Tumor-infiltrating immune cells can be conditioned by molecules released within the microenvironment to thwart antitumor immune responses, thereby facilitating tumor growth. Among immune cells, neutrophils play an important protumorigenic role by favoring neoangiogenesis and/or by suppressing antitumor immune responses. Tumor-derived oxysterols have recently been shown to favor tumor growth by inhibiting dendritic cell migration toward lymphoid organs. We report that tumor-derived oxysterols recruit protumor neutrophils in a liver X receptor (LXR)-independent, CXCR2-dependent manner, thus favoring tumor growth by promoting neoangiogenesis and immunosuppression. We demonstrate that interfering with the oxysterol-CXCR2 axis delays tumor growth and prolongs the overall survival of tumor-bearing mice. These results identify an unanticipated protumor function of the oxysterol-CXCR2 axis and a possible target for cancer therapy.

Recent data and publications suggest a promiscuous behaviour for GPR17, a class-A GPCR operated by different classes of ligands, such as uracil nucleotides, cysteinyl-leukotrienes and oxysterols. This observation, together with the ability of several class-A GPCRs to form homo- and hetero-dimers, is likely to unveil new pathophysiological roles and novel emerging pharmacological properties for some of these GPCRs, including GPR17. This receptor shares structural, phylogenetic and functional properties with some chemokine receptors, CXCRs. Both GPR17 and CXCR2 are operated by oxysterols, and both GPR17 and CXCR ligands have been demonstrated to have a role in orchestrating inflammatory responses and oligodendrocyte precursor cell differentiation to myelinating cells in acute and chronic diseases of the central nervous system. Here, by combining in silico modelling data with in vitro validation in (i) a classical reference pharmacological assay for GPCR activity and (ii) a model of maturation of primary oligodendrocyte precursor cells, we demonstrate that GPR17 can be activated by SDF-1, a ligand of chemokine receptors CXCR4 and CXCR7, and investigate the underlying molecular recognition mechanism. We also demonstrate that cangrelor, a GPR17 orthosteric antagonist, can block the SDF-1-mediated activation of GPR17 in a concentration-dependent manner. The ability of GPR17 to respond to different classes of GPCR ligands suggests that this receptor modifies its function depending on the extracellular mileu changes occurring under specific pathophysiological conditions and advocates it as a strategic target for neurodegenerative diseases with an inflammatory/immune component.

In endothelial cells, erythropoietin receptors (EPORs) mediate the protective, proliferative and angiogenic effects of EPO and its analogues, which act as EPOR agonists. Because hormonal receptors undergo functional changes upon chronic exposure to agonists and because erythropoiesis-stimulating agents (ESAs) are used for the long-term treatment of anemia, it is critical to determine the mechanism by which EPOR responsiveness is regulated at the vascular level after prolonged exposure to ESAs. Here, we investigated EPOR desensitization/resensitization in human umbilical vein endothelial cells (HUVECs) upon exposure to three ESAs with different pharmacokinetic profiles, epoetin alpha (EPOα), darbepoetin alpha (DarbEPO) and continuous EPOR activator (CERA). These agonists all induced activation of the transcription factor STAT-5, which is a component of the intracellular pathway associated with EPORs. STAT-5 activation occurred with either monophasic or biphasic kinetics for EPOα/DarbEPO and CERA, respectively. ESAs, likely through activation of the STAT-5 pathway, induced endothelial cell proliferation and stimulated angiogenesis in vitro, demonstrating a functional role for epoetins on endothelial cells. All epoetins induced EPOR desensitization with more rapid kinetics for CERA compared to EPOα and DarbEPO. However, the recovery of receptor responsiveness was strictly dependent on the type of epoetin, the agonist concentration and the time of exposure to the agonist. EPOR resensitization occurred with more rapid kinetics after exposure to low epoetin concentrations for a short period of desensitization. When the highest concentration of agonists was tested, the recovery of receptor responsiveness was more rapid with CERA compared to EPOα and was completely absent with DarbEPO. Our results demonstrate that these three ESAs regulate EPOR resensitization by very different mechanisms and that both the type of molecule and the length of EPOR stimulation are factors that are critical for the control of EPOR functioning in endothelial cells. The differences observed in receptor resensitization after stimulation with the structurally different ESAs are most likely due different control mechanisms of receptor turnover at the intracellular level.

