Changes Induced by P2X7 Receptor Stimulation of Human Glioblastoma Stem Cells in the Proteome of Extracellular Vesicles Isolated from Their Secretome.

Extracellular vesicles (EVs) are secreted from many tumors, including glioblastoma multiforme (GBM), the most common and lethal brain tumor in adults, which shows high resistance to current therapies and poor patient prognosis. Given the high relevance of the information provided by cancer cell secretome, we performed a proteomic analysis of microvesicles (MVs) and exosomes (EXOs) released from GBM-derived stem cells (GSCs). The latter, obtained from the brain of GBM patients, expressed P2X7 receptors (P2X7Rs), which positively correlate with GBM growth and invasiveness. P2X7R stimulation of GSCs caused significant changes in the EV content, mostly ex novo inducing or upregulating the expression of proteins related to cytoskeleton reorganization, cell motility/spreading, energy supply, protection against oxidative stress, chromatin remodeling, and transcriptional regulation. Most of the induced/upregulated proteins have already been identified as GBM diagnostic/prognostic factors, while others have only been reported in peripheral tumors. Our findings indicate that P2X7R stimulation enhances the transport and, therefore, possible intercellular exchange of GBM aggressiveness-increasing proteins by GSC-derived EVs. Thus, P2X7Rs could be considered a new druggable target of human GBM, although these data need to be confirmed in larger experimental sets.

Influence of Guanine-Based Purines on the Oxidoreductive Reactions Involved in Normal or Altered Brain Functions.

The production of reactive oxygen species (ROS) in the brain is homeostatically controlled and contributes to normal neural functions. Inefficiency of control mechanisms in brain aging or pathological conditions leads to ROS overproduction with oxidative neural cell damage and degeneration. Among the compounds showing therapeutic potential against neuro-dysfunctions induced by oxidative stress are the guanine-based purines (GBPs), of which the most characterized are the nucleoside guanosine (GUO) and the nucleobase guanine (GUA), which act differently. Indeed, the administration of GUO to in vitro or in vivo models of acute brain injury (ischemia/hypoxia or trauma) or chronic neurological/neurodegenerative disorders, exerts neuroprotective and anti-inflammatory effects, decreasing the production of reactive radicals and improving mitochondrial function via multiple molecular signals. However, GUO administration to rodents also causes an amnesic effect. In contrast, the metabolite, GUA, could be effective in memory-related disorders by transiently increasing ROS production and stimulating the nitric oxide/soluble guanylate cyclase/cGMP/protein kinase G cascade, which has long been recognized as beneficial for cognitive function. Thus, it is worth pursuing further studies to ascertain the therapeutic role of GUO and GUA and to evaluate the pathological brain conditions in which these compounds could be more usefully used.

Guanine inhibits the growth of human glioma and melanoma cell lines by interacting with GPR23.

Guanine-based purines (GBPs) exert numerous biological effects at the central nervous system through putative membrane receptors, the existence of which is still elusive. To shed light on this question, we screened orphan and poorly characterized G protein-coupled receptors (GPRs), selecting those that showed a high purinoreceptor similarity and were expressed in glioma cells, where GBPs exerted a powerful antiproliferative effect. Of the GPRs chosen, only the silencing of GPR23, also known as lysophosphatidic acid (LPA) 4 receptor, counteracted GBP-induced growth inhibition in U87 cells. Guanine (GUA) was the most potent compound behind the GPR23-mediated effect, acting as the endpoint effector of GBP antiproliferative effects. Accordingly, cells stably expressing GPR23 showed increased sensitivity to GUA. Furthermore, while GPR23 expression was low in a hypoxanthine-guanine phosphoribosyl-transferase (HGPRT)-mutated melanoma cell line showing poor sensitivity to GBPs, and in HGPRT-silenced glioma cells, GPR23-induced expression in both cell types rescued GUA-mediated cell growth inhibition. Finally, binding experiments using [3H]-GUA and U87 cell membranes revealed the existence of a selective GUA binding (KD = 29.44 ± 4.07 nM; Bmax 1.007 ± 0.035 pmol/mg prot) likely to GPR23. Overall, these data suggest GPR23 involvement in modulating responses to GUA in tumor cell lines, although further research needs to verify whether this receptor mediates other GUA effects.

