Combined Inhibition of Hedgehog and HDAC6: In Vitro and In Vivo Studies Reveal a New Role for Lysosomal Stress in Reducing Glioblastoma Cell Viability.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor in adults. The invasiveness and the rapid progression that characterize GBM negatively impact patients' survival. Temozolomide (TMZ) is currently considered the first-choice chemotherapeutic agent. Unfortunately, over 50% of patients with GBM do not respond to TMZ treatment, and the mutation-prone nature of GBM enables the development of resistance mechanisms. Therefore, efforts have been devoted to the dissection of aberrant pathways involved in GBM insurgence and resistance in order to identify new therapeutic targets. Among them, sphingolipid signaling, Hedgehog (Hh) pathway, and the histone deacetylase 6 (HDAC6) activity are frequently dysregulated and may represent key targets to counteract GBM progression. Given the positive correlation between Hh/HDAC6/sphingolipid metabolism in GBM, we decided to perform a dual pharmacological inhibition of Hh and HDAC6 through cyclopamine and tubastatin A, respectively, in a human GMB cell line and zebrafish embryos. The combined administration of these compounds elicited a more significant reduction of GMB cell viability than did single treatments in vitro and in cells orthotopically transplanted in the zebrafish hindbrain ventricle. We demonstrated, for the first time, that the inhibition of these pathways induces lysosomal stress which results in an impaired fusion of lysosomes with autophagosomes and a block of sphingolipid degradation in GBM cell lines. This condition, which we also recapitulated in zebrafish embryos, suggests an impairment of lysosome-dependent processes involving autophagy and sphingolipid homeostasis and might be instrumental in the reduction of GBM progression.

Secreted miR-153 Controls Proliferation and Invasion of Higher Gleason Score Prostate Cancer.

Prostate cancer (PC) is a male common neoplasm and is the second leading cause of cancer death in American men. PC is traditionally diagnosed by the evaluation of prostate secreted antigen (PSA) in the blood. Due to the high levels of false positives, digital rectal examination and transrectal ultrasound guided biopsy are necessary in uncertain cases with elevated PSA levels. Nevertheless, the high mortality rate suggests that new PC biomarkers are urgently needed to help clinical diagnosis. In a previous study, we have identified a network of genes, altered in high Gleason Score (GS) PC (GS ≥ 7), being regulated by miR-153. Until now, no publication has explained the mechanism of action of miR-153 in PC. By in vitro studies, we found that the overexpression of miR-153 in high GS cell lines is required to control cell proliferation, migration and invasion rates, targeting Kruppel-like factor 5 (KLF5). Moreover, miR-153 could be secreted by exosomes and microvesicles in the microenvironment and, once entered into the surrounding tissue, could influence cellular growth. Being upregulated in high GS human PC, miR-153 could be proposed as a circulating biomarker for PC diagnosis.

Galactocerebrosidase deficiency induces an increase in lactosylceramide content: A new hallmark of Krabbe disease?

Galactocerebrosidase (GALC) hydrolyses galactose residues from various substrates, including galactosylceramide, psychosine (galactosylsphingosine), and lactosylceramide. Its severe deficiency has been associated with the accumulation of psychosine, a toxic molecule with detergent-like features, which alters membrane structures and signalling pathways, inducing the death of oligodendrocytes and a sequence of events in the nervous system that explain the appearance of many clinical signs typical of Krabbe disease. Nevertheless, new evidence suggests the existence of other possible links among GALC action, myelination, and myelin stability, apart from psychosine release. In this study, we demonstrated that lactosylceramide metabolism is impaired in fibroblasts isolated from patients with Krabbe disease in the absence of psychosine accumulation. This event is responsible for the aberrant and constitutive activation of the AKT/prolin-rich AKT substrate of 40 kDa (PRAS40) signalling axis, inducing B cell lymphoma 2 (BCL2) overexpression and glycogen synthase kinase 3 beta (GSK-3ß) inhibition. In addition, nuclear factor E2-related factor 2 (NRF2) showed increased nuclear translocation. Due to the relevance of these molecular alterations in neurodegeneration, lactosylceramide increase should be evaluated as a novel marker of Krabbe disease, and because of its significant connections with signalling pathways.

