Tesi di Dottorato
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Item <> ruolo del GPER nelle arterie uterine e mesenteriche in gravidanza(2015-12-16) Tropea, Teresa; Canonaco, Marcello; Mandalà, MaurizioLow uteroplacental blood flow has been implicated as a cause of pregnancy hypertension and intrauterine fetal growth retardation. A successful pregnancy outcome requires a sufficient uteroplacental blood flow, which increases of many fold compared to the non pregnant levels. This significant increase is achieved in part by vasodilation of the uterine vasculature because pregnancy induces a physiological remodelling of the maternal uterine circulation. Estrogen may contribute to this effect although the mechanisms involved remain unclear. It is known that estrogen receptor-α and estrogen receptor-β in the uterine vascular endothelium are both functionally implicated in the regulation of the uterine blood flow but so far nothing is known about the novel G-protein coupled estrogen receptor (GPER) in the uterine vasculature. GPER has been identified in many vascular bed exerting its potential vasorelaxing effect. The aim of this study was to investigate the function of GPER in the regulation of the uterine vascular tone during pregnancy. Experiments were carried out on mesenteric arterioles and uterine radial arteries isolated from both non-pregnant (NP) and age-matched pregnant (P) Sprague Dawley rats to compare reproductive and systemic vasculature effect. Arterial segments were pressurized to 50 mmHg in the pressure myography, preconstricted of 40% with phenylephrine and then incubated with incremental doses (10-12-10-6 M) of the specific GPER agonist, G-1. A dose-response curve was obtained for G-1 in the 1 pM – 1 μM range with a maximum vasodilation of uterine arteries of 97,8 ± 2,5% in P vs 66,5 ± 3,7% in NP, p<0.001. G-1 vasorelaxing effect was similar in pregnant (45,5 ± 6,1%) and non-pregnant (53,6 ± 2,3%) mesenteric arteries. Pregnancy induced a significantly higher G-1 vasodilation only in uterine artery and its protein expression was also increased. G-1 effect was significantly reduced by the GPER antagonist, G-15. The NOS inhibitors, L-NAME+L-NNA or endothelium removal reduced the G-1 induced relaxation of uterine artery, suggesting an endothelium-dependent mechanism, involving cGMP pathway but not BKca channels. Immunohistochemistry revealed the GPER expression in the main uterine artery of both eNOS-/- and wild type pregnant mice. This receptor was functionally activated in a dose response manner with a significant less effect in knock out mice, showing the relevance of the NO in GPER pathway. GPER is also present in human chorionic plate arteries and its activation induces a slight vasodilation. These results demonstrate for the first time that GPER may have a role in regulating vascular tone, placental perfusion and normal fetal development, suggesting a potential therapeutic target in pregnant diseases.Item Estrogen Receptor beta, through Sp1, recruits a corepressor complex to the Estrogen Receptor alpha gene promoter in breast cancer cells(2012) Zito, Domenico; Sisci, Diego; Andò, SebastianoHuman Estrogen receptors alpha (α) and beta (β) are essential components of a complex signal transduction pathway that regulates mammary growth and development. Several studies have reported that normal breast tissues display a relative higher expression of ERβ than ERα, which drastically changes during breast tumorogenesis. Thus, it is reasonable to suggest that a dysregulation of the two estrogen receptor subtypes may induce breast cancer development. However, the molecular mechanisms involved in the potential opposing roles played by the two estrogen receptors on tumor cell growth still needs to be elucidated. In the present study, we have demonstrated that ERβ overexpression in breast cancer cells decreases cell proliferation and down-regulates ERα mRNA and protein content, with a concomitant repression of estrogen-regulated genes. Transient transfection experiments, using a vector containing the human ERα promoter region, showed that elevated levels of