Purinergic Signaling in Oral Tissues.

The role of the purinergic signal has been extensively investigated in many tissues and related organs, including the central and peripheral nervous systems as well as the gastrointestinal, cardiovascular, respiratory, renal, and immune systems. Less attention has been paid to the influence of purines in the oral cavity, which is the first part of the digestive apparatus and also acts as the body's first antimicrobial barrier. In this review, evidence is provided of the presence and possible physiological role of the purinergic system in the different structures forming the oral cavity including teeth, tongue, hard palate, and soft palate with their annexes such as taste buds, salivary glands, and nervous fibers innervating the oral structures. We also report findings on the involvement of the purinergic signal in pathological conditions affecting the oral apparatus such as Sjögren's syndrome or following irradiation for the treatment of head and neck cancer, and the use of experimental drugs interfering with the purine system to improve bone healing after damage. Further investigations are required to translate the results obtained so far into the clinical setting in order to pave the way for a wider application of purine-based treatments in oral diseases.

Adipose Stromal/Stem Cell-Derived Extracellular Vesicles: Potential Next-Generation Anti-Obesity Agents.

Over the last decade, several compounds have been identified for the treatment of obesity. However, due to the complexity of the disease, many pharmacological interventions have raised concerns about their efficacy and safety. Therefore, it is important to discover new factors involved in the induction/progression of obesity. Adipose stromal/stem cells (ASCs), which are mostly isolated from subcutaneous adipose tissue, are the primary cells contributing to the expansion of fat mass. Like other cells, ASCs release nanoparticles known as extracellular vesicles (EVs), which are being actively studied for their potential applications in a variety of diseases. Here, we focused on the importance of the con-tribution of ASC-derived EVs in the regulation of metabolic processes. In addition, we outlined the advantages/disadvantages of the use of EVs as potential next-generation anti-obesity agents.

Multipotent Stromal Cells from Subcutaneous Adipose Tissue of Normal Weight and Obese Subjects: Modulation of Their Adipogenic Differentiation by Adenosine A1 Receptor Ligands.

Adenosine A1 receptor (A1R) activation, stimulating lipogenesis and decreasing insulin resistance, could be useful for metabolic syndrome management in obese subjects. Since full A1R agonists induce harmful side-effects, while partial agonists show a better pharmacological profile, we investigated the influence of two derivatives of the full A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA), C1 and C2 behaving as A1R partial agonists in animal models, on the adipogenic differentiation of stromal/stem cells (ASCs) from human subcutaneous adipose tissue, which mainly contribute to increase fat mass in obesity. The ASCs from normal-weight subjects showed increased proliferation and A1R expression but reduced adipogenic differentiation compared to obese individual-derived ASCs. Cell exposure to CCPA, C1, C2 or DPCPX, an A1R antagonist, did not affect ASC proliferation, while mainly C2 and DPCPX significantly decreased adipogenic differentiation of both ASC types, reducing the activity of glycerol-3-phosphate dehydrogenase and the expression of PPARγ and FABP-4, all adipogenic markers, and phosphorylation of Akt in the phosphatidylinositol-3-kinase pathway, which plays a key-role in adipogenesis. While requiring confirmation in in vivo models, our results suggest that A1R partial agonists or antagonists, by limiting ASC differentiation into adipocytes and, thereby, fat mass expansion, could favor development/worsening of metabolic syndrome in obese subjects without a dietary control.

In Search of a Role for Extracellular Purine Enzymes in Bone Function.

Bone is one of the major tissues that undergoes continuous remodeling throughout life, thus ensuring both organic body growth during development and protection of internal organs as well as repair of trauma during adulthood. Many endogenous substances contribute to bone homeostasis, including purines. Their role has increasingly emerged in recent decades as compounds which, by interacting with specific receptors, can help determine adequate responses of bone cells to physiological or pathological stimuli. Equally, it is recognized that the activity of purines is closely dependent on their interconversion or metabolic degradation ensured by a series of enzymes present at extracellular level as predominantly bound to the cell membrane or, also, as soluble isoforms. While the effects of purines mediated by their receptor interactions have sufficiently, even though not entirely, been characterized in many tissues including bone, those promoted by the extracellular enzymes providing for purine metabolism have not been. In this review, we will try to circumstantiate the presence and the role of these enzymes in bone to define their close relationship with purine activities in maintaining bone homeostasis in normal or pathological conditions.

