Eleonora Da Pozzo

Brain-derived neurotrophic factor plasma levels in patients suffering from post-traumatic stress disorder

In both animals and humans, stress has been demonstrated to reduce the expression of the Brain-Derived Neurotrophic Factor (BDNF), a neurotrophin (NT) which promotes the proliferation, survival and differentiation of neurons. Although traumatic events have been found to be associated with lower BDNF plasma levels in affective disorders, no study has explored this parameter in patients with post-traumatic stress disorder (PTSD). We, therefore, measured BDNF plasma level in 18 patients with PTSD and in 18 healthy control subjects. Diagnoses were assessed by the Structured Clinical Interview for DSM-IV, while the specific symptoms were examined in the patients by means of the Impact of Event Scale for PTSD and the traumas experienced were assessed by using the Life Events Checklist. BDNF plasma levels were evaluated by means of a standardized Elisa method. The results, while showing significantly lower BDNF levels in PTSD patients, as compared with those of healthy subjects (p=0.001), although obtained in a small sample size, would suggest that this NT may be involved in the pathophysiology of PTSD.

Toward highly potent cancer agents by modulating the C-2 group of the arylthioindole class of tubulin polymerization inhibitors

New arylthioindole derivatives having different cyclic substituents at position 2 of the indole were synthesized as anticancer agents. Several compounds inhibited tubulin polymerization at submicromolar concentration and inhibited cell growth at low nanomolar concentrations. Compounds 18 and 57 were superior to the previously synthesized 5. Compound 18 was exceptionally potent as an inhibitor of cell growth: it showed IC₅₀ = 1.0 nM in MCF-7 cells, and it was uniformly active in the whole panel of cancer cells and superior to colchicine and combretastatin A-4. Compounds 18, 20, 55, and 57 were notably more potent than vinorelbine, vinblastine, and paclitaxel in the NCI/ADR-RES and Messa/Dx5 cell lines, which overexpress P-glycoprotein. Compounds 18 and 57 showed initial vascular disrupting effects in a tumor model of liver rhabdomyosarcomas at 15 mg/kg intravenous dosage. Derivative 18 showed water solubility and higher metabolic stability than 5 in human liver microsomes.

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.

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.

Combined inhibition of AKT/mTOR and MDM2 enhances Glioblastoma Multiforme cell apoptosis and differentiation of cancer stem cells

The poor prognosis of Glioblastoma Multiforme (GBM) is due to a high resistance to conventional treatments and to the presence of a subpopulation of glioma stem cells (GSCs). Combination therapies targeting survival/self-renewal signals of GBM and GSCs are emerging as useful tools to improve GBM treatment. In this context, the hyperactivated AKT/mammalian target of the rapamycin (AKT/mTOR) and the inhibited wild-type p53 appear to be good candidates. Herein, the interaction between these pathways was investigated, using the novel AKT/mTOR inhibitor FC85 and ISA27, which re-activates p53 functionality by blocking its endogenous inhibitor murine double minute 2 homologue (MDM2). In GBM cells, FC85 efficiently inhibited AKT/mTOR signalling and reactivated p53 functionality, triggering cellular apoptosis. The combined therapy with ISA27 produced a synergic effect on the inhibition of cell viability and on the reactivation of p53 pathway. Most importantly, FC85 and ISA27 blocked proliferation and promoted the differentiation of GSCs. The simultaneous use of these compounds significantly enhanced GSC differentiation/apoptosis. These findings suggest that FC85 actively enhances the downstream p53 signalling and that a combination strategy aimed at inhibiting the AKT/mTOR pathway and re-activating p53 signalling is potentially effective in GBM and in GSCs.

