Dipartimento di Farmacia e Scienze della Salute e della Nutrizione - Tesi di Dottorato

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Questa collezione raccoglie le Tesi di Dottorato afferenti al Dipartimento di Farmacia e Scienze della Salute e della Nutrizione dell'Università della Calabria.

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End date: 2019Subject: search.filters.subject.Biochimica

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    Study of the expression of a Small Leucin-Rich Proteoglycan, Asporin, in normal human osteoblasts and regulation by breast cancer cells
    (2014-11-17) Trombino, Giovanna Elvi; Sisci, Diego; Bellahcène, Akeila; Lanzino, Marilena
    Asporin (ASPN) is an extracellular matrix protein that belongs to the Small Leucine Rich Repeat proteoglycan (SLRP) family. Asporin is abundantly expressed in the articular cartilage of individuals with osteoarthritis. In the context of osteoarthritis, several studies have shown that asporin regulates cartilage matrix gene expression and cartilage formation by modulating the transforming growth factor-β (TGF- β) signaling pathway. Asporin directly binds to TGF‐β and inhibits TGF-β-mediated expression of cartilage matrix genes. Previous studies in our laboratory, showed that Asporin inhibits TGF- β-1-mediated SMAD2 phosphorylation in breast cancer cells as well as migration and epithelial to mesenchymal transition in A549 human lung cancer cells. The present study was undertaken to investigate whether asporin secretion could indirectly mediate the ability of metastatic breast cancer cells to regulate osteoblastic differentiation. The Wnt antagonist sclerostin (SOST) is a potent inhibitor of bone formation. We considered the possibility that the balance between ASPN and SOST present in the ECM may create a specific environment favorable to aggressive breast cancer cell growth. Results: Breast cancer cells do not produce ASPN themselves but they regulate its expression in osteoblasts. Normal human osteoblasts have been cultured in presence of MCF7 and MDA-MB-231 serum-free conditioned medium. Immunoblot analysis and real time PCR, revealed a significant increase in ASPN expression and secretion in osteoblasts treated with MCF7-conditioned medium, while the opposite effect was observed with MDA-MB-231-conditioned medium. We investigated the role of MCF7 and MDAMB231 conditioned media in osteoblast differentiation and mineralization through alkaline phospatase and Runx2 expression. Our results showed the ability of MCF7 conditioned medium to induce the osteoblast differentiation and mineralization compared to the MDA-MB-231 conditioned medium treatment. Osteoblasts treated with MCF7 conditioned medium and challenged with recombinant SOST showed a significant reduction in their differentiation potential through the decrease of ASPN expression. Contrarily to non-metastatic MCF-7 breast cancer cells, MDA-MB-231 metastatic breast cancer cells inhibited the secretion of ASPN by osteoblasts through the overexpression of SOST. The result is the reduction of osteoblast differentiation and mineralization that can create a specific environment favorable to aggressive breast cancer cell growth
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    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, Sebastiano
    Human 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 growth
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    Preparazione di nuovi biomateriali: membrane zeolitiche ibride e cristalli per adsorbimento di farmaci di interesse oncologico
    (2012-11-20) Riccio, Ilaria Iolanda; Sisci, Diego; Tavolaro, Adalgisa; Aquila, Saveria; Tavolaro, Palmira
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    Bisphenol A induces gene expression changes an proliferative effects through GPER in breast cancer celles and cancer-associated fibroblasts
    (2012) Pupo, Marco; Maggiolini, Marcello; Sisci, Diego
    Bisphenol A (BPA) is the principal constituent of baby bottles, reusable water bottles, metal cans, and plastic food containers. BPA exerts estrogen-like activity by interacting with the classical estrogen receptors (ER and ER ) and through the G protein-coupled receptor (GPR30/GPER). In this regard, recent studies have shown that GPER was involved in the proliferative effects induced by BPA in both normal and tumor cells. In this study we evaluated the transduction pathways through which BPA influences cell proliferation and migration in human breast cancer cells and cancer-associated fibroblasts (CAFs), that lack the classical ERs. Specific pharmacological inhibitors and gene-silencing procedures showed that BPA induces the expression of the GPER target genes c􀌻FOS, EGR􀌻1, and CTGF through the GPER/EGFR/ERK transduction signaling in SKBR3 breast cancer cells and CAFs. Moreover, we observed that GPER is required for growth effects and migration stimulated by BPA in both cell types. Our results indicate that GPER is involved in the biological action elicited by BPA in breast cancer cells and CAFs. Hence, GPER-mediated signaling should be included among the transduction mechanisms through which BPA may stimulate cancer progression