Investigating the Role of Guanosine on Human Neuroblastoma Cell Differentiation and the Underlying Molecular Mechanisms.

Neuroblastoma arises from neural crest cell precursors failing to complete the process of differentiation. Thus, agents helping tumor cells to differentiate into normal cells can represent a valid therapeutic strategy. Here, we evaluated whether guanosine (GUO), a natural purine nucleoside, which is able to induce differentiation of many cell types, may cause the differentiation of human neuroblastoma SH-SY5Y cells and the molecular mechanisms involved. We found that GUO, added to the cell culture medium, promoted neuron-like cell differentiation in a time- and concentration-dependent manner. This effect was mainly due to an extracellular GUO action since nucleoside transporter inhibitors reduced but not abolished it. Importantly, GUO-mediated neuron-like cell differentiation was independent of adenosine receptor activation as it was not altered by the blockade of these receptors. Noteworthy, the neuritogenic activity of GUO was not affected by blocking the phosphoinositide 3-kinase pathway, while it was reduced by inhibitors of protein kinase C or soluble guanylate cyclase. Furthermore, the inhibitor of the enzyme heme oxygenase-1 but not that of nitric oxide synthase reduced GUO-induced neurite outgrowth. Interestingly, we found that GUO was largely metabolized into guanine by the purine nucleoside phosphorylase (PNP) enzyme released from cells. Taken together, our results suggest that GUO, promoting neuroblastoma cell differentiation, may represent a potential therapeutic agent; however, due to its spontaneous extracellular metabolism, the role played by the GUO-PNP-guanine system needs to be further investigated.

Proteomic Characterization of Two Extracellular Vesicle Subtypes Isolated from Human Glioblastoma Stem Cell Secretome by Sequential Centrifugal Ultrafiltration.

Extracellular vesicles (EVs) released from tumor cells are actively investigated, since molecules therein contained and likely transferred to neighboring cells, supplying them with oncogenic information/functions, may represent cancer biomarkers and/or druggable targets. Here, we characterized by a proteomic point of view two EV subtypes isolated by sequential centrifugal ultrafiltration technique from culture medium of glioblastoma (GBM)-derived stem-like cells (GSCs) obtained from surgical specimens of human GBM, the most aggressive and lethal primary brain tumor. Electron microscopy and western blot analysis distinguished them into microvesicles (MVs) and exosomes (Exos). Two-dimensional electrophoresis followed by MALDI TOF analysis allowed us to identify, besides a common pool, sets of proteins specific for each EV subtypes with peculiar differences in their molecular/biological functions. Such a diversity was confirmed by identification of some top proteins selected in MVs and Exos. They were mainly chaperone or metabolic enzymes in MVs, whereas, in Exos, molecules are involved in cell-matrix adhesion, cell migration/aggressiveness, and chemotherapy resistance. These proteins, identified by EVs from primary GSCs and not GBM cell lines, could be regarded as new possible prognostic markers/druggable targets of the human tumor, although data need to be confirmed in EVs isolated from a greater GSC number.

Adenine-Based Purines and Related Metabolizing Enzymes: Evidence for Their Impact on Tumor Extracellular Vesicle Activities.

Extracellular vesicles (EVs), mainly classified as small and large EVs according to their size/origin, contribute as multi-signal messengers to intercellular communications in normal/pathological conditions. EVs are now recognized as critical players in cancer processes by promoting transformation, growth, invasion, and drug-resistance of tumor cells thanks to the release of molecules contained inside them (i.e., nucleic acids, lipids and proteins) into the tumor microenvironment (TME). Interestingly, secretion from donor cells and/or uptake of EVs/their content by recipient cells are regulated by extracellular signals present in TME. Among those able to modulate the EV-tumor crosstalk, purines, mainly the adenine-based ones, could be included. Indeed, TME is characterized by high levels of ATP/adenosine and by the presence of enzymes deputed to their turnover. Moreover, ATP/adenosine, interacting with their own receptors, can affect both host and tumor responses. However, studies on whether/how the purinergic system behaves as a modulator of EV biogenesis, release and functions in cancer are still poor. Thus, this review is aimed at collecting data so far obtained to stimulate further research in this regard. Hopefully, new findings on the impact of adenine purines/related enzymes on EV functions may be exploited in tumor management uncovering novel tumor biomarkers and/or druggable targets.

