mentioned observations, OHT but not ICI clearly induced a marked expression of FasL and the cleavage of both caspase-8 and caspase-3. Interestingly, the apoptotic effects of OHT in H295R cells may be consequent to the enhanced levels of ER beta which stimulate the expression of FasL interacting with activating protein (AP)-1 sites located within its promoter sequence. In conclusion, we have demonstrated that H295R cells are able to transform androgens to estrogens that activate an autocrine mechanism, mediated by their own receptors, and contribute to regulate the proliferation of these cells. Moreover, this study points towards a role for ER beta as an important mediator of the repressive effects exerted by antiestrogens on H295R cells; however, further studies are needed to clarify its role in the control of adrenocortical cell proliferation and on the potential benefits of antiestrogens for treatment of adrenocortical cancer.

The red wine phenolics piceatannol and myricetin act as agonists for estrogen receptor alpha in human breast cancer cells.

Previous epidemiological reports have suggested that red wine intake is associated with beneficial health effects due to the ability of certain phytochemical components to exert estrogen-like activity. It has been also documented that estrogens induce the proliferation of hormone-dependent breast cancer cells by binding to and transactivating estrogen receptor (ER) alpha, which in turn interacts with responsive DNA sequences located within the promoter region of target genes. In order to provide further insight into the positive association between wine consumption and the incidence of breast carcinoma in postmenopausal women, we have evaluated the estrogenic properties of two abundant wine-derived compounds, named piceatannol (PIC) and myricetin (MYR), using as model systems the hormone-sensitive MCF7 and the endocrine-independent SKBR3 breast cancer cells. On the basis of our experimental evidence PIC and MYR may contribute to the estrogenicity of red wine since: (1) they transactivate endogenous ER alpha; (2) they activate the agonist-dependent activation function (AF) 2 of ER alpha and ER beta in the context of the Gal4 chimeric proteins; (3) they rapidly induce the nuclear immunodetection of ER alpha; (4) they regulate the expression of diverse estrogen target genes; (5) they compete with 17beta-estradiol for binding to ER alpha and ER beta; and--as a biological counterpart of the aforementioned abilities--(6) they exert stimulatory effects on the proliferation of MCF7 cells. Hence, the estrogenic activity of PIC and MYR might be considered at least as a potential factor in the association of red wine intake and breast tumors, particularly in postmenopausal women.

Immunolocalization of estrogen receptor beta in the epididymis of mature and immature pigs.

A growing body of evidence suggests a role of estrogens in the male reproduction via their specific estrogen receptors (ERalpha/ERbeta). Estrogen receptor distribution along the genital tract tissues has been described in different species, but it is unknown in the pig. Therefore, the aim of the present study was to localize ERbeta in the epididymis of mature and immature pigs (aged 2 and 18 months, respectively). Immunohistochemistry was carried out on paraffin-embedded tissues using a mouse anti-human monoclonal IgG against ERbeta as the primary antibody, and a goat anti-mouse biotinylated IgG as the secondary antibody. Avidin-biotin-peroxidase complex was then applied followed by diaminobenzidine. In immature pigs, the epithelial cells from the caput, corpus and cauda epididymis showed no or very weak immunoreactivity for ERbeta, whereas they were all strongly immmunoreactive in mature pigs. A various intensity of immunostaining from weak to strong in the smooth muscle cells as well as in the connective tissue cells were detected in the epididymis of both, young and adult pigs. This is the first report on the cellular localization of ERbeta protein in porcine epidydimis. The present study demonstrated that (1) irrespectively of the epididymal region, the epithelial cells of caput, corpus and cauda epididymis of mature pigs revealed a strong immunoreactivity for ERbeta, and (2) ERbeta expression in the epididymal epithelium is regulated by puberty. Finally, although the biological activity of ERbeta has not yet been established, the results of the present study suggest its involvement in estrogen modulation of pig epididymal function.

Xenoestrogens and the induction of proliferative effects in breast cancer cells via direct activation of oestrogen receptor alpha.