Apoptosis Therapy in Cancer: The First Single-molecule Co-activating p53 and the Translocator Protein in Glioblastoma

In the complex scenario of cancer, treatment with compounds targeting multiple cell pathways has been emerging. In Glioblastoma Multiforme (GBM), p53 and Translocator Protein (TSPO), both acting as apoptosis inducers, represent two attractive intracellular targets. On this basis, novel indolylglyoxylyldipeptides, rationally designed to activate TSPO and p53, were synthesized and biologically characterized. The new compounds were able to bind TSPO and to reactivate p53 functionality, through the dissociation from its physiological inhibitor, murine double minute 2 (MDM2). In GBM cells, the new molecules caused Δψm dissipation and inhibition of cell viability. These effects resulted significantly higher with respect to those elicited by the single target reference standards applied alone, and coherent with the synergism resulting from the simultaneous activation of TSPO and p53. Taken together, these results suggest that TSPO/MDM2 dual-target ligands could represent a new attractive multi-modal opportunity for anti-cancer strategy in GBM.

The GABAA-BZR Complex as Target for the Development of Anxiolytic Drugs

Anxiety disorders have been linked to alterations in γ-aminobutyric acid (GABA) neurotransmission. GABA interacts with the ligand-gated ion channels, GABAA receptor (GABAA-R) subtypes, and regulates the flow of chloride into the cell, causing neuron hyperpolarization. GABAA-Rs are assembled from a family of 19 homologous subunit gene products and form mostly hetero-oligomeric pentamers. The major isoforms of the GABAA-Rs contain α, β and γ subunits and show a regional heterogeneity that is associated with distinct physiological effects. A variety of allosteric ligands can modulate the response to GABA by binding at different sites on the GABAA-R complex. The best characterized binding site is the benzodiazepine (BZ) one, which is located at the α/γ subunit interface. BZs are commonly used in therapy for their effects as anxiolytic, anticonvulsants, myorelaxants and hypnotics. The broad range of pharmacological effects of classical BZs are mediated by the selective activation of different GABAA-R subtypes: the α1 subunit containing BZ receptor (BZ-R) mediates sedation, the α2 and α3 subunit containing BZ-R mediates anxiolysis and myorelaxation, and the α5 subunit containing BZ-R mediates cognitive impairment. Based on the current understanding of the diversity of the GABAA-R family, different approaches have been employed to develop drugs that target the GABAA/BZ-R complex with selective anxiolytic action and improved profiles. In this review, we present current knowledge about the role of the GABAA/BZ-R complex in anxiety disorders, new insights into the molecular biology of the receptor complex, and the importance of this target in the development of new therapeutic agents in anxiety.

Peripheral Benzodiazepine Receptor: Characterization in Human T-Lymphoma Jurkat Cells

Peripheral benzodiazepine receptor (PBR) has been considered a promising drug target for cancer therapy, and several ligands have been developed for this purpose. Human T-lymphoma Jurkat cells have been considered as lacking PBR and are often used as negative control to prove the specificity of PBR ligands effects. It is surprising that we evidenced PBR protein expression in this cell line by means of Western blotting and immunocytochemistry assays using specific anti-PBR antibodies. PBR intracellular localization was evidenced in mitochondria and nuclei, as demonstrated by confocal and electron microscopy. The binding of the [3H]4′-chloro derivative of diazepam [3H]7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864) and the isoquinoline carboxamide derivative [3H]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3 isoquinolinecarboxamide (PK11195) evidenced a single class of binding sites with an unusual affinity constant (Kd) of 1.77 ± 0.30 and 2.20 ± 0.20 μM, respectively. The pharmacological profile of the classic ligands showed that PK11195 was the most potent inhibitor in the radioligand binding assays followed by Ro5-4864 and diazepam, whereas clonazepam, a specific ligand for the central-type receptor, showed a Ki >1.0 × 10–4 M. By a combined strategy of reverse transcriptase-polymerase chain reaction and Southern blot experiments, we succeeded in isolating and cloning the full-length Jurkat PBR cDNA, called JuPBR. The JuPBR gene showed two single-nucleotide polymorphisms resulting in the two substitutions, Ala147 → threonine and His162 → arginine, of PBR amino acidic sequence. In conclusion, for the first time, we demonstrated PBR expression in Jurkat cells: the protein bound classic PBR ligands with micromolar affinity constants and presented a modified amino acidic sequence consequent to the detection of two gene polymorphisms.

