P Gabelloni

Oxytocin receptor polymorphisms and adult attachment style in patients with depression

Much evidence of an association between specific attachment styles and depression prompted us to investigate, in depressive disorders, the potential role of polymorphisms within the gene encoding the receptor of the main neurohormone involved in attachment processes, oxytocin. For this purpose, two single nucleotide polymorphisms (SNPs), 6930G>A (rs53576) and 9073G>A (rs2254298), within the oxytocin receptor gene (OXTR), were studied in a cohort of 185 patients with major depression (50.3%) or bipolar I or II disorders (49.7%) and 192 matched healthy controls. A positive association between the GG genotype of OXTR SNPs (6930G>A or 9073G>A) and unipolar depression was demonstrated. In this group, GG individuals showed high scores on Attachment Style Questionnaire factors that have been previously associated with depression. Moreover, the GG genotype was also associated with high levels of adult separation anxiety. These findings support the involvement of the oxytocinergic system in the mechanisms that underlie depression and specific adult attachment styles.

The Spontaneous Ala147Thr Amino Acid Substitution within the Translocator Protein Influences Pregnenolone Production in Lymphomonocytes of Healthy Individuals

The de novo production of steroids and neurosteroids begins in mitochondria by the conversion of cholesterol to pregnenolone through cytochrome P450 side-chain cleavage (CYP11A1) enzymatic activity. The C-terminal amino acid domain of the translocator protein (TSPO) has been demonstrated to bind cholesterol, thereby determining its mitochondrial translocation. The goal of the present study was to investigate the effect of the Ala147Thr single-nucleotide polymorphism localized in this TSPO region on pregnenolone production in healthy volunteers. Pregnenolone production was evaluated in a peripheral cell model, represented by circulating lymphomonocytes. First, CYP11A1 expression, both at mRNA and protein level, was demonstrated. Pregnenolone production varied among genotype groups. Comparison of pregnenolone mean values revealed that Thr147 homozygous or heterozygous individuals had significantly lower pregnenolone levels compared with Ala147 homozygous individuals. These findings suggested a dominant effect of the minor allelic variant Thr147 to produce this first metabolite of the steroidogenesis pathway. Interestingly, Ala147 homozygous individuals exhibited significant higher levels of circulating cholesterol-rich low-density lipoproteins with respect to heterozygous individuals. In conclusion, our results demonstrate that the Ala147Thr spontaneous amino acid substitution within TSPO is able to affect pregnenolone production; this should encourage further studies to investigate its potential role in polygenic dyslipidemias.

Inhibition of metalloproteinases derived from tumours: new insights in the treatment of human glioblastoma

Glioblastoma multiforme is the most commonly diagnosed malignant primary brain tumour in adults. Invasive behaviour is the pathological hallmark of malignant gliomas; consequently, its inhibition has been suggested as a therapeutic strategy. Tumour cell-derived gelatinases (matrix metalloproteinase-2, matrix metalloproteinase-9) can be considered prime factors in glioma invasiveness: their expression correlates with the progression and the degree of malignancy. Thus, broad spectrum matrix metalloproteinase inhibitors (MMP inhibitors) have been included in clinical trials. In the present study, the invasiveness, viability and progression of the human glioma cell line U87MG were investigated following treatment with N-O-isopropyl sulfonamido-based hydroxamates (compounds 1 and 2) as MMP-2 inhibitors used at nanomolar concentration. A standard broad spectrum MMP-inhibitor belonging to the classical tertiary sulfonamido-based hydroxamates family (CGS_27023A) was used too. The compounds 1 and 2 resulted in potent inhibition of cell invasiveness (P<0.0001) without affecting viability. In some clinical trials, the combined therapy of temozolomide (an alkylating agent used in glioma treatment) plus marimastat (a broad spectrum MMP inhibitor) has provided evidence of the importance of MMPs to tumor progression and invasiveness. On this basis, the effect on U87MG cells of a combined treatment with temozolomide, plus each of the two MMP inhibitors at nanomolar concentration, was investigated. The obtained data demonstrated the inhibition of cell invasiveness and viability after treatment. These results can help in developing clinical combined therapy using MMP inhibitors that, at low doses, increase the anticancer efficacy of chemotherapeutic drugs, probably without causing the side effects typical of broad-spectrum MMP inhibitors.

Human Glioblastoma Multiforme: p53 Reactivation by a Novel MDM2 Inhibitor

Cancer development and chemo-resistance are often due to impaired functioning of the p53 tumor suppressor through genetic mutation or sequestration by other proteins. In glioblastoma multiforme (GBM), p53 availability is frequently reduced because it binds to the Murine Double Minute-2 (MDM2) oncoprotein, which accumulates at high concentrations in tumor cells. The use of MDM2 inhibitors that interfere with the binding of p53 and MDM2 has become a valid approach to inhibit cell growth in a number of cancers; however little is known about the efficacy of these inhibitors in GBM. We report that a new small-molecule inhibitor of MDM2 with a spirooxoindolepyrrolidine core structure, named ISA27, effectively reactivated p53 function and inhibited human GBM cell growth in vitro by inducing cell cycle arrest and apoptosis. In immunoincompetent BALB/c nude mice bearing a human GBM xenograft, the administration of ISA27 in vivo activated p53, inhibited cell proliferation and induced apoptosis in tumor tissue. Significantly, ISA27 was non-toxic in an in vitro normal human cell model and an in vivo mouse model. ISA27 administration in combination with temozolomide (TMZ) produced a synergistic inhibitory effect on GBM cell viability in vitro, suggesting the possibility of lowering the dose of TMZ used in the treatment of GBM. In conclusion, our data show that ISA27 releases the powerful antitumor capacities of p53 in GBM cells. The use of this MDM2 inhibitor could become a novel therapy for the treatment of GBM patients.

