Preclinical evaluation of bozepinib in bladder cancer cell lines: modulation of the NPP1 enzyme.

Bladder cancer (BC) is the most common cancer of the urinary tract. Bozepinib (BZP), a purine-derived molecule, is a potential compound for the treatment of cancer. Purinergic signaling consists of the activity of nucleosides and nucleotides present in the extracellular environment, modulating a variety of biological actions. In cancer, this signaling is mainly controlled by the enzymatic cascade involving the NTPDase/E-NPP family and ecto-5'-nucleotidase/CD73, which hydrolyze extracellular adenosine triphosphate (ATP) to adenosine (ADO). The aim of this work is to evaluate the activity of BZP in the purinergic system in BC cell lines and to compare its in vitro antitumor activity with cisplatin, a chemotherapeutic drug widely used in the treatment of BC. In this study, two different BC cell lines, grade 1 RT4 and the more aggressive grade 3 T24, were used along with a human fibroblast cell line MRC-5, a cell used to predict the selectivity index (SI). BZP shows strong antitumor activity, with notable IC50 values (8.7 ± 0.9 µM for RT4; 6.7 ± 0.7 µM for T24), far from the SI for cisplatin (SI for BZP: 19.7 and 25.7 for RT4 and T24, respectively; SI for cisplatin: 1.7 for T24). BZP arrests T24 cells in the G2/M phase of the cell cycle, inducing early apoptosis. Moreover, BZP increases ATP and ADP hydrolysis and gene/protein expression of the NPP1 enzyme in the T24 cell line. In conclusion, BZP shows superior activity compared to cisplatin against BC cell lines in vitro.

Tumor-neutrophil crosstalk promotes in vitro and in vivo glioblastoma progression.

Introduction: The tumor microenvironment (TME) of glioblastoma (GB) is characterized by an increased infiltration of immunosuppressive cells that attenuate the antitumor immune response. The participation of neutrophils in tumor progression is still controversial and a dual role in the TME has been proposed. In this study, we show that neutrophils are reprogrammed by the tumor to ultimately promote GB progression. Methods: Using in vitro and in vivo assays, we demonstrate the existence of bidirectional GB and neutrophil communication, directly promoting an immunosuppressive TME. Results and discussion: Neutrophils have shown to play an important role in tumor malignancy especially in advanced 3D tumor model and Balb/c nude mice experiments, implying a time- and neutrophil concentration-dependent modulation. Studying the tumor energetic metabolism indicated a mitochondria mismatch shaping the TME secretome. The given data suggests a cytokine milieu in patients with GB that favors the recruitment of neutrophils, sustaining an anti-inflammatory profile which is associated with poor prognosis. Besides, glioma-neutrophil crosstalk has sustained a tumor prolonged activation via NETs formation, indicating the role of NFκB signaling in tumor progression. Moreover, clinical samples have indicated that neutrophil-lymphocyte ratio (NLR), IL-1ß, and IL-10 are associated with poor outcomes in patients with GB. Conclusion: These results are relevant for understanding how tumor progression occurs and how immune cells can help in this process.

UTP increases wound healing in the self assembled skin substitute (SASS).

The therapeutic potential of purinergic signaling has been explored for a wide variety of diseases, including those related to the skin. In this study, we used the self-assembled skin substitutes (SASS), a highly functional reconstructed human skin model, which shares many properties with normal human skin, to study the impact of purinergic receptors agonists, such as ATP, UTP and a P2Y receptor antagonist, Reactive Blue 2 during wound healing. After treating the wounded skins, we evaluated the wound area, reepithelialization, length of migrating tongues toward the wound, quality of the skins through the cytokeratin 10 and laminin-5 expression, epidermal and dermal cell proliferation. In addition, the expression of the main ectoenzymes capable of hydrolyzing nucleotides were investigated through the wounded SASS regions: unwounded region, wound margin, intermediate region and migrating epidermal tongue. After 3 days, under the UTP treatment, the wounded SASS showed an increase in the reepithelialization and in the proliferation of keratinocytes and fibroblasts, without altering the quality of the skin. We also identified the presence of the ectoenzymes NTPDase1 and NPP1 in the reconstructed human skin model, suggesting their involvement in wound healing. Considering the need for new therapies capable of promoting healing in complex wounds, although these results are still preliminary, they suggest the involvement of extracellular nucleotides in human skin healing and the importance to understand their role in this mechanism. New experiments it will be necessary to determine the mechanisms by which the purinergic signaling is involved in the skin wound healing.

Epidermal melanocytes metabolize extracellular nucleotides by purinergic enzymes.

The human epidermal melanocyte (hEM) are melanin-producing cells that provide skin pigmentation and protection against ultraviolet radiation. Although purinergic signaling is involved in skin biology and pathology, the presence of NTPDase members, as well as the rate of nucleotides degradation by melanocytes were not described yet. Therefore, in this study, we analyzed the expression of ectonucleotidases in hEM derived from discarded foreskin of male patients. The expression of purinergic enzymes was confirmed by mRNA and flow cytometry. Among the ectonucleotidases, ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1) and ecto-5´-nucleotidase were the ectoenzymes with higher expressions. The hydrolysis rate for ATP, ADP, and AMP was low in comparison to other primary cells already investigated. The amount of ATP in the culture medium was increased after a scratch wound and decreased to basal levels in 48 h, while the NTPDase1 and P2X7 expressions increased. Therefore, it is possible to suggest that after cell injury, the ATP released by hEM into the extracellular space will be hydrolyzed by ectonucleotidases as the NTPDase1 that will control the levels of nucleotides in the skin micro-environment.