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    Chenodeoxycholic acid (CDCA) through TGR5-dependent CREB-signaling activation enhances Cyclin D1 expression and promotes human endometrial cancer cell proliferation
    (2011) Avena, Paola; Casaburi, Ivan; Sisci, Diego
    Increasing epidemiologic data in humans as well as many in vitro investigative reports suggest that overweight and obesity are important risk factors for type-I EC which is the most common malignancy in women and accounts for 80% of all ECs. The strongest support for mechanisms to link obesity and cancer risk involves the metabolic and endocrine effects of obesity and the alterations that they induce in the production of peptide and steroid hormones. One way in which fat might exert its effect is stimulation of bile acids (BAs) synthesis and secretion. Bile acids (BAs) are amphipathic detergents that are synthesized in the liver and stored in the gallbladder. An important physiological role of BAs is to facilitate the uptake of lipids together with the fat-soluble vitamins A, D, E and K from the intestine. BAs facilitate these absorptive processes through their detergent properties, which allow the emulsification of lipids. Moreover, BAs have evolved over the past few years from being considered as simple lipid solubilizers to complex metabolic integrators. Beyond the orchestration of bile acids, lipid and glucose metabolism by the nuclear receptor farnesoid X receptor (FXR), BAs also act as signaling molecules through the non-genomic pathway activated by a BAdedicated G protein-coupled receptor (GPCR) TGR5 (also known as BG37 or MBAR). Despite the efficient enterohepatic recirculation of bile acids, a small amount of them can spill over into the systemic circulation particularly during high fat diet. Since in the obese women a strong correlation with endometrial cancer does exist, we investigated the biological effects of different concentrations of the primary bile acid CDCA in a human endometrial type-I cancer cell line, Ishikawa. Ishikawa cell proliferation was determined by [3H]-thymidine incorporation assay after 72 hours of treatment with different concentrations of CDCA. The effects of CDCA on the expression of the cell cycle regulatory proteins were evaluated by RT-PCR and Western blotting assays. Transient transfection method and mutagenesis studies were performed to functionally characterize the Cyclin D1 promoter while ChIP assay was to highlight the direct involvement of the ciselements in CDCA-dependent Cyclin D1 up-regulation. In addition, the effects of impaired expression and function of the proteins involved in CDCA activated signaling were assessed by using small interfering RNAs (siRNAs) technology. Our results indicate that low concentrations (< 30μM) of CDCA were able to stimulate Ishikawa cell growth as evidenced by [3H]Thymidine incorporation and cell cycle analysis by inducing a significant increase in Cyclin D1(CD1) protein and mRNA expression. In contrast, according to previous findings, high doses (> 50μ) of CDCA showed cytotoxic effects concomitantly with an increase of CDK inhibitor p21WAF1/CIP1 expression through a p53-indipendent pathway. Moreover, mutagenesis studies and ChIP analysis revealed that the CDCA-induced CD1 expression requires a cyclic AMP-responsive element (CRE) binding protein motif within the CD1 proximal promoter. Silencing of a cell surface bile acid sensor TGR5 and/or CREB gene expression by RNA interference reversed the CDCA-dependent induction of CD1 expression and proliferation in Ishikawa cells. In conclusion, extrahepatic spillover of BAs following high fat diet particularly in overweight and obese women, could be involved in the onset and/or maintenance of endometrial cancer by stimulation of TGR5 that activates ERK signaling that in turn induces the recruitment of the transcription factor CREB to the Cyclin D1 gene promoter enhancing cell proliferation
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    Modelli sperimentali cellulari e molecolari per la valutazione della tossicità di xenobiotici
    (2013-12-02) Peta, Valentina; Sisc, Diego; Indiveri, Cesare