Choline and nicotine increase glioblastoma cell proliferation by binding and activating α7- and α9- containing nicotinic receptors.

Glioblastomas (GBMs), the most frequent and aggressive human primary brain tumours, have altered cell metabolism, and one of the strongest indicators of malignancy is an increase in choline compounds. Choline is also a selective agonist of some neuronal nicotinic acetylcholine receptor (nAChR) subtypes. As little is known concerning the expression of nAChR in glioblastoma cells, we analysed in U87MG human grade-IV astrocytoma cell line and GBM5 temozolomide-resistant glioblastoma cells selected from a cancer stem cell-enriched culture, molecularly, pharmacologically and functionally which nAChR subtypes are expressed and,whether choline and nicotine can affect GBM cell proliferation. We found that U87MG and GBM5 cells express similar nAChR subtypes, and choline and nicotine increase their proliferation rate and activate the anti-apoptotic AKT and pro-proliferative ERK pathways. These effects are blocked by the presence of non-cell-permeable peptide antagonists selective for α7- and α9-containing nicotinic receptors. siRNA-mediated silencing of α7 or α9 subunit expression also selectively prevents the effects of nicotine and choline on GBM cell proliferation. Our findings indicate that nicotine and choline activate the signalling pathways involved in the proliferation of GBM cells, and that these effects are mediated by α7 and α9-containing nAChRs. This suggests that these nicotinic receptors may contribute to the aggressive behaviour of this tumor and may indicate new therapeutic strategies against high-grade human brain tumours.

Modifications at C(5) of 2-(2-Pyrrolidinyl)-Substituted 1,4-Benzodioxane Elicit Potent α4ß2 Nicotinic Acetylcholine Receptor Partial Agonism with High Selectivity over the α3ß4 Subtype.

A series of diastereomeric 2-(2-pyrrolidinyl)-1,4-benzodioxanes bearing a small, hydrogen-bonding substituent at the 7-, 6-, or 5-position of benzodioxane have been studied for α4ß2 and α3ß4 nicotinic acetylcholine receptor affinity and activity. Analogous to C(5)H replacement with N and to a much greater extent than decoration at C(7), substitution at benzodioxane C(5) confers very high α4ß2/α3ß4 selectivity to the α4ß2 partial agonism. Docking into the two receptor structures recently determined by cryo-electron microscopy and site-directed mutagenesis at the minus ß2 side converge in indicating that the limited accommodation capacity of the ß2 pocket, compared to that of the ß4 pocket, makes substitution at C(5) rather than at more projecting C(7) position determinant for this pursued subtype selectivity.

Behavioural and pharmacological profiles of zebrafish administrated pyrrolidinyl benzodioxanes and prolinol aryl ethers with high affinity for heteromeric nicotinic acetylcholine receptors.