Pros and Cons of Pharmacological Manipulation of cGMP-PDEs in the Prevention and Treatment of Breast Cancer.

The cyclic nucleotides, cAMP and cGMP, are ubiquitous second messengers responsible for translating extracellular signals to intracellular biological responses in both normal and tumor cells. When these signals are aberrant or missing, cells may undergo neoplastic transformation or become resistant to chemotherapy. cGMP-hydrolyzing phosphodiesterases (PDEs) are attracting tremendous interest as drug targets for many diseases, including cancer, where they regulate cell growth, apoptosis and sensitization to radio- and chemotherapy. In breast cancer, PDE5 inhibition is associated with increased intracellular cGMP levels, which is responsible for the phosphorylation of PKG and other downstream molecules involved in cell proliferation or apoptosis. In this review, we provide an overview of the most relevant studies regarding the controversial role of PDE inhibitors as off-label adjuvants in cancer therapy.

Guanosine-Mediated Anxiolytic-Like Effect: Interplay with Adenosine A1 and A2A Receptors.

Acute or chronic administration of guanosine (GUO) induces anxiolytic-like effects, for which the adenosine (ADO) system involvement has been postulated yet without a direct experimental evidence. Thus, we aimed to investigate whether adenosine receptors (ARs) are involved in the GUO-mediated anxiolytic-like effect, evaluated by three anxiety-related paradigms in rats. First, we confirmed that acute treatment with GUO exerts an anxiolytic-like effect. Subsequently, we investigated the effects of pretreatment with ADO or A1R (CPA, CCPA) or A2AR (CGS21680) agonists 10 min prior to GUO on a GUO-induced anxiolytic-like effect. All the combined treatments blocked the GUO anxiolytic-like effect, whereas when administered alone, each compound was ineffective as compared to the control group. Interestingly, the pretreatment with nonselective antagonist caffeine or selective A1R (DPCPX) or A2AR (ZM241385) antagonists did not modify the GUO-induced anxiolytic-like effect. Finally, binding assay performed in hippocampal membranes showed that [3H]GUO binding became saturable at 100-300 nM, suggesting the existence of a putative GUO binding site. In competition experiments, ADO showed a potency order similar to GUO in displacing [3H]GUO binding, whereas AR selective agonists, CPA and CGS21680, partially displaced [3H]GUO binding, but the sum of the two effects was able to displace [3H]GUO binding to the same extent of ADO alone. Overall, our results strengthen previous data supporting GUO-mediated anxiolytic-like effects, add new evidence that these effects are blocked by A1R and A2AR agonists and pave, although they do not elucidate the mechanism of GUO and ADO receptor interaction, for a better characterization of GUO binding sites in ARs.

Adult mesenchymal stem cells: is there a role for purine receptors in their osteogenic differentiation?

The role played by mesenchymal stem cells (MSCs) in contributing to adult tissue homeostasis and damage repair thanks to their differentiation capabilities has raised a great interest, mainly in bone regenerative medicine. The growth/function of these undifferentiated cells of mesodermal origin, located in specialized structures (niches) of differentiated organs is influenced by substances present in this microenvironment. Among them, ancestral and ubiquitous molecules such as adenine-based purines, i.e., ATP and adenosine, may be included. Notably, extracellular purine concentrations greatly increase during tissue injury; thus, MSCs are exposed to effects mediated by these agents interacting with their own receptors when they act/migrate in vivo or are transplanted into a damaged tissue. Here, we reported that ATP modulates MSC osteogenic differentiation via different P2Y and P2X receptors, but data are often inconclusive/contradictory so that the ATP receptor importance for MSC physiology/differentiation into osteoblasts is yet undetermined. An exception is represented by P2X7 receptors, whose expression was shown at various differentiation stages of bone cells resulting essential for differentiation/survival of both osteoclasts and osteoblasts. As well, adenosine, usually derived from extracellular ATP metabolism, can promote osteogenesis, likely via A2B receptors, even though findings from human MSCs should be implemented and confirmed in preclinical models. Therefore, although many data have revealed possible effects caused by extracellular purines in bone healing/remodeling, further studies, hopefully performed in in vivo models, are necessary to identify defined roles for these compounds in favoring/increasing the pro-osteogenic properties of MSCs and thereby their usefulness in bone regenerative medicine.