Nucleotides and cysteinyl-leukotrienes (CysLTs) are unrelated signaling molecules inducing multiple effects through separate G-protein-coupled receptors: the P2Y and the CysLT receptors. Here we show that GPR17, a Gi-coupled orphan receptor at intermediate phylogenetic position between P2Y and CysLT receptors, is specifically activated by both families of endogenous ligands, leading to both adenylyl cyclase inhibition and intracellular calcium increases. Agonist-response profile, as determined by [(35)S]GTPgammaS binding, was different from that of already known CysLT and P2Y receptors, with EC(50) values in the nanomolar and micromolar range, for CysLTs and uracil nucleotides, respectively. Both rat and human receptors are highly expressed in the organs typically undergoing ischemic damage, that is, brain, heart and kidney. In vivo inhibition of GPR17 by either CysLT/P2Y receptor antagonists or antisense technology dramatically reduced ischemic damage in a rat focal ischemia model, suggesting GPR17 as the common molecular target mediating brain damage by nucleotides and CysLTs. In conclusion, the deorphanization of GPR17 revealed a dualistic receptor for two endogenous unrelated ligand families. These findings may lead to dualistic drugs of previously unexplored therapeutic potential.

Adult mesenchymal stem cells (MSCs) play a crucial role in the maintenance of tissue homeostasis and in regenerative processes. Among the different MSC types, the gingiva-derived mesenchymal stem cells (GMSCs) have arisen as a promising tool to promote the repair of damaged tissues secreting trophic mediators that affect different types of cells involved in regenerative processes. Tumour necrosis factor (TNF)-α is one of the key mediators of inflammation that could affect tissue regenerative processes and modify the MSC properties in in-vitro applications. To date, no data have been reported on the effects of TNF-α on GMSC trophic activities and how its modulation with anti-inflammatory agents from natural sources could modulate the GMSC properties.

The epithelial-mesenchymal transition (EMT) is a complex process in which cell phenotype switches from the epithelial to mesenchymal one. The deregulations of this process have been related with the occurrence of different diseases such as lung cancer and fibrosis. In the last decade, several efforts have been devoted in understanding the mechanisms that trigger and sustain this transition process. Adenosine is a purinergic signaling molecule that has been involved in the onset and progression of chronic lung diseases and cancer through the A2B adenosine receptor subtype activation, too. However, the relationship between A2BAR and EMT has not been investigated, yet. Herein, the A2BAR characterization was carried out in human epithelial lung cells. Moreover, the effects of receptor activation on EMT were investigated in the absence and presence of transforming growth factor-beta (TGF-β1), which has been known to promote the transition. The A2BAR activation alone decreased and increased the expression of epithelial markers (E-cadherin) and the mesenchymal one (Vimentin, N-cadherin), respectively, nevertheless a complete EMT was not observed. Surprisingly, the receptor activation counteracted the EMT induced by TGF-β1. Several intracellular pathways regulate the EMT: high levels of cAMP and ERK1/2 phosphorylation has been demonstrated to counteract and promote the transition, respectively. The A2BAR stimulation was able to modulated these two pathways, cAMP/PKA and MAPK/ERK, shifting the fine balance toward activation or inhibition of EMT. In fact, using a selective PKA inhibitor, which blocks the cAMP pathway, the A2BAR-mediated EMT promotion were exacerbated, and conversely the selective inhibition of MAPK/ERK counteracted the receptor-induced transition. These results highlighted the A2BAR as one of the receptors involved in the modulation of EMT process. Nevertheless, its activation is not enough to trigger a complete transition, its ability to affect different intracellular pathways could represent a mechanism at the basis of EMT maintenance/inhibition based on the extracellular microenvironment. Despite further investigations are needed, herein for the first time the A2BAR has been related to the EMT process, and therefore to the different EMT-related pathologies.