RATIONALE: Prolinol aryl ethers and their rigidified analogues pyrrolidinyl benzodioxanes have a high affinity for mammalian α4ß2 nicotinic acetylcholine receptors (nAChRs). Electrophysiological studies have shown that the former are full agonists and the latter partial agonists or antagonists of human α4ß2 receptors, but their in vivo effects are unknown. OBJECTIVES AND METHODS: As α4ß2 nAChRs play an important role in the cognition and the rewarding effects of nicotine, we tested the effects of two full agonists and one antagonist on spatial learning, memory and attention in zebrafish using a T-maze task and virtual object recognition test (VORT). The effect of a partial agonist in reducing nicotine-induced conditioned place preference (CPP) was also investigated. RESULTS: In comparison with the vehicle alone, the full agonists MCL-11 and MCL-28 induced a significant cognitive enhancement as measured by the reduced running time in the T-maze and increased attention as measured by the increased discrimination index in the VORT. MCL-11 was 882 times more potent than nicotine. The two compounds were characterised by an inverted U-shaped dose-response curve, and their effects were blocked by the co-administration of the antagonist MCL-117, which alone had no effect. The partial agonist MCL-54 induced CPP and had an inverted U-shaped dose-response curve similar to that of nicotine but blocked the reinforcing effect of co-administered nicotine. Binding studies showed that all of the compounds have a higher affinity for heteromeric [3H]-epibatidine receptors than [125I]-αBungarotoxin receptors. MCL-11 was the most selective of heteromeric receptors. CONCLUSIONS: These behavioural studies indicate that full agonist prolinol aryl ethers are very active in increasing spatial learning, memory and attention in zebrafish. The benzodioxane partial agonist MCL-54 reduced nicotine-induced CPP, and the benzodioxane antagonist MCL-117 blocked all agonist-induced activities.

Persistent cognitive and affective alterations at late withdrawal stages after long-term intermittent exposure to tobacco smoke or electronic cigarette vapour: Behavioural changes and their neurochemical correlates.

Smoking cessation induces a withdrawal syndrome associated with anxiety, depression, and impaired neurocognitive functions, but much less is known about the withdrawal of e-cigarettes (e-CIG). We investigated in Balb/c mice the behavioural and neurochemical effects of withdrawal for up to 90 days after seven weeks' intermittent exposure to e-CIG vapour or cigarette smoke (CIG). The withdrawal of e-CIG and CIG induced early behavioural alterations such as spatial memory deficits (spatial object recognition task), increased anxiety (elevated plus maze test) and compulsive-like behaviour (marble burying test) that persisted for 60-90 days. Notably, attention-related (virtual object recognition task) and depression-like behaviours (tail suspension and sucrose preference tests) appeared only 15-30 days after withdrawal and persisted for as long as up to 90 days. At hippocampal level, the withdrawal-induced changes in the levels of AMPA receptor GluA1 and GluA2/3 subunits, PSD 95 protein, corticotropin-releasing factor (Crf) and Crf receptor 1 (CrfR1) mRNA were biphasic: AMPA receptor subunit and PSD95 protein levels initially remained unchanged and decreased after 60-90 days, whereas Crf/CrfR1 mRNA levels initially increased and then markedly decreased after 60 days. These late reductions correlated with the behavioural impairments, particularly the appearance of depression-like behaviours. Our findings show that major behavioural and neurochemical alterations persist or even first appear late after the withdrawal of chronic CIG smoke or e-CIG vapour exposure, and underline importance of conducting similar studies of humans, including e-CIG vapers.

Detrimental and protective action of microglial extracellular vesicles on myelin lesions: astrocyte involvement in remyelination failure.

Microglia are highly plastic immune cells which exist in a continuum of activation states. By shaping the function of oligodendrocyte precursor cells (OPCs), the brain cells which differentiate to myelin-forming cells, microglia participate in both myelin injury and remyelination during multiple sclerosis. However, the mode(s) of action of microglia in supporting or inhibiting myelin repair is still largely unclear. Here, we analysed the effects of extracellular vesicles (EVs) produced in vitro by either pro-inflammatory or pro-regenerative microglia on OPCs at demyelinated lesions caused by lysolecithin injection in the mouse corpus callosum. Immunolabelling for myelin proteins and electron microscopy showed that EVs released by pro-inflammatory microglia blocked remyelination, whereas EVs produced by microglia co-cultured with immunosuppressive mesenchymal stem cells promoted OPC recruitment and myelin repair. The molecular mechanisms responsible for the harmful and beneficial EV actions were dissected in primary OPC cultures. By exposing OPCs, cultured either alone or with astrocytes, to inflammatory EVs, we observed a blockade of OPC maturation only in the presence of astrocytes, implicating these cells in remyelination failure. Biochemical fractionation revealed that astrocytes may be converted into harmful cells by the inflammatory EV cargo, as indicated by immunohistochemical and qPCR analyses, whereas surface lipid components of EVs promote OPC migration and/or differentiation, linking EV lipids to myelin repair. Although the mechanisms through which the lipid species enhance OPC maturation still remain to be fully defined, we provide the first demonstration that vesicular sphingosine 1 phosphate stimulates OPC migration, the first fundamental step in myelin repair. From this study, microglial EVs emerge as multimodal and multitarget signalling mediators able to influence both OPCs and astrocytes around myelin lesions, which may be exploited to develop novel approaches for myelin repair not only in multiple sclerosis, but also in neurological and neuropsychiatric diseases characterized by demyelination.