Involvement of P2X7 Receptors in the Osteogenic Differentiation of Mesenchymal Stromal/Stem Cells Derived from Human Subcutaneous Adipose Tissue.

The ionotropic P2X7 receptor (P2X7R) is involved in bone homeostasis but its role in osteogenesis is controversial. Thus, we investigated the expression of P2X7R and the effects exerted by its modulation in mesenchymal stromal cells from human subcutaneous adipose tissue (S-ASCs), which have potential therapeutic application in bone regenerative medicine. We found that undifferentiated S-ASCs expressed P2X7R and its functional splice variants P2X7AR and P2X7BR. Cell stimulation by P2X7R agonist BzATP (100 µM) neither modified proliferation nor caused membrane pore opening while increasing intracellular Ca2+ levels and migration. The P2X7R antagonist A438079 reversed these effects. However, 25-100 µM BzATP, administered to S-ASCs undergoing osteogenic differentiation, dose-dependently decreased extracellular matrix mineralization and expression of osteogenic transcription factors Runx2, alkaline phosphatase and osteopontin. These effects were not coupled to cell proliferation reduction or to cell death increase, but were associated to decrease in P2X7AR and P2X7BR expression. In contrast, expression of P2X7R, especially P2X7BR isoform, significantly increased during the osteogenic process. Noteworthy, the antagonist A438079, administered alone, at first restrained cell differentiation, enhancing it later. Accordingly, A438079 reversed BzATP effects only in the second phase of S-ASCs osteogenic differentiation. Apyrase, a diphosphohydrolase converting ATP/ADP into AMP, showed a similar behavior. Altogether, findings related to A438079 or apyrase effects suggest an earlier and prevailing pro-osteogenic activity by endogenous ATP and a later one by adenosine derived from endogenous ATP metabolism. Conversely, P2X7R pharmacological stimulation by BzATP, mimicking the effects of high ATP levels occurring during tissue injuries, depressed receptor expression/activity impairing MSC osteogenic differentiation.

Upregulation of Epithelial-To-Mesenchymal Transition Markers and P2X7 Receptors Is Associated to Increased Invasiveness Caused by P2X7 Receptor Stimulation in Human Glioblastoma Stem Cells.

Glioblastoma (GBM) stem cells (GSCs), which contribute to GBM unfavorable prognosis, show high expression levels of ATP/P2X7 receptors (P2X7R). Here, we reported that cells exposure to 2'(3')-O-(4-benzoylbenzoyl)-ATP (BzATP), a P2X7R agonist, up-regulated the expression of markers associated to epithelial-to-mesenchymal transition (EMT), a process likely contributing to GSC malignancy, and increased GSC migration/invasiveness like the known EMT inducer, Transforming Growth Factor ß1 (TGFß1). These effects were coupled to phosphorylation of SMAD2, a downstream effector in the TGFß pathway, suggesting its involvement in P2X7R-mediated activity in GSCs. All BzATP effects, including a decrease in the caspase 3/7 activity in GSC medium, were mostly counteracted by the P2X7R antagonist A438079. Finally, BzATP increased the subunit expression of two main human P2X7R splice variants, the full-length P2X7A and the truncated P2X7B, lacking the carboxylic tail, which have different functional properties depending on their arrangement. Since up-regulation of A/B subunits might favor their assembly into a heterotrimeric P2X7R with great sensitivity towards agonists and cell energy support, this is in line with increased EMT markers expression, cell migration/invasion and GSC survival observed following P2X7R stimulation. As in GBM microenvironment extracellular ATP levels may activate P2X7R, our data suggest a P2X7R role in GBM recurrence/invasiveness.

Uncovering the Signaling Pathway behind Extracellular Guanine-Induced Activation of NO System: New Perspectives in Memory-Related Disorders.