A high cell proliferation rate, invasiveness and resistance to chemotherapy are the main features of glioblastoma (GBM). GBM aggressiveness has been widely associated both with a minor population of cells presenting stem-like properties (cancer stem-like cells, CSCs) and with the ability of tumor cells to acquire a mesenchymal phenotype (epithelial-mesenchymal transition, EMT). Carnosol (CAR), a natural inhibitor of MDM2/p53 complex, has been attracted attention for its anti-cancer effects on several tumor types, including GBM. Herein, the effects of CAR on U87MG-derived CSC viability and stemness features were evaluated. CAR decreased the rate of CSC formation and promoted the CSC apoptotic cell death through p53 functional reactivation. Moreover, CAR was able to control the TNF-α/TGF-β-induced EMT, counteracting the effects of the cytokine on EMT master regulator genes (Slug, Snail, Twist and ZEB1) and modulating the activation of miR-200c, a key player in the EMT process. Finally, CAR was able to increase the temozolomide (TMZ) anti-proliferative effects. These findings demonstrate that CAR affected the different intracellular mechanism of the complex machinery that regulates GBM stemness. For the first time, the diterpene was highlighted as a promising lead for the development of agents able to decrease the stemness features, thus controlling GBM aggressiveness.

Increasing evidence suggests that intestinal dysfunctions may represent early events in Alzheimer's disease and contribute to brain pathology. This study examined the relationship between onset of cognitive impairment and colonic dysfunctions in a spontaneous AD model before the full development of brain pathology. SAMP8 mice underwent Morris water maze and assessment of faecal output at four, six and eight months of age. In vitro colonic motility was examined. Faecal and colonic Aβ, tau proteins, α-synuclein and IL-1β were assessed by ELISA. Colonic citrate synthase activity was assessed by spectrophotometry. Colonic NLRP3, caspase-1 and ASC expression were evaluated by Western blotting. Colonic eosinophil density and claudin-1 expression were evaluated by immunohistochemistry. The effect of Aβ on NLRP3 signalling and mitochondrial function was tested in cultured cells. Cognitive impairment and decreased faecal output occurred in SAMP8 mice from six months. When compared with SAMR1, SAMP8 animals displayed: (1) impaired in vitro colonic contractions; (2) increased enteric AD-related proteins, IL-1β, active-caspase-1 expression and eosinophil density; and (3) decreased citrate synthase activity and claudin-1 expression. In THP-1 cells, Aβ promoted IL-1β release, which was abrogated upon incubation with caspase-1 inhibitor or in ASC-/- cells. Aβ decreased mitochondrial function in THP-1 cells. In SAMP8, enteric AD-related proteins deposition, inflammation and impaired colonic excitatory neurotransmission, occurring before the full brain pathology development, could contribute to bowel dysmotility and represent prodromal events in AD.

The aim of this study was to investigate the potential of surface plasmon resonance (SPR) spectroscopy for the measurement of real-time ligand-binding affinities and kinetic parameters for GPR17, a G protein-coupled receptor (GPCR) of major interest in medicinal chemistry as potential target in demyelinating diseases. The receptor was directly captured, in a single-step, from solubilized membrane extracts on the sensor chip through a covalently bound anti-6x-His-antibody and retained its ligand binding activity for over 24 h. Furthermore, our experimental setup made possible, after a mild regeneration step, to remove the bound receptor without damaging the antibody, and thus to reuse many times the same chip. Two engineered variants of GPR17, designed for crystallographic studies, were expressed in insect cells, extracted from crude membranes and analyzed for their binding with two high affinity ligands: the antagonist Cangrelor and the agonist Asinex 1. The calculated kinetic parameters and binding constants of ligands were in good agreement with those reported from activity assays and highlighted a possible functional role of the N-terminal residues of the receptor in ligand recognition and binding. Validation of SPR results was obtained by docking and molecular dynamics of GPR17-ligands interactions and by functional in vitro studies. The latter allowed us to confirm that Asinex 1 behaves as GPR17 receptor agonist, inhibits forskolin-stimulated adenylyl cyclase pathway and promotes oligodendrocyte precursor cell maturation and myelinating ability.