ERβ down-regulated basal ERα promoter activity. Furthermore, site-directed mutagenesis and deletion analysis revealed that the proximal GC-rich motifs at −223 and -214 are critical for the ERβ-induced ERα down-regulation in breast cancer cells. This occurred through ERβ-Sp1 proteinprotein interactions within the ERα promoter region and the recruitment of a corepressor complex containing the nuclear receptor corepressor NCoR, accompanied by hypoacetylation of histone H4 and displacement of RNA polymerase II. Silencing of NCoR gene expression by RNA interference reversed the down-regulatory effects of ERβ on ER α gene expression and cell proliferation Collectively, these results suggest a novel mechanism by which overexpression of ERβ, through NCoR, is able to down regulate ERα expression, thus repressing ERα’s main role on breast cancer cell growthItem DAX-1 at the connection between Androgen Receptor and Aromatase: a novel mechanism in the inhibition of estrogen-dependent cancer cell proliferation(2012) Maris, Pamela; Sisci, Diego; Lanzino, MarilenaItem PPARγ ligands as novel agents able to inhibit breast tumor growth and progression(2011) Vizza, Donatella; Sisci, Diego; Bonofiglio, DanielaItem Molecular Mechanisms of FXR-mediated Growth Inhibition in Hormone-dependent Cancers(2011) Panza, Salvatore; Sisci, Diego; Catalano, StefaniaItem <> estrogen receptor α is the key regulator of the bifunctional role of FoxO3a transcription factor in breast cancer motility and invasiveness(2013-12-02) Cesario, Maria Grazia; Sisci, DiegoThe role of the Forkhead box class O (FoxO)3a transcription factor in breast cancer migration and invasion is controversial. Here we show that FoxO3a overexpression decreases motility, invasiveness, and anchorage-independent growth in estrogen receptor α-positive (ERα+) cancer cells while eliciting opposite effects in ERα-silenced cells and in ERα-negative (ERα−) cell lines, demonstrating that the nuclear receptor represents a crucial switch in FoxO3a control of breast cancer cell aggressiveness. In ERα+ cells, FoxO3a-mediated events were paralleled by a significant induction of Caveolin-1 (Cav1), an essential constituent of caveolae negatively associated to tumor invasion and metastasis. Cav1 induction occurs at the transcriptional level through FoxO3a binding to a Forkhead responsive core sequence located at position −305/−299 of the Cav1 promoter. 17β-estradiol (E2) strongly emphasized FoxO3a effects on cell migration and invasion, while ERα and Cav1 silencing were able to reverse them, demonstrating that both proteins are pivotal mediators of these FoxO3a controlled processes. In vivo, an immunohistochemical analysis on tissue sections from patients with ERα+ or ERα− invasive breast cancers or in situ ductal carcinoma showed that nuclear FoxO3a inversely (ERα+) or directly (ERα−) correlated with the invasive phenotype of breast tumors. In conclusion, FoxO3a role in breast cancer motility and invasion depends on ERα status, disclosing a novel aspect of the well-established FoxO3a/ERα interplay. Therefore FoxO3a might become a pursuable target to be suitably exploited in combination therapies either in ERα+ or ERα− breast tumors.Item Different medicinal chemistry approaches towards the identification of novel targets in breast cancer(2013-12-02) Pisano, Assunta; Bartolino, Roberto; Gabriele, Bartolo; Sindona, Giovanni; Maggiolini, MarcelloG protein-coupled receptors (GPCRs) belong to the largest family of cellsurface molecules representing the targets of approximately 40% of current medicinal drugs (Overington, J.P et al 2006). GPCRs are ubiquitous in mammalian (Bockaert, J. et al. 1999), regulate several physiological processes and play an important role in multiple diseases ranging from cardiovascular dysfunction, depression, pain, obesity to cancer (Rosenbaum D.M. et al. 2011). One member of this superfamily, named GPR30/GPER, mediates estrogen signaling in different cell contexts, leading to gene expression changes and relevant biological responses (Filardo E.J et al. 2000, Bologa C.G.et al. 2006, Maggiolini M. and Picard D. 2010). GPER acts