    The aim of my thesis was to find two different experimental models to study cellular and molecular xenobiotics toxicity. In the first part of the present work we studied the interaction between two different plasma membrane transporters (OCTN2 and OCTN1), mercury reagents and heavy metals. Mercury and heavy metals in general cause toxic effects in many tissues interacting with protein cysteine (Cys) thiols. Transport systems represent critical targets of mercurials. Indeed, the majority of transport systems of higher eukaryotes containseveral Cys residues. One of the most up to date method of studying transport is the reconstitution of transportersin proteoliposomes. This method has been used as a useful approach to test the effect of HgCl2, methylmercury(MeHg) and Cadmium on the carnitine (OCTN2) transporter, extracted from rat kidney brush border membranes and reconstituted in liposomes by removing the detergent withhydrophobic chromatography columns, and the human organic cation transporter (OCTN1) overexpressed in E. coli, purified by Ni-chelating chromatography and reconstituted in liposomes by detergent removal with a batch-wise procedure.Transport was measured as [3H]carnitine uptake into proteoliposomescontaining carnitine (antiport reaction) in the case of rat OCTN2 and as [14C]tetraethylammonium uptake in the case of human OCTN1. Mercurials and heavy metals strongly inhibited the transport. Inhibition was reversed by1,4-dithioerythritol (DTE), L-cysteine (Cys), and N-acetyl-Lcysteine (NAC) indicating that it was caused by covalent reactionof mercurials and heavy metals with Cys residue(s) of OCTN2 and OCTN1.The presence of substrate prevented the inhibition in rat OCTN2 transporter indicating that the mercurial binding residue (Cys) is in the substrate binding site. No substrate protection was found in the case of the human OCTN1, so probably mercurial and heavy metal binding residue is away from substrate binding site. For the human OCTN1 we also tested the effect of chemical reagents which are known to form mixeddisulphides with proteins SH residues, MTS reagents. MTSEA exerted inhibition of transport very similar to those observed for the heavy metals and as the toxic compounds. To ascertain the involvement of Cys residues in the interaction of the human OCTN1 with the xenobiotics and to identify the possible target of the reagents, 7 mutants were prepared in which the seven cysteines present in the transmembrane domains or in the extracellular loop were mutated to alanine. An additional mutant lacking two Cys residues has also been constructed (C50A/C136A). We studies the dose-response curves of the transporter for each inhibitor, the mercury reagents showed similar behavior, both HgCl2 and MeHg strongly inhibited the WT and the mutants C81A, C113A, C236A, C270A and C374A. While a clear shift of the curves towards higher concentrations of the compounds was observed in the case of mutants C50A and C136A indicating a decrease of affinity of these mutants for the mercury reagents. The experiment on the double mutant C50A/C136A showed a nearly complete lack of inhibition by the two reagents demonstrating the two Cys residues were indeed the target of the mercury compounds. The homology model of the human OCTN1 confirms the experimental data obtained in this work, in fact the model shows that the two cysteine residues (Cys-50 and Cys-136) are exposed to the extracellular site of the plasma membrane and are accessible to sulfhydryl groups reagents. In the present work we studied, also, biomarkers expression and nephrotoxic effect induced by drugs in human kidney primary cells model. Kidney is a primary target of drug-induced toxicity. Toxic effects on the kidney related to drugs are both common and expected, given the kidney's roles in plasma filtration and maintenance of metabolic homeostasis. As such, glomerular, tubular and renal interstitial cells frequently are exposed to concentrations of drugs, which can induce changes in kidney function and structure. We tested three different drugs: 2-Bromoethanamine an analgesic, cisplatin a chemotherapeutic agent and cyclosporine a immunosuppressive agent. We found that after the addition of these drugs on human primary kidney cells there is an increase in the expression of two different biomarkers: Osteopontin (OPN) a distal tubule biomarker and KIM-1 a membrane glycoprotein expressed by proximal tubule cells after kidney injury. We also found the toxic concentrations after 24 hours of exposition to the three different drugs, we quantified the percentual of necrotic and apoptotic cells and we studied the toxicological effect of these drugs on cellular organelles like mitochondria. In conclusion proteoliposomes represent a suitable molecular model for studying the interaction of plasma membrane transport and toxic compounds, such as mercurials, and human primary cell culture is a valuable tool to study cell toxicity mechanisms of different drugs. Both the experimental models are a novel and potentially important tools in drug discovery and in the understanding of toxicity mechanism of xenobiotics compounds.
