Rosa Sirianni

Role of estrogen receptors and g protein-coupled estrogen receptor in regulation of hypothalamus-pituitary-testis axis and spermatogenesis.

Male reproductive function is under the control of both gonadotropins and androgens through a negative feedback loop that involves the hypothalamus, pituitary, and testis known as hypothalamus–pituitary–gonadal axis (HPG). Indeed, estrogens also play an important role in regulating HPG axis but the study on relative contribution to the inhibition of gonadotropins secretion exerted by the amount of estrogens produced within the hypothalamus and/or the pituitary or by the amount of circulating estrogens is still ongoing. Moreover, it is known that the maintenance of spermatogenesis is controlled by gonadotropins and testosterone, the effects of which are modulated by a complex network of locally produced factors, including estrogens. Physiological effects of estrogens are mediated by the classical nuclear estrogen receptor alpha and estrogen receptor beta, which mediate both genomic and rapid signaling events. In addition, estrogens induce rapid non-genomic responses through a membrane-associated G protein-coupled estrogen receptor (GPER). Ours and other studies reported that, in the testis, GPER is expressed in both normal germ cells and somatic cells and it is involved in mediating the estrogen action in spermatogenesis controlling proliferative and/or apoptotic events. Interestingly, GPER expression has been revealed also in the hypothalamus and pituitary. However, its role in mediating estrogen rapid actions in this context is under investigation. Recent studies indicate that GPER is involved in modulating gonadotropin-releasing hormone (GnRH) release as well as gonadotropins secretion. In this review, we will summarize the current knowledge concerning the role of estrogen/estrogen receptors molecular pathways in regulating GnRH, follicle-stimulating hormone, and luteinizing hormone release at the hypothalamic and pituitary levels in males as well as in controlling specific testicular functions such as spermatogenesis, focusing our attention mainly on estrogen signaling mediated by GPER.

Oleuropein and hydroxytyrosol inhibit MCF-7 breast cancer cell proliferation interfering with ERK1/2 activation.

The growth of many breast tumors is stimulated by estradiol (E2), which activates a classic mechanism of regulation of gene expression and signal transduction pathways inducing cell proliferation. Polyphenols of natural origin with chemical similarity to estrogen have been shown to interfere with tumor cell proliferation. The aim of this study was to investigate whether hydroxytyrosol (HT) and oleuropein (OL), two polyphenols contained in extra-virgin olive oil, can affect breast cancer cell proliferation interfering with E2-induced molecular mechanisms. Both HT and OL inhibited proliferation of MCF-7 breast cancer cells. Luciferase gene reporter experiments, using a construct containing estrogen responsive elements able to bind estrogen receptor alpha (ERalpha) and the study of the effects of HT or OL on ERalpha expression, demonstrated that HT and OL are not involved in ERalpha-mediated regulation of gene expression. However, further experiments pointed out that both OL and HT determined a clear inhibition of E2-dependent activation of extracellular regulated kinase1/2 belonging to the mitogen activating protein kinase family. Our study demonstrated that HT and OL can have a chemo-preventive role in breast cancer cell proliferation through the inhibition of estrogen-dependent rapid signals involved in uncontrolled tumor cell growth.

17β-Estradiol activates rapid signaling pathways involved in rat pachytene spermatocytes apoptosis through GPR30 and ERα