Novel N-aryl nicotinamide derivatives: Taking stock on 3,6-diazabicyclo[3.1.1]heptanes as ligands for neuronal acetylcholine receptors.

We designed the synthesis of a small library of 3-substituted-3,6-diazabicyclo[3.1.1]heptanes whose affinity on neuronal nicotinic receptors (nAChRs) was evaluated. Among the synthesized compounds, the 5-(3,6-diazabicyclo[3.1.1]heptane-3-yl)-N-(2-fluorophenyl)nicotinamide 43 proved to be the most interesting compound with α4ß2Ki value of 10 pM and a very high α7/α4ß2 selectivity. Furthermore, compounds 35, 39 and 43 elicited a selective partial agonist activity for α4ß2 nAChR subtype. Finally, in this paper we also report the conclusions on the 3,6-diazabicyclo[3.1.1]heptanes as ligands for nAChRs, resulting from our consolidated structure activity relationship (SAR) studies on this template.

Increased sensitivity to Δ9-THC-induced rewarding effects after seven-week exposure to electronic and tobacco cigarettes in mice.

Cigarette (CIG) smoking often precedes the use of illegal drugs. Electronic-cigarettes (e-CIGs) have been promoted as a means of stopping smoking and reducing the harmful effects of CIGs on the population. However, although e-CIGs eliminate some of the morbidity associated with combustible tobacco, they are still nicotine-delivery devices. In order to study whether the nicotine delivered via e-CIG acts as "a gateway drug" to the use of cannabis, we analysed the behavioural and molecular effects of 7 weeks' pre-exposure to air (AIR), e-CIGs or CIGs on addiction-related conditioned place preference (CPP) in mice using a sub-threshold (0.01 mg/kg) dose of delta-9-tetrahydrocannabinol (Δ9-THC), the principal psychoactive constituent of cannabis. After 8 and 66 days of withdrawal, this Δ9-THC dose was ineffective in inducing CPP in mice pre-exposed to pump-driven AIR, but very effective in mice pre-exposed to e-CIGs or CIGs. Exposure to e-CIGs or CIGs increases the expression of ΔFosB in the nucleus accumbens (NAc), which remains high during short-term e-CIG or CIG withdrawal and long-term CIG withdrawal and is not influenced by treatment with Δ9-THC. At the end of e-CIG or CIG exposure and during withdrawal, the mice also had a higher AMPA receptors GluA1/GluA2-3 ratio in the NAc. Chronic nicotine exposure increases sensitivity to rewarding effects of Δ9-THC in mice and produces long-lasting neurobiological changes regardless of the delivery method (CIG vs. e-CIG). The exposure to passive tobacco smoke or e-CIG vapours can similarly increase vulnerability to the effects of cannabis and possibly other drugs of abuse.

HDAC8 regulates canonical Wnt pathway to promote differentiation in skeletal muscles.

