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BACKGROUND AND PURPOSE: N6-Isopentenyladenosine (i6A) is a modified nucleoside exerting in vitro and in vivo antiproliferative effects. We previously demonstrated that the i6A action is correlated to farnesyl pyrophosphate synthase (FPPS) expression and activity, a key enzyme involved in the mevalonate (MVA) pathway, which is found aberrant in brain cancer. To develop new anti-glioma strategies, we looked for other valuable compounds exhibiting improved activity as compared to i6A. EXPERIMENTAL APPROACH: we designed and synthesized i6A derivatives characterized by the introduction of diverse chemical moieties in the N6 position of adenosine and tested for their efficacy in U87 and primary derived patient's glioma cell model. NMR based structural analysis, molecular docking calculations and siRNA mediated knock-down helped to clarify the molecular basis of their action, targeting FPPS protein. KEY RESULTS: We demonstrated that CM223, the i6A derivative including a benzyl moiety in N6 position of adenine, is endowed with a marked activity in selectively targeting glioma cells but not normal human astrocytes (NHA). This is achieved by the induction of intrinsic pathways of apoptosis and inhibition of proliferation along a FPPS-dependent protein prenylation blocking, which counteract the oncogenic signaling mediated by the epidermal growth factor receptor (EGFR). CONCLUSION AND IMPLICATIONS: This biological effect together with structural data on interaction of CM223 with FPPS, gain additional evidence on the correlation of the i6A/CM223 antitumoral activity with FPPS modulation. Because the MVA pathway is becoming an important promising target, CM223 and derivatives should be considered interesting active molecules in antiglioma pharmacological research.

N6-Isopentenyladenosine (iPA) is a cytokinin identified in plants but also present in a free form or as a modified nucleoside bound to selenocysteine tRNA in human cells. It is an adenosine modified by an isopentenyl chain which derives from dimethylallil pyrophosphate (DMAPP), an intermediate of the mevalonate pathway. iPA is required for efficient translational decoding of selenoproteins, and can modulate a variety of biological processes including cell cycle progression, DNA synthesis and apoptosis (1). Recently, it has been shown that iPA can exhibit immunomodulatory and anti-inflammatory properties by activating NK cells and modulating cytokine production in a way depending on the concentration used (2). In order to further investigate the anti-inflammatory properties of iPA and its possible mechanisms of action, we analyzed its ability to inhibit TNFα-induced inflammation, either in normal human bronchial epithelial cells or in a model of exacerbated inflammation represented by bronchial cells derived from a Cystic Fibrosis (CF) patient bearing the ΔF508 mutation. Results showed that iPA inhibited IL-8 and RANTES release in both type of cells in a different manner. The analysis of the key enzymes of the STAT3 and NF-κB signalling pathways showed that iPA decreased the phosphorylation of STAT3 enzyme and markedly increased the expression of the direct NF-κB inhibitor, IκBα. These results were corroborated analyzing directly the NF-κB activity in HEK 293/T cells transfected with a NF-κB reporter plasmid. In these cells, iPA was also able to decrease IκBα levels. Of interest, we found that iPA also increased the expression of the antioxidant selenoprotein glutathione peroxidase only in CF cells. Altogether these data suggest that iPA can negatively regulate inflammation with a general mechanism of action involving the inhibition of NF-κB pathway but also propose that, in the presence of an altered inflammatory response such as in CF disease, iPA might act by modulating expression and/or synthesis of glutathione peroxidase., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

