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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    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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    Mechanisms of RA action in steroidogenic tissues and pro-apoptotic effects of combined treatment of breast tumoral cell lines with 9-cis retinoic acid and rosiglitazione
    (2008-11-20) Pingitore, Attilio; Bonofiglio, Daniela; Sisci, Diego
    Vitamin A (Retinol) plays a central role in many essential biological processes such as vision, immunity, reproduction, growth, development, control of cellular proliferation and differentiation. The main active forms of retinol, not primary involved in vision, are alltrans retinoic acid and 9-cis retinoic acid, both able to act at nuclear level by binding their receptors RAR and RXR and modulating many physiological processes. However, the nuclear action of vitamin A derivatives is not the only mechanism of retinoic acid (RA) acting on cells. RA is able to modify covalently proteins via a post-translational modification, named retinoylation that has been shown to occur at physiological concentration on pre-existing proteins and localized mainly in the mitochondrial compartment. The present study has been focused on the non genomic action of RA on steroidogenic tissues, testes and adrenal glands, giving further details on the ability of RA to influence protein activity and therefore cell physiology. In particular RA effects on mitochondria from the adrenal glands and the 2-oxoglutarate carrier protein from testes and TM-3 Leydig cell line were studied, providing new data on the peculiarity of steroidogenic tissues to incorporate RA at dietary levels and demonstrating how the shuttling of reducing equivalent across the mitochondrial membrane is influenced by RA treatment. Looking for the biochemical mechanism of RA action on the Adenine Nucleotide Translocator, that exchanges ATP for ADP between mitochondria and cytosol, for the first time, it was possible to demonstrate how the activity of this carrier protein is positively modulated by the Coenzyme A, a fundamental component of the retynoilating buffer. At pharmacological levels, retinoids are also active compounds in the treatment of cancer due to the capability to promote cell differentiation and their pro-apoptotic activity. In this latter concern, the mechanisms of nutriceutical concentration of 9-cis RA, together with nanomolar concentration of the selective PPARγ ligand, rosiglitazone, to promote apoptosis in breast cancer cell lines, have been investigated. The data lay the basis for a potential use of the combined therapy with low doses of both BRL and 9-cis RA as novel therapeutic tool particularly for breast cancer patients who develop resistance to antiestrogen therapy