Histone deacetylase 8 (HDAC8) is a class 1 histone deacetylase and a member of the cohesin complex. HDAC8 is expressed in smooth muscles, but its expression in skeletal muscle has not been described. We have shown for the first time that HDAC8 is expressed in human and zebrafish skeletal muscles. Using RD/12 and RD/18 rhabdomyosarcoma cells with low and high differentiation potency, respectively, we highlighted a specific correlation with HDAC8 expression and an advanced stage of muscle differentiation. We inhibited HDAC8 activity through a specific PCI-34051 inhibitor in murine C2C12 myoblasts and zebrafish embryos, and we observed skeletal muscles differentiation impairment. We also found a positive regulation of the canonical Wnt signaling by HDAC8 that might explain muscle differentiation defects. These findings suggest a novel mechanism through which HDAC8 expression, in a specific time window of skeletal muscle development, positively regulates canonical Wnt pathway that is necessary for muscle differentiation.

Potent Antiglioblastoma Agents by Hybridizing the Onium-Alkyloxy-Stilbene Based Structures of an α7-nAChR, α9-nAChR Antagonist and of a Pro-Oxidant Mitocan.

Adenocarcinoma and glioblastoma cell lines express α7- and α9α10-containing nicotinic acetylcholine receptors (nAChRs), whose activation promotes tumor cell growth. On these cells, the triethylammoniumethyl ether of 4-stilbenol MG624, a known selective antagonist of α7 and α9α10 nAChRs, has antiproliferative activity. The structural analogy of MG624 with the mitocan RDM-4'BTPI, triphenylphosphoniumbutyl ether of pterostilbene, suggested us that molecular hybridization among their three substructures (stilbenoxy residue, alkylene linker, and terminal onium) and elongation of the alkylene linker might result in novel antitumor agents with higher potency and selectivity. We found that lengthening the ethylene bridge in the triethylammonium derivatives results in more potent and selective toxicity toward adenocarcinoma and glioblastoma cells, which was paralleled by increased α7 and α9α10 nAChR antagonism and improved ability of reducing mitochondrial ATP production. Elongation of the alkylene linker was advantageous also for the triphenylphosphonium derivatives resulting in a generalized enhancement of antitumor activity, associated with increased mitotoxicity.

Sphingosine-1-Phosphate (S1P) Impacts Presynaptic Functions by Regulating Synapsin I Localization in the Presynaptic Compartment.

Growing evidence indicates that sphingosine-1-P (S1P) upregulates glutamate secretion in hippocampal neurons. However, the molecular mechanisms through which S1P enhances excitatory activity remain largely undefined. The aim of this study was to identify presynaptic targets of S1P action controlling exocytosis. Confocal analysis of rat hippocampal neurons showed that S1P applied at nanomolar concentration alters the distribution of Synapsin I (SynI), a presynaptic phosphoprotein that controls the availability of synaptic vesicles for exocytosis. S1P induced SynI relocation to extrasynaptic regions of mature neurons, as well as SynI dispersion from synaptic vesicle clusters present at axonal growth cones of developing neurons. S1P-induced SynI relocation occurred in a Ca(2+)-independent but ERK-dependent manner, likely through the activation of S1P3 receptors, as it was prevented by the S1P3 receptor selective antagonist CAY1044 and in neurons in which S1P3 receptor was silenced. Our recent evidence indicates that microvesicles (MVs) released by microglia enhance the metabolism of endogenous sphingolipids in neurons and stimulate excitatory transmission. We therefore investigated whether MVs affect SynI distribution and whether endogenous S1P could be involved in the process. Analysis of SynI immunoreactivity showed that exposure to microglial MVs induces SynI mobilization at presynaptic sites and growth cones, whereas the use of inhibitors of sphingolipid cascade identified S1P as the sphingolipid mediating SynI redistribution. Our data represent the first demonstration that S1P induces SynI mobilization from synapses, thereby indicating the phosphoprotein as a novel target through which S1P controls exocytosis. SIGNIFICANCE STATEMENT: Growing evidence indicates that the bioactive lipid sphingosine and its metabolite sphingosine-1-P (S1P) stimulate excitatory transmission. While it has been recently clarified that sphingosine influences directly the exocytotic machinery by activating the synaptic vesicle protein VAMP2 to form SNARE fusion complexes, the molecular mechanism by which S1P promotes neurotransmission remained largely undefined. In this study, we identify Synapsin I, a presynaptic phosphoprotein involved in the control of availability of synaptic vesicles for exocytosis, as the key target of S1P action. In addition, we provide evidence that S1P can be produced at mature axon terminals as well as at immature growth cones in response to microglia-derived signals, which may be important to stabilize nascent synapses and to restore or potentiate transmission.