Aim of the present study was to investigate whether estrogens were able to directly activate rapid signaling pathways controlling spermatogenesis in rat pachytene spermatocytes (PS). Classically, estrogens act by binding to estrogen receptors (ERs) alpha and beta. Recently, it has been demonstrated that rapid estrogen action can also be activated through the G-protein-coupled receptor (GPR)-30. Herein, we demonstrated that rat PS express ER alpha, ER beta and GPR30. Treatment of PS with estradiol (E2), the selective GPR30 agonist G1 and the selective ER alpha agonist PPT determined activation of ERK1/2 which are part of GPR30 signaling cascade. ERK1/2 activation in response to E2 and G1 was correlated to an increased phosphorylation of c-Jun. All treatments failed to induce these responses in the presence of EGFR inhibitor AG1478, ERK inhibitor PD98059 and ER inhibitor ICI182780. mRNA expression of cell cycle regulators cyclin A1 and B1 was downregulated by E2 and G1 while an up-regulation of proapoptotic factor Bax was observed in the same conditions. These data demonstrate that E2, working through both ER alpha and/or GPR30, activates in PS the rapid EGFR/ERK/c-Jun pathway, modulating the expression of genes involved in the balance between cellular proliferation and apoptosis.

Potential of olive oil phenols as chemopreventive and therapeutic agents against cancer: A review of in vitro studies

Olive oil is a common component of Mediterranean dietary habits. Epidemiological studies have shown how the incidence of various diseases, including certain cancers, is relatively low in the Mediterranean basin compared to that of other European or North America countries. Current knowledge indicates that the phenolic fraction of olive oil has antitumor effects. In addition to the ability to be chemopreventive, with its high antioxidant activity, the antitumor effects of olive oil phenols (OO-phenols) has been studied because of their capacity to inhibit proliferation and promote apoptosis in several tumor cell lines, by diverse mechanisms. This review will summarize and discuss the most recent relevant results on the antitumor effect of OO-phenols on leukemia tumor cells, colorectal carcinoma cells, and breast cancer (BC) cells. In particular, very recent data will be reported and discussed showing the molecular signaling pathways activated by OO-phenols in different histopathological BC cell types, suggesting the potential use of OO-phenols as adjuvant treatment against several subsets of BC. Data summarized here represent a good starting point for more extensive studies for better insight into the molecular mechanisms induced by OO-phenols and to increase the availability of chemopreventive or therapeutic drugs to fight cancer.

Insulin-Like Growth Factor-I, Regulating Aromatase Expression through Steroidogenic Factor 1, Supports Estrogen-Dependent Tumor Leydig Cell Proliferation

The aim of this study was to investigate the role of estrogens in Leydig cell tumor proliferation. We used R2C rat Leydig tumor cells and testicular samples from Fischer rats with a developed Leydig tumor. Both experimental models express high levels of aromatase and estrogen receptor alpha (ERalpha). Treatment with exogenous 17beta-estradiol (E(2)) induced proliferation of R2C cells and up-regulation of cell cycle regulators cyclin D1 and cyclin E, the expression of which was blocked by addition of antiestrogens. These observations led us to hypothesize an E(2)/ERalpha-dependent mechanism for Leydig cell tumor proliferation. In determining the molecular mechanism responsible for aromatase overexpression, we found that total and phosphorylated levels of transcription factors cyclic AMP-responsive element binding protein and steroidogenic factor 1 (SF-1) were higher in tumor samples. Moreover, we found that tumor Leydig cells produce high levels of insulin-like growth factor I (IGF-I), which increased aromatase mRNA, protein, and activity as a consequence of increased total and phosphorylated SF-1 levels. Specific inhibitors of IGF-I receptor, protein kinase C, and phosphatidylinositol 3-kinase determined a reduction in SF-1 expression and in IGF-I-dependent SF-1 recruitment to the aromatase PII promoter. The same inhibitors also inhibited aromatase expression and activity and, consequently, R2C cell proliferation. We can conclude that one of the molecular mechanisms determining Leydig cell tumorigenesis is an excessive estrogen production that stimulates a short autocrine loop determining cell proliferation. In addition, cell-produced IGF-I amplifies estrogen signaling through an SF-1-dependent up-regulation of aromatase expression. The identification of this molecular mechanism will be helpful in defining new therapeutic approaches for Leydig cell tumors.

Aromatase expression in prepuberal Sertoli cells: effect of thyroid hormone.