Mounting evidence suggests that the guanine-based purines stand out as key player in cell metabolism and in several models of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Guanosine (GUO) and guanine (GUA) are extracellular signaling molecules derived from the breakdown of the correspondent nucleotide, GTP, and their intracellular and extracellular levels are regulated by the fine-tuned activity of two major enzymes, purine nucleoside phosphorylase (PNP) and guanine deaminase (GDA). Noteworthy, GUO and GUA, seem to play opposite roles in the modulation of cognitive functions, such as learning and memory. Indeed GUO, despite exerting neuroprotective, anti-apoptotic and neurotrophic effects, causes a decay of cognitive activities, whereas GUA administration in rats results in working memory improvement (prevented by L-NAME pre-treatment). This study was designed to investigate, in a model of SH-SY5Y neuroblastoma cell line, the signal transduction pathway activated by extracellular GUA. Altogether, our results showed that: (i) in addition to an enhanced phosphorylation of ASK1, p38 and JNK, likely linked to a non-massive and transient ROS production, the PKB/NO/sGC/cGMP/PKG/ERK cascade seems to be the main signaling pathway elicited by extracellular GUA; (ii) the activation of this pathway occurs in a pertussis-toxin sensitive manner, thus suggesting the involvement of a putative G protein coupled receptor; (iii) the GUA-induced NO production, strongly reduced by cell pre-treatment with L-NAME, is negatively modulated by the EPAC-cAMP-CaMKII pathway, which causes the over-expression of GDA that, in turn, reduces the levels of GUA. These molecular mechanisms activated by GUA may be useful to support our previous observation showing that GUA improves learning and memory functions through the stimulation of NO signaling pathway, and underscore the therapeutic potential of oral administration of guanine for treating memory-related disorders.

A New Investigational Perspective for Purines Against Glioblastoma Invasiveness.

BACKGROUND: Glioblastoma Multiforme (GBM) is the most common and lethal brain malignancy. Recent evidence suggests that the presence of stem-like cells (GSCs) inside the tumor with high self-renewal, resistance to chemotherapy and invasiveness/migration potential is associated with poor GBM prognosis. GSC aggressiveness seems to be linked to an important process involved in tumorigenesis and cancer metastasis called Epithelial-to-Mesenchymal Transition (EMT), which is responsible for several biochemical changes and the acquisition of a more mesenchymal phenotype by GSCs, that enhance their migration, invasiveness and resistance to apoptosis. OBJECTIVE: Since previous reports demonstrated that purines, interacting with their own receptors, exerted anti-tumor effects in GBM and derived cells, we tried to investigate the ability of these compounds to reduce tumor cell migration/invasion acting on EMT-associated genes/activators and/or signal pathways. METHODS: Search in the literature of relevant articles related to the objective. RESULTS: Papers examining the activity of purines on EMT signaling pathways/markers in GSCs are still few whereas literature is more abundant as for other kinds of tumors. CONCLUSION: Considering the significance of EMT in GBM aggressiveness and the promising involvement of purines in this process, we think that further research in this regard may open the way towards a new therapeutic approach for the control of GBM invasiveness/recurrence.

The Role of Wnt Signal in Glioblastoma Development and Progression: A Possible New Pharmacological Target for the Therapy of This Tumor.

Wnt is a complex signaling pathway involved in the regulation of crucial biological functions such as development, proliferation, differentiation and migration of cells, mainly stem cells, which are virtually present in all embryonic and adult tissues. Conversely, dysregulation of Wnt signal is implicated in development/progression/invasiveness of different kinds of tumors, wherein a certain number of multipotent cells, namely "cancer stem cells", are characterized by high self-renewal and aggressiveness. Hence, the pharmacological modulation of Wnt pathway could be of particular interest, especially in tumors for which the current standard therapy results to be unsuccessful. This might be the case of glioblastoma multiforme (GBM), one of the most lethal, aggressive and recurrent brain cancers, probably due to the presence of highly malignant GBM stem cells (GSCs) as well as to a dysregulation of Wnt system. By examining the most recent literature, here we point out several factors in the Wnt pathway that are altered in human GBM and derived GSCs, as well as new molecular strategies or experimental drugs able to modulate/inhibit aberrant Wnt signal. Altogether, these aspects serve to emphasize the existence of alternative pharmacological targets that may be useful to develop novel therapies for GBM.