Sphingosine Kinase 2 and Ceramide Transport as Key Targets of the Natural Flavonoid Luteolin to Induce Apoptosis in Colon Cancer Cells.

The plant flavonoid luteolin exhibits different biological effects, including anticancer properties. Little is known on the molecular mechanisms underlying its actions in colorectal cancer (CRC). Here we investigated the effects of luteolin on colon cancer cells, focusing on the balance between ceramide and sphingosine-1-phosphate (S1P), two sphingoid mediators with opposite roles on cell fate. Using cultured cells, we found that physiological concentrations of luteolin induce the elevation of ceramide, followed by apoptotic death of colon cancer cells, but not of differentiated enterocytes. Pulse studies revealed that luteolin inhibits ceramide anabolism to complex sphingolipids. Further experiments led us to demonstrate that luteolin induces an alteration of the endoplasmic reticulum (ER)-Golgi flow of ceramide, pivotal to its metabolic processing to complex sphingolipids. We report that luteolin exerts its action by inhibiting both Akt activation, and sphingosine kinase (SphK) 2, with the consequent reduction of S1P, an Akt stimulator. S1P administration protected colon cancer cells from luteolin-induced apoptosis, most likely by an intracellular, receptor-independent mechanism. Overall this study reveals for the first time that the dietary flavonoid luteolin exerts toxic effects on colon cancer cells by inhibiting both S1P biosynthesis and ceramide traffic, suggesting its dietary introduction/supplementation as a potential strategy to improve existing treatments in CRC.

Proteomic analysis of human glioblastoma cell lines differently resistant to a nitric oxide releasing agent.

Glioblastoma multiforme is the most aggressive astrocytoma characterized by the development of resistant cells to various cytotoxic stimuli. Nitric oxide (NO) is able to overcome tumor resistance in PTEN mutated rat C6 glioma cells due to its ability to inhibit cell growth by influencing the intracellular distribution of ceramide. The aim of this study is to monitor the effects of NO donor PAPANONOate on ceramide trafficking in human glioma cell lines, CCF-STTG1 (PTEN-mutated, p53-wt) and T98G (PTEN-harboring, p53-mutated), together with the assessment of their differential molecular signature by 2D-DIGE and MALDI mass spectrometry. In the CCF-STTG1 cell line, the results indicate that treatment with PAPANONOate decreased cell proliferation (<50%) and intracellular trafficking of ceramide, assessed by BODIPY-C5Cer, while these events were not observed in the T98G cell line. Proteomic results suggest that CCF-STTG1 cells are characterized by an increased expression of proteins involved in NO-associated ER stress (i.e. protein disulfide-isomerase A3, calreticulin, 78 kDa glucose-regulated protein), which could compromise ceramide delivery from ER to Golgi, leading to ceramide accumulation in ER and partial growth arrest. Conversely, T98G cell lines, resistant to NO exposure, are characterized by increased levels of cytosolic antioxidant proteins (i.e. glutathione-S-transferase P, peroxiredoxin 1), which might buffer intracellular NO. By providing differential ceramide distribution after NO exposure and differential protein expression of two high grade glioma cell lines, this study highlights specific proteins as possible markers for tumor aggressiveness. This study demonstrates that, in two different high grade glioma cell lines, NO exposure results in a different ceramide distribution and protein expression. Furthermore, this study highlights specific proteins as possible markers for tumor aggressiveness.

Glucolipotoxicity impairs ceramide flow from the endoplasmic reticulum to the Golgi apparatus in INS-1 ß-cells.