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC). Treatment options for TNBC patients are limited and further insights into disease aetiology are needed to develop better therapeutic approaches. microRNAs' ability to regulate multiple targets could hold a promising discovery approach to pathways relevant for TNBC aggressiveness. Thus, we address the role of miRNAs in controlling three signalling pathways relevant to the biology of TNBC, and their downstream phenotypes.

The use of platinum-based anticancer drugs, such as cisplatin, oxaliplatin, and carboplatin, is a common frontline option in cancer management, but they have debilitating side effects and can lead to drug resistance. Combination therapy with other chemotherapeutic agents, such as capecitabine and gemcitabine, has been explored. One approach to overcome these limitations is the modification of traditional Pt(II) drugs to obtain new molecules with an improved pharmacological profile, such as Pt(IV) prodrugs. The design, synthesis, and characterization of two novel Pt(IV) prodrugs based on oxaliplatin bearing the anticancer drugs gemcitabine or capecitabine in the axial positions have been reported. These complexes were able to dissociate into their constituents to promote cell death and induce apoptosis and cell cycle blockade in a representative colorectal cancer cell model. Specifically, the complex bearing gemcitabine resulted in being the most active on the HCT116 colorectal cancer cell line with an IC50 value of 0.49 ± 0.04. A pilot study on the encapsulation of these complexes in biocompatible PLGA-PEG nanoparticles is also included to confirm the retention of the pharmacological properties and cellular drug uptake, opening up to the possible delivery of the studied complexes through their nanoformulation.

G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors, and are the target of at least one-third of the current therapeutic drugs on the market. Along their life cycle, GPCRs are accompanied by a range of specialized GPCR-interacting proteins (GIPs), which take part in receptor proper folding, targeting to the appropriate subcellular compartments and in receptor signaling tasks, and also in receptor regulation processes, such as desensitization and internalization. The direction of protein-protein interactions and multi-protein complexes formation is crucial in understanding protein function and their implication in pathological events. Although several methods have been already developed to assay protein complexes, some of them are quite laborious, expensive, and, more important, they do not generate fully quantitative results. Herein, we show a rapid immunoenzymatic assay to quantify GPCR interactionswith its signaling proteins. The recently de-orphanized GPCR, GPR17, was chosen as a GPCR prototype to optimize the assay. In a GPR17 transfected cell line and primary oligodendrocyte precursor cells, GPR17 interaction with proteins involved in the typical GPCR regulation, such as desensitization and internalization machinery, was investigated. The obtained results were validated by co-immunoprecipitation experiments, confirming this new method as a rapid and quantitative assay to study protein-protein interactions.

The steroidogenic 18 kDa translocator protein (TSPO) is an emerging, attractive therapeutic tool for several pathological conditions of the nervous system. Here, 13 high affinity TSPO ligands belonging to our previously described N,N-dialkyl-2-phenylindol-3-ylglyoxylamide (PIGA) class were evaluated for their potential ability to affect the cellular Oxidative Metabolism Activity/Proliferation index, which is used as a measure of astrocyte well-being. The most active PIGA ligands were also assessed for steroidogenic activity in terms of pregnenolone production, and the values were related to the metabolic index in rat and human models. The results showed a positive correlation between the increase in the Oxidative Metabolism Activity/Proliferation index and the pharmacologically induced stimulation of steroidogenesis. The specific involvement of steroid molecules in mediating the metabolic effects of the PIGA ligands was demonstrated using aminoglutethimide, a specific inhibitor of the first step of steroid biosynthesis. The most promising steroidogenic PIGA ligands were the 2-naphthyl derivatives that showed a long residence time to the target, in agreement with our previous data. In conclusion, TSPO ligand-induced neurosteroidogenesis was involved in astrocyte well-being.