Ala147thr substitution in translocator protein is associated with adult separation anxiety in patients with depression

Departments of Human Morphology and Applied Biology, Psychiatry, Neurobiology, Pharmacology and Biotechnology, Biology, University of Pisa, Pisa and Psychiatry, University of Naples SUN, Naples, Italy Correspondence to Professor Stefano Pini, MD, Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy Tel: + 39 050 993559; fax: + 39 05

Agonist-induced desensitization/resensitization of human G protein-coupled receptor 17: a functional cross-talk between purinergic and cysteinyl-leukotriene ligands.

G protein-coupled receptor (GPR) 17 is a P2Y-like receptor that responds to both uracil nucleotides (as UDP-glucose) and cysteinyl-leukotrienes (cysLTs, as LTD4). By bioinformatic analysis, two distinct binding sites have been hypothesized to be present on GPR17, but little is known on their putative cross-regulation and on GPR17 desensitization/resensitization upon agonist exposure. In this study, we investigated in GPR17-expressing 1321N1 cells the cross-regulation between purinergic- and cysLT-mediated responses and analyzed GPR17 regulation after prolonged agonist exposure. Because GPR17 receptors couple to Gi proteins and adenylyl cyclase inhibition, both guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding and the cAMP assay have been used to investigate receptor functional activity. UDP-glucose was found to enhance LTD4 potency in mediating activation of G proteins and vice versa, possibly through an allosteric mechanism. Both UDP-glucose and LTD4 induced a time- and concentration-dependent GPR17 loss of response (homologous desensitization) with similar kinetics. GPR17 homologous desensitization was accompanied by internalization of receptors inside cells, which occurred in a time-dependent manner with similar kinetics for both agonists. Upon agonist removal, receptor resensitization occurred with the typical kinetics of G protein-coupled receptors. Finally, activation of GPR17 by UDP-glucose (but not vice versa) induced a partial heterologous desensitization of LTD4-mediated responses, suggesting that nucleotides have a hierarchy in producing desensitizing signals. These findings suggest a functional cross-talk between purinergic and cysLT ligands at GPR17. Because of the recently suggested key role of GPR17 in brain oligodendrogliogenesis and myelination, this cross-talk may have profound implications in fine-tuning cell responses to demyelinating and inflammatory conditions when these ligands accumulate at lesion sites.

Synthesis and biological evaluation in U87MG glioma cells of (ethynylthiophene)sulfonamido-based hydroxamates as matrix metalloproteinase inhibitors

Matrix metalloproteinases (MMPs) are important factors in gliomas since these enzymes facilitate invasion into the surrounding brain and participate in neovascularization. In particular, the gelatinases (MMP-2 and MMP-9), and more recently MMP-25, have been shown to be highly expressed in gliomas and have been associated with disease progression. Thus, inhibition of these MMPs may represent a promising non-cytotoxic approach to glioma treatment. We report herein the synthesis and biological evaluation of a series of 4-butylphenyl(ethynylthiophene)sulfonamido-based hydroxamates. Among the new compounds tested, a promising derivative, 5a, was identified, which exhibits nanomolar inhibition of MMP-2, MMP-9, and MMP-25, but weak inhibitory activity toward other members of the MMP family. This compound also exhibited anti-invasive activity of U87MG glioblastoma cells at nanomolar concentrations, without affecting cell viability.

Agonist-induced desensitisation/resensitisation of human GPR17: a functional cross talk between purinergic and cysteinyl-leukotriene ligands

Abstracts of the Fourth Joint German-Italian Purine Club Meeting 2011s of the Fourth Joint German-Italian Purine Club Meeting 2011 Conference organization Conference chairperson Christa E. Müller Conference coordinator Heidi Zinecker Scientific coordination Italian Chapter of Purine Club German Chapter of Purine Club Maria P. Abbracchio (Milan) Joachim Deitmer (Kaiserslautern) Pier A. Borea (Ferrara) Peter Illes (Leipzig) Pier Leopoldo Capecchi (Siena) Karl-Norbert Klotz (Würzburg) Stefania Ceruti (Milan) Friedrich Koch-Nolte (Hamburg) Renata Ciccarelli (Chieti) Georg Reiser (Magdeburg) Patrizia Popoli (Rome) Günther Schmalzing (Aachen) Anna Maria Pugliese (Florence) Torsten Schöneberg (Leipzig) Letizia Trincavelli (Pisa) Jürgen Schrader (Düsseldorf) Rosaria Volpini (Camerino) Norbert Sträter (Leipzig) Organizing Committee Ivar von Kügelgen (Bonn) Christa E. Müller (Bonn) Karen Nieber (Leipzig) Alexander Pfeifer (Bonn) Anke C. Schiedel (Bonn) Christian Steinhäuser (Bonn) Herbert Zimmermann (Frankfurt) Heidi Zinecker (Bonn) Business Secretariat (University of Bonn) Michael Hemmersbach Beate Ponatowski Anke C. Schiedel Heidi Zinecker Contact: e-mail: purineclub@uni-bonn.de visit: www.purineclub.uni-bonn.de call: +49 228 732697 Purinergic Signalling (2012) 8:107–175 DOI 10.1007/s11302-011-9257-4