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    Dynamic effects of retinoic acid and its isomers on cancer and physiology
    (2010-11-23) Perri, Mariarita; Bonofiglio, Daniela; Sisci, Diego
    In the search for new cancer chemo-preventive compounds, hundreds of naturally occurring molecules have been evaluated. Among these, antioxidants appear to be very promising. In this contest, over the last decade retinoids, natural and synthetic substances structurally related to vitamin A, are often used as part of a combined therapy and have been object of intense investigation. However, clinical trials have shown that retinoids can also be deleterious and are associated with the activation of proto-oncogenes, leading to an increased incidence of neoplasias. In fact, retinoic acid (RA) partition is regulated by cognate intracellular lipid binding proteins (iLBPs): cellular retinoic acid binding protein II (CRABP-II) delivers RA to RARs, while fatty acid binding protein 5 (FABP5) shuttles the RA to PPARβ/δ. In cells with high CRABP-II/FABP5 ratio, RA functions through RAR acting as a pro-apoptotic agent, while signaling through PPARβ/δ promotes survival in those cells highly expressing FABP5. So that, in some tissues RA promotes cell survival and hyperplasia. The apparently conflicting data regarding the pro-oxidant/ anti-oxidant and proliferative/anti-proliferative potential of different retinoids molecules, stimulated us to investigate the effect of RA on cell proliferation and its mechanisms in two different tumor Leydig cell lines (MLTC-1 and R2C) using as normal phenotype counterpart the Leydig TM-3 cell line. Our previous data demonstrated how pharmacological doses of RA induce cell death via the apoptotic pathway in Leydic TM-3 cell line. Recently dose-response treatment of TM-3, MLTC-1 and R2C with RA at nutraceutic/physiological doses, promotes cell proliferation accompanied by stimulation of antioxidant enzymes activity (CAT, GST), decreases p21 levels and fosters cell cycle progression via activation of the IP3K/Akt pathway in the cancer cell line, while administration of pharmacological doses of RA still results in apoptosis. Interestingly treatment with 500 nM of RA resulted in cytosolic vacuolization, hallmark of the autophagic process. Autophagy is a major cellular pathway for the degradation of long-lived proteins and organelles in eukaryotic cells. A large number of intracellular/extracellular stimuli, including amino acid starvation, testosterone production and invasion of microorganisms are able to induce autophagic response. In addition, retinoic acid is also implicated in a post-translation modification called retinoylation that modify, in vitro, the activity of the mitochondrial carrier oxo-chetoglutarate (OCG). Moreover, retinoids are often used as part of a combined therapy, their action is prevalently mediated by two types of receptor RAR and RXR. This latter, is also called master coordinator due to its versatility to heterodimering with several nuclear receptor. Thus, we have elucidated the molecular mechanism by which combined treatment with rosiglitazone (BRL) and 9 cis retinoic acid (9cRA) at nanomolar doses triggers apoptotic events in breast cancer cells, suggesting potential therapeutic uses for these compounds, demonstrating an up-regulation of tumor suppressor gene p53 and its activity is due to the NFkB site, giving emphasis to the potential use of the combined therapy with low doses of both BRL and 9cRA as novel therapeutic tool particularly for breast cancer patients who develop resistance to anti-estrogen therapy. Recently, 9cRA was found as endogenous in pancreas highlighted its rule in both glucose stimulated insulin secretion (GSIS) mechanism and glucose homeostasis, establishing it as autocoid hormone with a unique physiological function among retinoids, and broaden insight into mechanisms of GSIS