Protein responses to extracellular cues are governed by gene transcription, mRNA degradation and translation, and protein degradation. In order to understand how these time-dependent processes cooperate to generate dynamic responses, we analyzed the response of human mammary cells to the epidermal growth factor (EGF). Integrating time-dependent transcript and protein data into a mathematical model, we inferred for several proteins their pre-and post-stimulus translation and degradation coefficients and found that they exhibit complex, time-dependent variation. Specifically, we identified strategies of protein production and degradation acting in concert to generate rapid, transient protein bursts in response to EGF. Remarkably, for some proteins, for which the response necessitates rapidly decreased abundance, cells exhibit a transient increase in the corresponding degradation coefficient. Our model and analysis allow inference of the kinetics of mRNA translation and protein degradation, without perturbing cells, and open a way to understanding the fundamental processes governing time-dependent protein abundance profiles.

Epigenetic regulation may contribute to the beneficial effects of physical activity against age-related neurodegeneration. For example, epigenetic alterations of the gene encoding for α-synuclein (SNCA) have been widely explored in both brain and peripheral tissues of Parkinson's disease samples. However, no data are currently available about the effects of physical exercise on SNCA epigenetic regulation in ageing healthy subjects. The present paper explored whether, in healthy individuals, age and physical activity are related to blood intron1-SNCA (SNCAI1 ) methylation, as well as further parameters linked to such epigenetic modification (total, oligomeric α-synuclein and DNA methyltransferase concentrations in the blood). Here, the SNCAI1 methylation status increased with ageing, and consistent with this result, low α-synuclein levels were found in the blood. The direct relationship between SNCAI1 methylation and α-synuclein levels was observed in samples characterized by blood α-synuclein concentrations of 76.3 ng/mg protein or lower (confidence interval (CI) = 95%). In this selected population, higher physical activity reduced the total and oligomeric α-synuclein levels. Taken together, our data shed light on ageing- and physical exercise-induced changes on the SNCA methylation status and protein levels of α-synuclein.

Breast cancer is the most common cancer in women worldwide. The tumor microenvironment contributes to tumor progression by inducing cell dissemination from the primary tumor and metastasis. TGFβ signaling is involved in breast cancer progression and is specifically elevated during metastatic transformation in aggressive breast cancer. In this study, we performed genomewide correlation analysis of TGFBR2 expression in a panel of 51 breast cancer cell lines and identified that MET is coregulated with TGFBR2. This correlation was confirmed at the protein level in breast cancer cell lines and human tumor tissues. Flow cytometric analysis of luminal and basal-like breast cancer cell lines and examination of 801 tumor specimens from a prospective cohort of breast cancer patients using reverse phase protein arrays revealed that expression of TGFBR2 and MET is increased in basal-like breast cancer cell lines, as well as in triple-negative breast cancer tumor tissues, compared to other subtypes. Using real-time cell analysis technology, we demonstrated that TGFβ1 triggered hepatocyte growth factor (HGF)-induced and MET-dependent migration in vitro. Bioinformatic analysis predicted that TGFβ1 induces expression of C-ets-1 as a candidate transcription factor regulating MET expression. Indeed, TGFβ1-induced expression of ETS1 and breast cancer cell migration was blocked by knockdown of ETS1. Further, we identified that MET is a direct target of miR-128-3p and that this miRNA is negatively regulated by TGFβ1. Overexpression of miR-128-3p reduced MET expression and abrogated HGF-induced cell migration of invasive breast cancer cells. In conclusion, we have identified that TGFβ1 regulates HGF-induced and MET-mediated cell migration, through positive regulation of C-ets-1 and negative regulation of miR-128-3p expression in basal-like breast cancer cell lines and in triple-negative breast cancer tissue.