TSPO over-expression increases motility, transmigration and proliferation properties of C6 rat glioma cells

Gliomas are one of the most malignant cancers. The molecular bases regulating the onset of such tumors are still poorly understood. The translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, is a mitochondrial permeability transition (MPT)-pore protein robustly expressed in gliomas and involved in the regulation of apoptosis and cell proliferation. TSPO expression levels have been correlated with tumor malignancy. Here we describe the production of C6 rat glioma cells engineered to over-express the TSPO protein with the aim of providing the first direct evidence of a correlation between TSPO expression level and glioma cell aggressiveness. We observed that TSPO potentiates proliferation, motility and transmigration capabilities as well as the ability to overcome contact-induced cell growth inhibition of glioma cells. On the whole, these data demonstrate that TSPO density influences metastatic potential of glioma cells. Since several data suggest that TSPO ligands may act as chemotherapeutic agents, in this paper we also demonstrate that TSPO ligand-induced cell death is dependent on TSPO density. These findings suggest that the use of TSPO ligands as chemotherapeutic agents could be effective on aggressive tumor cells with a high TSPO expression level.

Trazodone treatment protects neuronal-like cells from inflammatory insult by inhibiting NF-κB, p38 and JNK

Growing evidence suggests that alterations of the inflammatory/immune system contribute to the pathogenesis of major depression and that inflammatory processes may influence the antidepressant treatment response. Depressed patients exhibit increased levels of inflammatory markers in both the periphery and brain, and high co-morbidity exists between depression and diseases associated with inflammatory alterations. Trazodone (TDZ) is a triazolopyridine derivative that belongs to the class of serotonin receptor antagonists and reuptake inhibitors. Although the trophic and protective properties of classic antidepressants have extensively been exploited, the effects of TDZ remain to be fully elucidated. In this study, the pharmacological activities of TDZ on human neuronal-like cells were investigated under both physiological and inflammatory conditions. An in vitro inflammatory model was established using lipopolysaccharide (LPS) and tumour necrosis factor-α (TNF-α), which efficiently mimic the stress-related changes in neurotrophic and pro-inflammatory genes. Our results showed that TDZ significantly increased the mRNA expression of both brain-derived nerve factor (BDNF) and cAMP response element-binding protein (CREB) and decreased the cellular release of the pro-inflammatory cytokine interferon gamma (IFN-γ) in neuronal-like cells. In contrast, neuronal cell treatment with LPS and TNF-α decreased the expression of CREB and BDNF and increased the expression of nuclear factor kappa B (NF-κB), a primary transcription factor that functions in inflammatory response initiation. Moreover, the two agents induced the release of pro-inflammatory cytokines (i.e., interleukin-6 and IFN-γ) and decreased the production of the anti-inflammatory cytokine interleukin-10. TDZ pre-treatment completely reversed the decrease in cell viability and counteracted the decrease in BDNF and CREB expression mediated by LPS-TNF-α. In addition, the production of inflammatory mediators was inhibited, and the release of interleukin-10 was restored to control levels. Furthermore, the intracellular signalling mechanism regulating TDZ-elicited effects was specifically investigated. TDZ induced extracellular signal-regulated kinase (ERK) phosphorylation and inhibited constitutive p38 activation. Moreover, TDZ counteracted the activation of p38 and c-Jun NH2-terminal kinase (JNK) elicited by LPS-TNF-α, suggesting that the neuro-protective role of TDZ could be mediated by p38 and JNK. Overall, our results demonstrated that the protective effects of TDZ under inflammation in neuronal-like cells function by decreasing pro-inflammatory signalling and by enhancing anti-inflammatory signalling.