Accumulating evidence suggests that glucolipotoxicity, arising from the combined actions of elevated glucose and free fatty acid levels, acts as a key pathogenic component in type II diabetes, contributing to ß-cell dysfunction and death. Endoplasmic reticulum (ER) stress is among the molecular pathways and regulators involved in these negative effects, and ceramide accumulation due to glucolipotoxicity can be associated with the induction of ER stress. Increased levels of ceramide in ER may be due to enhanced ceramide biosynthesis and/or decreased ceramide utilization. Here, we studied the effect of glucolipotoxic conditions on ceramide traffic in INS-1 cells in order to gain insights into the molecular mechanism(s) of glucolipotoxicity. We showed that glucolipotoxicity inhibited ceramide utilization for complex sphingolipid biosynthesis, thereby reducing the flow of ceramide from the ER to Golgi. Glucolipotoxicity impaired both vesicular- and CERT-mediated ceramide transport through (1) the decreasing of phospho-Akt levels which in turn possibly inhibits vesicular traffic, and (2) the reducing of the amount of active CERT mainly due to a lower protein levels and increased protein phosphorylation to prevent its localization to the Golgi. In conclusion, our findings provide evidence that glucolipotoxicity-induced ceramide overload in the ER, arising from a defect in ceramide trafficking may be a mechanism that contributes to dysfunction and/or death of ß-cells exposed to glucolipotoxicity.

Autocrine/paracrine sphingosine-1-phosphate fuels proliferative and stemness qualities of glioblastoma stem cells.

Accumulating reports suggest that human glioblastoma contains glioma stem-like cells (GSCs) which act as key determinants driving tumor growth, angiogenesis, and contributing to therapeutic resistance. The proliferative signals involved in GSC proliferation and progression remain unclear. Using GSC lines derived from human glioblastoma specimens with different proliferative index and stemness marker expression, we assessed the hypothesis that sphingosine-1-phosphate (S1P) affects the proliferative and stemness properties of GSCs. The results of metabolic studies demonstrated that GSCs rapidly consume newly synthesized ceramide, and export S1P in the extracellular environment, both processes being enhanced in the cells exhibiting high proliferative index and stemness markers. Extracellular S1P levels reached nM concentrations in response to increased extracellular sphingosine. In addition, the presence of EGF and bFGF potentiated the constitutive capacity of GSCs to rapidly secrete newly synthesized S1P, suggesting that cooperation between S1P and these growth factors is of central importance in the maintenance and proliferation of GSCs. We also report for the first time that S1P is able to act as a proliferative and pro-stemness autocrine factor for GSCs, promoting both their cell cycle progression and stemness phenotypic profile. These results suggest for the first time that the GSC population is critically modulated by microenvironmental S1P, this bioactive lipid acting as an autocrine signal to maintain a pro-stemness environment and favoring GSC proliferation, survival and stem properties.

Sphingolipids: key regulators of apoptosis and pivotal players in cancer drug resistance.

Drug resistance elicited by cancer cells still constitutes a huge problem that frequently impairs the efficacy of both conventional and novel molecular therapies. Chemotherapy usually acts to induce apoptosis in cancer cells; therefore, the investigation of apoptosis control and of the mechanisms used by cancer cells to evade apoptosis could be translated in an improvement of therapies. Among many tools acquired by cancer cells to this end, the de-regulated synthesis and metabolism of sphingolipids have been well documented. Sphingolipids are known to play many structural and signalling roles in cells, as they are involved in the control of growth, survival, adhesion, and motility. In particular, in order to increase survival, cancer cells: (a) counteract the accumulation of ceramide that is endowed with pro-apoptotic potential and is induced by many drugs; (b) increase the synthesis of sphingosine-1-phosphate and glucosylceramide that are pro-survivals signals; (c) modify the synthesis and the metabolism of complex glycosphingolipids, particularly increasing the levels of modified species of gangliosides such as 9-O acetylated GD3 (αNeu5Ac(2-8)αNeu5Ac(2-3)ßGal(1-4)ßGlc(1-1)Cer) or N-glycolyl GM3 (αNeu5Ac (2-3)ßGal(1-4)ßGlc(1-1)Cer) and de-N-acetyl GM3 (NeuNH(2)ßGal(1-4)ßGlc(1-1)Cer) endowed with anti-apoptotic roles and of globoside Gb3 related to a higher expression of the multidrug resistance gene MDR1. In light of this evidence, the employment of chemical or genetic approaches specifically targeting sphingolipid dysregulations appears a promising tool for the improvement of current chemotherapy efficacy.