Microglial cells are a component of the innate immune system in the brain that support cell-to-cell communication via secreted molecules and extracellular vesicles (EVs). EVs can be divided into two major populations: large (LEVs) and small (SEVs) EVs, carrying different mediators, such as proteins, lipids, and miRNAs. The microglia EVs cargo crucially reflects the status of parental cells and can lead to both beneficial and detrimental effects in many physiopathological states. Herein, a workflow for the extraction and characterization of SEVs and LEVs from human C20 and HMC3 microglia cell lines derived, respectively, from adult and embryonic microglia is reported. EVs were gathered from the culture media of the two cell lines by sequential ultracentrifugation steps and their biochemical and biophysical properties were analyzed by Western blot, transmission electron microscopy, and dynamic light scattering. Although the C20- and HMC3-derived EVs shared several common features, C20-derived EVs were slightly lower in number and more polydispersed. Interestingly, C20- but not HMC3-SEVs were able to interfere with the proliferation of U87 glioblastoma cells. This correlated with the different relative levels of eight miRNAs involved in neuroinflammation and tumor progression in the C20- and HMC3-derived EVs, which in turn reflected a different basal activation state of the two cell types. Our data fill a gap in the community of microglia EVs, in which the preparations from human cells have been poorly characterized so far. Furthermore, these results shed light on both the differences and similarities of EVs extracted from different human microglia cell models, underlining the need to better characterize the features and biological effects of EVs for therein useful and correct application.

Small-molecules acting as positive allosteric modulators (PAMs) of the A2B adenosine receptor (A2B AR) could potentially represent a novel therapeutic strategy for pathological conditions characterised by altered bone homeostasis, including osteoporosis. We investigated a library of compounds (4-13) exhibiting different degrees of chemical similarity with three indole derivatives (1-3), which have been recently identified by us as PAMs of the A2B AR able to promote mesenchymal stem cell differentiation and bone formation. Evaluation of mineralisation activity of 4-13 in the presence and in the absence of the agonist BAY60-6583 allowed the identification of lead compounds with therapeutic potential as anti-osteoporosis agents. Further biological characterisation of one of the most performing compounds, the benzofurane derivative 9, confirmed that such a molecule behaves as PAM of the A2B AR.

We address the contribution of kinase domain structure and catalytic activity to membrane trafficking of TrkA receptor tyrosine kinase. We conduct a systematic comparison between TrkA-wt, an ATP-binding defective mutant (TrkA-K544N) and other mutants displaying separate functional impairments of phosphorylation, ubiquitination, or recruitment of intracellular partners. We find that only K544N mutation endows TrkA with restricted membrane mobility and a substantial increase of cell surface pool already in the absence of ligand stimulation. This mutation is predicted to drive a structural destabilization of the αC helix in the N-lobe by molecular dynamics simulations, and enhances interactions with elements of the actin cytoskeleton. On the other hand, a different TrkA membrane immobilization is selectively observed after NGF stimulation, requires both phosphorylation and ubiquitination to occur, and is most probably related to the signaling abilities displayed by the wt but not mutated receptors. In conclusion, our results allow to distinguish two different TrkA membrane immobilization modes and demonstrate that not all kinase-inactive mutants display identical membrane trafficking.

Intracellular pathogens, such as Chlamydia trachomatis, have been recently shown to induce degradation of p53 during infection, thus impairing the protective response of the host cells. Therefore, p53 reactivation by disruption of the p53-MDM2 complex could reduce infection and restore pro-apoptotic effect of p53. Here, we report the identification of a novel MDM2 inhibitor with potential antitumoural and antibacterial activity able to reactivate p53. A virtual screening was performed on an in-house chemical library, previously synthesised for other targets, and led to the identification of a hit compound with a benzo[a]dihydrocarbazole structure, RM37. This compound induced p53 up-regulation in U343MG glioblastoma cells by blocking MDM2-p53 interaction and reduced tumour cell growth. NMR studies confirmed its ability to dissociate the MDM2-p53 complex. Notably, RM37 reduced Chlamydia infection in HeLa cells in a concentration-dependent manner and ameliorated the inflammatory status associated with infection.