Inflammatory conditions and infections in selected organs increase the risk of cancer. In the tumor microenvironment, smoldering inflammation contributes to proliferation and survival of malignant cells, angiogenesis, metastasis and subversion of adaptive immunity. The human immunodeficiency virus (HIV) infection is characterized by increased risk of several solid tumors due to its inherent nature of weakening the immune system. Recent observations point to a lower incidence of some cancers in patients treated with protease inhibitor (PI) cocktail such as HAART (Highly Active Anti-Retroviral Therapy). Human Immunodeficiency Type-1 Virus protease inhibitors (HIV-1-PIs) originally designed to block selectively the aspartic protease of HIV-1, also shown the ability to modulate a variety of biological functions, including the immune response, by mechanisms largely independent from their anti-viral activity. Herein, we investigate the effects of PIs on differentiation programs of monocytes toward: (a) dendritic cells (DC); (b) Tumor Associated Macrophages-like cells (TAM-like). Differentiation of human circulating monocytes in the presence of PIs led to generation of DC with atypical phenotype, including low level of Cd1a, and DC-SIGN, a receptor that enables DC to bind HIV-1 virions in tissues, and carry them to lymphonodes. Moreover, DC generated in the presence of ritonavir also fail to terminally differentiate, and secrete lower amounts of IL- 12 and IL-15, in response to bacterial endotoxin (LPS). This phenomenon parallels their inability to prime NK cells, and become resistant to NK-mediated cytotoxicity. The exposure of monocytes to certains PIs determines generation of Tumour Associated Macrophages-Like cells with an atypical phenotype, including higher level of the co-stimulatory molecules CD86, and lower expression of ILT3, a receptor playing an imunosuppressive role. Accordingly, in response to LPS, TAM-like cells generated in the presence of PIs, secrete lower amount of MM9 and VEGF, a phenomenon accompanying their ability to release more GM-CSF. Altogether, these findings demonstrate the ability of PIs to modulate the differentiation programs of human monocytes. The remakable property of certain PIs to modulate phenotypes and functionalities of DC and TAM, might open novel perspectives for immune-intervention aimed to manipulate the cancer inflammatory milieu.

Alzheimer's disease (AD), is the most common neurodegenerative disorder of the aging population resulting in progressive cognitive and functional decline. Accumulation of amyloid plaques around neuronal cells is considered a critical pathogenetic event and, in most cases, a hallmark of the pathology. In the attempt to identify anti-AD drug candidates, hundreds of molecules targeting Aβ peptides have been screened. Peptide molecules have been widely explored, appreciating chemical stability, biocompatibility, and low production cost. More recently, many anti-Aβ(1-42) monoclonal antibodies have been developed, given the excellent potential of immunotherapy for treating or preventing AD. Antibodies are versatile ligands that bind a large variety of molecules with high affinity and specificity; however, their extensive therapeutic application is complex and requires huge economic investments. Novel approaches to identify alternative antibody formats are considered with great interest. In this context, taking advantage of the favorable peptide properties and the availability of Aβ-antibodies structural data, we followed an innovative research approach to identify short peptide sequences on the model of the binding sites of Aβ(1-42)/antibodies. WAibH and SYSTPGK were designed as mimics of solanezumab and aducanumab, respectively. Circular dichroism and nuclear magnetic resonance analysis reveal that the antibody-derived peptides interact with Aβ(1-42) in the soluble monomeric form. Moreover, AFM microscopy imaging shows that WAibH and SYSTPGK are capable of controlling the Aβ(1-42) aggregation. The strategy to identify WAibH and SYSTPGK is innovative and can be widely applied for new anti-Aβ antibody mimicking peptides.

Over the past years, several lines of evidence support a therapeutic potential of Cannabis derivatives and in particular phytocannabinoids. ?9-THC and cannabidiol (CBD) are the most abundant phytocannabinoids in Cannabis plants and therapeutic application for both compounds have been suggested. However, CBD is recently emerging as a therapeutic agent in numerous pathological conditions since devoid of the psychoactive side effects exhibited instead by ?9-THC. In this review, we highlight the pharmacological activities of CBD, its cannabinoid receptor-dependent and -independent action, its biological effects focusing on immunomodulation, angiogenetic properties, and modulation of neuronal and cardiovascular function. Furthermore, the therapeutic potential of cannabidiol is also highlighted, in particular in nuerological diseases and cancer.

Humulus lupulus (Hop) contains numerous metabolites with anticancer potential. Despite this, their immunomodulatory activity, and in particular of bitter acids, is unknown. In this study, we demonstrated that a Hop pellet extract fraction containing bitter acids possesses immunomodulatory activity by enhancing Natural Killer (NK) cells function. After fractionation by semi-preparative Liquid Chromatography, three different fractions were obtained. The phytocomplex and the fractions were tested to verify the ability to modulate the NK compartment. Cytofluorimetric analysis revealed that a fraction containing bitter acids was able to up-regulate of NKG2D and NKp44 activating receptors. A further simplification yield a fraction mainly composed by Humulinones and Cohulupone derivatives, that at the concentration of 0.1 µg/mL induced selective activation of Nkp44 receptor and enhanced the cytolytic activity of NK cells against leukemia cell line K562. Those results point out the possible use of Hop bitter acids as natural immunostimulants and adjuvants in chemotherapy protocols.