Extracellular sphingosine-1-phosphate: a novel actor in human glioblastoma stem cell survival.

Glioblastomas are the most frequent and aggressive intracranial neoplasms in humans, and despite advances and the introduction of the alkylating agent temozolomide in therapy have improved patient survival, resistance mechanisms limit benefits. Recent studies support that glioblastoma stem-like cells (GSCs), a cell subpopulation within the tumour, are involved in the aberrant expansion and therapy resistance properties of glioblastomas, through still unclear mechanisms. Emerging evidence suggests that sphingosine-1-phosphate (S1P) a potent onco-promoter able to act as extracellular signal, favours malignant and chemoresistance properties in GSCs. Notwithstanding, the origin of S1P in the GSC environment remains unknown. We investigated S1P metabolism, release, and role in cell survival properties of GSCs isolated from either U87-MG cell line or a primary culture of human glioblastoma. We show that both GSC models, grown as neurospheres and expressing GSC markers, are resistant to temozolomide, despite not expressing the DNA repair protein MGMT, a major contributor to temozolomide-resistance. Pulse experiments with labelled sphingosine revealed that both GSC types are able to rapidly phosphorylate the long-chain base, and that the newly produced S1P is efficiently degraded. Of relevance, we found that S1P was present in GSC extracellular medium, its level being significantly higher than in U87-MG cells, and that the extracellular/intracellular ratio of S1P was about ten-fold higher in GSCs. The activity of sphingosine kinases was undetectable in GSC media, suggesting that mechanisms of S1P transport to the extracellular environment are constitutive in GSCs. In addition we found that an inhibitor of S1P biosynthesis made GSCs sensitive to temozolomide (TMZ), and that exogenous S1P reverted this effect, thus involving extracellular S1P as a GSC survival signal in TMZ resistance. Altogether our data implicate for the first time GSCs as a pivotal source of extracellular S1P, which might act as an autocrine/paracrine signal contributing to their malignant properties.

Microvesicles released from microglia stimulate synaptic activity via enhanced sphingolipid metabolism.

Microvesicles (MVs) released into the brain microenvironment are emerging as a novel way of cell-to-cell communication. We have recently shown that microglia, the immune cells of the brain, shed MVs upon activation but their possible role in microglia-to-neuron communication has never been explored. To investigate whether MVs affect neurotransmission, we analysed spontaneous release of glutamate in neurons exposed to MVs and found a dose-dependent increase in miniature excitatory postsynaptic current (mEPSC) frequency without changes in mEPSC amplitude. Paired-pulse recording analysis of evoked neurotransmission showed that MVs mainly act at the presynaptic site, by increasing release probability. In line with the enhancement of excitatory transmission in vitro, injection of MVs into the rat visual cortex caused an acute increase in the amplitude of field potentials evoked by visual stimuli. Stimulation of synaptic activity occurred via enhanced sphingolipid metabolism. Indeed, MVs promoted ceramide and sphingosine production in neurons, while the increase of excitatory transmission induced by MVs was prevented by pharmacological or genetic inhibition of sphingosine synthesis. These data identify microglia-derived MVs as a new mechanism by which microglia influence synaptic activity and highlight the involvement of neuronal sphingosine in this microglia-to-neuron signalling pathway.