Aromatase activity has recently been assumed as a Sertoli cell functional maturation marker since it is maximally expressed in prepuberal age then it dramatically decreases at puberty and is virtually absent in adult age. Neonatal hypothyroidism is associated with a prolonged proliferation of Sertoli cells. This immature stage persists concomitantly with a dramatic enhancement of aromatase activity reversed by triiodothyronine (T3) either in vivo or in vitro administration. Therefore, in the present study, after immunolocalisation of aromatase in the cytoplasm of cultured Sertoli cells as well as in testis section, we investigate the regulatory effects of T3 in the same cells just at the age when aromatase activity is reported to be maximally expressed. In this aim, the effects of thyroid hormone have been evaluated in 2-weeks-old rats, in basal condition and upon stimulation with dibutyryl cyclic AMP [(Bu)(2)cAMP] by simultaneously analysing three functional levels of aromatase, mRNA expression; protein content; enzymatic activity. Western-blot analysis of Sertoli cell extracts revealed a protein, which co-migrated with a 55 kDa protein detected in human placenta used a positive control. The presence of a functional P450 aromatase protein in purified Sertoli cells was confirmed by the measurement [3H]H(2)O released after incubation with [1beta-(3)H]androst-4-3,17-dione. At the dose used, T3 down-regulates basal aromatase activity, while aromatase mRNA expression was apparently not inhibited. It is noteworthy that aromatase content pattern evaluated by Western blot analysis did not tightly parallel the aromatase activity pattern which clearly displays the inhibitory effects of T3, in basal condition ad upon (Bu)(2)cAMP stimulation, simulating FSH stimulation. The detection of mRNA altered transcript coding for putative protein lacking both aromatic and heme-binding regions upon T3 treatment and unable to convert androgens into estrogens, provides a reasonable explanation for the observed discrepancies between aromatase protein pattern, P450arom mRNA levels and aromatase activity. The authors conclude that although the altered transcript induced by prolonged exposure to T3 is a mechanism by which T3 may down regulate aromatase activity, it cannot be ruled out a direct effect of this hormone at the transcription levels since a recognisable emisite for potential TR(s) binding is located in the promoter region of aromatase gene. Thus a further investigation on T3 modulator role on aromatase gene promoter should be pursued even utilising higher doses of T3.

Oleuropein and hydroxytyrosol activate GPER/ GPR30‐dependent pathways leading to apoptosis of ER‐negative SKBR3 breast cancer cells

SCOPE We have previously demonstrated that oleuropein (OL) and hydroxytyrosol (HT) reduce 17β-estradiol-mediated proliferation in MCF-7 breast cancer (BC) cells without affecting the classical genomic action of estrogen receptor (ER), but activating instead the ERK1/2 pathway. Here, we hypothesized that this inhibition could be mediated by a G-protein-coupled receptor named GPER/GPR30. Using the ER-negative and GPER-positive SKBR3 BC cells as experimental model, we investigated the effects of OL and HT on GPER-mediated activation of downstream pathways. METHODS AND RESULTS Docking simulations and ligand-binding studies evidenced that OL and HT are able to bind GPER. MTT cell proliferation assays revealed that both phenols reduced SKBR3 cell growth; this effect was abolished silencing GPER. Focusing on OL and HT GPER-mediated pathways, using Western blot analysis we showed a sustained ERK1/2 activation triggering an intrinsic apoptotic pathway. CONCLUSION Showing that OL and HT work as GPER inverse agonists in ER-negative and GPER-positive SKBR3 BC cells, we provide novel insights into the potential of these two molecules as tools in the therapy of this subtype of BC.