by transactivating the Epidermal Growth Factor Receptor (EGFR), which thereafter induces the increase of intracellular cyclic AMP (cAMP), calcium mobilization and the activation of the phosphatidylinositol 3-kinase (PI3K) and the mitogen-activated protein kinases (MAPKs) (Maggiolini M. and Picard D. 2010). Moreover, the GPER-mediated transduction pathways activated by estrogens trigger the expression of a typical gene signature, including the expression of cfos and the gene encoding the connective tissue growth factor (CTGF), which are involved in the proliferation and migration of diverse cell types (Lappano R. et al 2012a, Madeo A. and Maggiolini M. 2010). On the basis of these findings, the first objective of the present study was the characterization of GPER from different points of view: GPERmediated signaling pathways and biological functions, selective ligands and molecular characterization of the receptors. In particular, the research project focused on:1. the transduction pathways by which the environmental contaminant Bisphenol A (BPA) influences cell proliferation and migration of human breast cancer cells and cancer-associated fibroblasts (CAFs); 2. the characterization of novel carbazole derivatives as GPER agonists in ER-negative breast cancer cells; 3. the isolation and characterization of GPER in estrogen-sensitive cancer cells by Mass Spectrometry. Additionally, the second section of this doctoral thesis was focused on the evaluation of the cytotoxic activity of novel synthesized compounds, given the interest and the need to discover new molecules against cancer. In particular, novel titanocene-complexes were studied evaluating their ability to elicit repressive effects on the growth of estrogen-sensitive breast cancer cells.Item Synthesis and biological evaluation of polyheterocyclic derivatives endowed with antiviral or anticancer activity(2006-11-10) Grande, Fedora; Garofalo, Antonio; Sindona, GiovanniItem Inhibition of estrogen-dependent cyclin D1 expression by androgen receptor in MCF-7 breast cancer cell(2007-11-30) Casaburi, Ivan; Andò, SebastianoItem Ruolo del sistema endocannabinoide nei meccanismi di neuroprotezione da 17β-estradiolo in un modello sperimentale di ischemia cerebrale focale(2013-10-28) Mazzei,Cinzia; Bagetta,GiacintoIn questo studio sono stati determinati i livelli endogeni dell’endocannabinoide finora meglio caratterizzato, anandamide (AEA) e l’attività degli enzimi responsabili, rispettivamente, della sua sintesi e degradazione, la NAPE-PLD e la FAAH, nella corteccia e nello striato di ratti sottoposti ad occlusione dell’arteria cerebrale media (MCAo) di 2 ore. È stato osservato che il contenuto di AEA nello striato di ratti sottoposti a MCAo, ma non nella corteccia, era significativamente incrementato (all’incirca di 3 volte rispetto ai ratti controllo, P < 0.01) e questo incremento era accompagnato parallelamente da un aumentata attività della NAPE-PLD (di circa 1.7 volte rispetto ai ratti controllo, P < 0.01) e da una ridotta attività (~ 0.6 volte; P<0.05) ed espressione della FAAH (~0.7 volte; P< 0.05). Questi effetti indotti dalla MCAo venivano ulteriormente potenziati da un ora di riperfusione, mentre il legame dell’AEA al recettore cannabinoide CB1 e al recettore vanilloide TRPV1 non erano influenzati in maniera significativa dall’insulto ischemico. Inoltre, il trattamento con l’antagonista del recettore CB1, SR141716, e non quello con l’agonista R-(+)- WIN55,212-2, ha dimostrato di ridurre significativamente (33%; P<0.05) il volume cerebrale d’infarto dopo 22 ore di riperfusione; mentre la somministrazione di una dose neuroprotettiva di 17β-estradiolo (0.20 mg/kg, i.p.), che era in grado di ridurre il volume d’infarto del 43%, si dimostrava capace anche di ridurre l’effetto dell’ischemia cerebrale sul sistema endocannabinoide in maniera recettore estrogenico dipendente. In conclusione, abbiamo dimostrato che il sistema endocannabinoide è implicato nella fisiopatologia del danno cerebrale tMCAo indotto e che la modulazione farmacologica di questo sistema endogeno da parte dell’estradiolo conferisce neuroprotezione.