Environmental contamination with a variety of industrial products has been associated with developmental and reproductive abnormalities in wildlife species. Increasing evidence has suggested that bisphenol A (BPA) and 4-nonylphenol (NPH), two major endocrine-disrupting chemicals, might be responsible for adverse effects on humans as a consequence of ubiquitous use together with potential oestrogen-like activity. To provide insight into the oestrogen-like nature of BPA and NPH, their ability to activate a reporter gene construct via an oestrogen response element in the hormone-dependent breast cancer cell lines MCF7 and T47D was ascertained. Both compounds transactivated the endogenous oestrogen receptor (ER) alpha in a direct fashion since the anti-oestrogen 4-hydroxytamoxifen abolished the response. In addition, using steroid-receptor-negative HeLa cells engineered to express ERalpha and ERbeta and the hormone-binding domains of both ERalpha and ERbeta, BPA and NPH confirmed the direct transcriptional activity. Interestingly these properties were supported in MCF7 cells by the ability to autoregulate ERalpha expression as well as to induce its nuclear compartmentalization. We therefore evaluated by reverse transcriptase polymerase chain reaction the expression of oestrogen-controlled genes such as cathepsin D and TFF1 (formerly pS2), which were increased by both chemicals tested. The agonistic effects exhibited in all assays performed prompted the evaluation of a more complex biological response such as the proliferation of MCF7 and T47D cells. The same concentration of xenoestrogens eliciting substantial transcriptional activity significantly stimulated the proliferation of both breast cancer cell lines, although with a reduced effectiveness with respect to the natural hormone 17beta-oestradiol. The results indicate that the biological action of environmental oestrogen such as BPA and NPH should be taken into account for the potential impact on human disease-like hormone-dependent breast cancer. However, further studies are needed to clarify their bioavailability and metabolism as well as whether compound mixtures could produce noticeable effects by synergistic activity.

Breast cancer: from estrogen to androgen receptor.

To investigate the link existing between androgens and human breast cancer, the hormonal milieu present in pre- and post-menopausal women has been translated in an in vitro model utilizing a hormone dependent breast cancer cell line MCF-7 exposed to DHEA, DHEAS, androstenediol, T, DHT with or w/o E(2). DHEAS and androstenediol stimulate the growth of MCF-7 cell line but reduce cell proliferation induced by E(2) (1 nM). T and DHT (1-100 nM) instead inhibit MCF-7 cell proliferation independently on E(2) presence. When we focused our study on the most powerful androgen, DHT alone (100 nM) consistently inhibits MCF-7 cell proliferation by 50% of the basal growth rate and counteracts E(2) proliferative action by 68%. These data correlate well with cell cycle analysis showing an enhanced number of cells in G(0)/G(1) phase after 6 days of DHT treatment. Upon prolonged DHT exposure, Western blotting analysis shows a markedly increased AR content, while immunohistochemistry indicates that it was mostly translocated into the nucleus. So we assumed that the enhanced activation of the AR might inhibit MCF-7 cells proliferation. This assumption is corroborated by the fact that the inhibitory effects induced by DHT on MCF-7 cell proliferation are abrogated in the presence of hydroxyflutamide. Therefore to better investigate the role of AR in inhibiting E(2) action at genomic level, MCF-7 cells were transiently cotransfected with the reporter plasmid XETL carrying firefly luciferase sequence under the control of an estrogen responsive element and the full length AR or with an AR carrying a mutation (Cis 574-->Arg 574) which abolishes its binding to DNA. The over-expression of the AR markedly decreases E(2) signalling which furthermore appears inhibited by simultaneous exposure to DHT but reversed by addition of hydroxyflutamide. The inhibitory effect was no longer noticeable when MCF-7 cells were cotransfected with XETL and the mutant AR. Taken together these data demonstrate that gonadal androgens antagonize MCF-7 proliferation induced by E(2). This seems to be related to the inhibitory effects of the over-expressed AR on E(2) genomic action.

The direct proliferative stimulus of dehydroepiandrosterone on MCF7 breast cancer cells is potentiated by overexpression of aromatase.

In women after menopause aromatization of adrenal androgens represents the main source of estrogens, which may promote the development of hormone-dependent breast tumor. Several studies have attempted to determine the cell type within carcinomas that is responsible for 'in situ' estrogen biosynthesis and whether the amount produced may sustain relevant biological effects. Here we show P450arom mRNA and protein expression together with immunocytochemical localization of aromatase in the epithelial MCF7 breast cancer cell line. Moreover, we demonstrate that the enhanced aromatization of dehydroepiandrosterone in aromatase transfected MCF7 cells confers biological advantages such as proliferative stimulation similar to that induced by estradiol. Our results suggest that aromatase inhibitors may be particularly effective in the treatment of hormone-dependent breast cancer disease in postmenopausal women.