Inhibition of Cyclooxygenase-2 Down-regulates Aromatase Activity and Decreases Proliferation of Leydig Tumor Cells

Our recent studies have revealed that estrogens stimulate an autocrine mechanism determining Leydig tumor cell proliferation. Estrogen overproduction is due to an elevated steroidogenic factor-1 (SF-1) expression and cAMP-response element-binding protein (CREB) phosphorylation, both inducing aromatase overexpression. Although we have shown that increased SF-1 expression depends mainly on higher local insulin-like growth factor I production, the mechanisms and factors determining increased CREB activation in Leydig tumor cells are not completely understood. In this study, we investigated the role of cyclooxygenase-2 (COX-2) in CREB dependent-aromatase expression in Leydig tumor cells. We found that COX-2 is expressed in rat and human Leydigiomas as well as in the rat Leydig tumor cell line R2C, but not in normal testis. Our data indicate that in R2C cells the COX-2-derived prostaglandin E2 (PGE2) binds the PGE2 receptor EP4 and activates protein kinase A (PKA) and ultimately CREB. Inhibitors for COX-2 (NS398), EP4 (AH23848), and PKA (H89) decreased aromatase expression and activity as a consequence of a decreased phosphorylated CREB recruitment to the PII promoter of the aromatase gene. The COX-2/PGE2/PKA pathway also seems to be involved in aromatase post-translational activation, an observation that requires further studies. The reduction in aromatase activity was responsible for a drop in estrogen production and subsequent reduction in cyclin E expression resulting in a decrease in tumor Leydig cell proliferation. Furthermore, COX-2 silencing caused a significant decrease in CREB phosphorylation, aromatase expression, and R2C cell proliferation. These novel findings clarify the mechanisms involved in the growth of Leydig cell tumors and should be taken into account in determining new therapeutic approaches.

Synthesis and cytotoxic activity evaluation of 2,3-thiazolidin-4-one derivatives on human breast cancer cell lines

It is well known that resveratrol (RSV) displayed cancer-preventing and anticancer properties but its clinical application is limited because of a low bioavailability and a rapid clearance from the circulation. Aim of this work was to synthesize pharmacologically active resveratrol analogs with an enhanced structural rigidity and bioavailability. In particular, we have synthesized a library of 2,3-thiazolidin-4-one derivatives in which a thiazolidinone nucleus connects two aromatic rings. Some of these compounds showed strong inhibitory effects on breast cancer cell growth. Our results indicate that some of thiazolidin-based resveratrol derivatives may become a new potent alternative tool for the treatment of human breast cancer.

17β-Estradiol activates GPER- and ESR1-dependent pathways inducing apoptosis in GC-2 cells, a mouse spermatocyte-derived cell line

In mammals, spontaneous apoptosis is observed particularly in differentiating spermatogonia and in spermatocytes. 17β-Estradiol (E2) in primary rat pachytene spermatocytes (PS) binds estrogen receptor α (ESR1) and GPER to activate EGFR/ERK/c-Jun pathway leading to up regulation of proapoptotic factor bax. Aim of this study was to clarify the effector pathway(s) controlling spermatocytes apoptosis using as model GC-2 cells, an immortalized mouse pachytene spermatocyte-derived cell line, which reproduces primary cells responses to E2. In fact, in GC-2 cells we observed that ESR1 and GPER activation caused rapid ERK and c-Jun phosphorylation, bax up-regulation, events associated with apoptosis. We further investigated the apoptotic mechanism demonstrating that E2, as well as ESR1 and GPER specific agonists, induced sustained ERK, c-Jun and p38 phosphorylation, Cytochrome c release, caspase 3 and endogenous substrate Poly (ADP-ribose) polymerase (PARP) activation and increased expression of cell cycle inhibitor p21. When ESR1 or GPER expression was silenced, E2 was still able to decrease cell proliferation, only the concomitant silencing abolished E2 effect. These results indicate that GC-2 cells are a valid cell model to study E2-dependent apoptosis in spermatocytes and show that E2, activating both ESR1 and GPER, is able to induce an ERK1/2, c-Jun and p38-dependent mitochondrion apoptotic pathway in this cell type.