Leishmania infantum NTPDase1 and NTPDase2 play an important role in infection and nitric oxide production in macrophages.

Leishmania infantum, the causative agent of American Visceral Leishmaniasis (VL), is known for its ability to modulate the host immune response to its own favor. Ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) represents a family of enzymes that hydrolyze nucleotides and are involved in nucleotide-dependent biological processes. L. infantum has two ENTPDases, namely LiNTPDase1 and LiNTPDase2. Here, we used genetic tools to overexpress or abolish the expression of LiNTPDase1 and -2 to assess their role in parasite growth in culture and macrophage infection. While LiNTPDase1 or 2-overexpressing clones showed no morphological or growth changes in promastigotes, LiNTPDase2 overexpression increased macrophage adhesion and infection by 50% and 30%, respectively. The individual LiNTPDase1 and 2 knockout mutants showed lag in growth profile, which was reversed by the addition of adenine and guanine to the culture media. Moreover, the morphology of the knockout mutants even in supplemented media was changed to an amastigote-like form. The double knockout of both genes was lethal and a mechanism of compensation of deletion of one isoform was detected in these mutants. Correspondingly, the absence of LiNTPDase1 or LiNTPDase2 led to a dramatic reduction in in vitro infection (∼90%). Interestingly, nitric oxide production was decreased in both knockout mutants during infection, which suggests that both LiNTPDases can inhibit macrophage responses against the parasite. Overall, our results show important roles of LiNTPDase1 and -2 concerning in vitro macrophage infection and reinforce their use as potential targets to control Leishmania infections.

Bioscaffold developed with decellularized human amniotic membrane seeded with mesenchymal stromal cells: assessment of efficacy and safety profiles in a second-degree burn preclinical model.

Therapies to deep burn injuries remain a global challenge. Human amniotic membrane (hAM) is a biomaterial that has been increasingly explored by the field of regenerative medicine. A decellularized hAM (DhAM) can be used as scaffold for mesenchymal stromal cells (MSCs) to grow without the loss of their stemness potential, allowing its application as cell therapy for wound healing. In this work, we associated DhAM with adipose-derived MSCs (DhAM + AD-MSCs), as a therapy strategy for second-degree burns in a preclinical model. Animals with induced second-degree burns were divided into four groups: control, which consists of a non-adherent gauze; a synthetic commercial dressing as the positive control (Control+); DhAM; and DhAM plus rat AD-MSCs (DhAM + AD-MSCs), followed by detailed and long term analysis (5 weeks). The macroscopical analysis showed the healing improvement in the wound area after the DhAM + AD-MSC treatment. Histological analysis also showed no alteration in the animal organs and a regular epithelial progression in comparison to the control. This observation was also confirmed by the analysis of suprabasal layers in the neoepidermis with CK10, showing a stratified and differentiated epithelium, when compared to Control and Control+. A strong CD73 (ecto-5'-nucleotidase) labeling was observed in the first 2 weeks postburn in dermis and epidermis. The expression in dermis was stronger in the second week in the middle of the wound, when comparing the Control+ with DhAM + AD-MSCs (p= 0.0238). In the epidermis the expression of CD73 was increased in all regions when compared to the control. This data suggests the involvement of this protein on wound healing. A low CD11b labeling was observed in DhAM + AD-MSCs treatment group mainly in the last treatment week, in comparison to Control and Control+ (p< 0.0001), which indicates a reduction in the inflammatory process. MSCs through CD73 can release high concentrations of adenosine, an immunosuppressive molecule, suggesting that this could be the mechanism by which the inflammation was better modulated in the DhAM + AD-MSCs group. The results obtained with this preclinical model confirm the effectiveness and safety of this low-cost and highly available dressing for future clinical application as a therapy for burn treatments.

Neutrophils: fast and furious-the nucleotide pathway.

Nucleotide signaling is a key element of the neutrophil activation pathway. Neutrophil recruitment and migration to injured tissues is guided by purinergic receptor sensitization, mostly induced by extracellular adenosine triphosphate (ATP) and its hydrolysis product, adenosine (ADO), which is primarily produced by the CD39-CD73 axis located at the neutrophil cell surface. In inflammation unrelated to cancer, neutrophil activation via purinergic signaling aims to eliminate antigens and promote an immune response with minimal damage to healthy tissues; however, an antagonistic response may be expected in tumors. Indeed, alterations in purinergic signaling favor the accumulation of extracellular ATP and ADO in the microenvironment of solid tumors, which promote tumor progression by inducing cell proliferation, angiogenesis, and escape from immune surveillance. Since neutrophils and their N1/N2 polarization spectrum are being considered new components of cancer-related inflammation, the participation of purinergic signaling in pro-tumor activities of neutrophils should also be considered. However, there is a lack of studies investigating purinergic signaling in human neutrophil polarization and in tumor-associated neutrophils. In this review, we discussed the human neutrophil response elicited by nucleotides in inflammation and extrapolated its behavior in the context of cancer. Understanding these mechanisms in cancerous conditions may help to identify new biological targets and therapeutic strategies, particularly regarding tumors that are refractory to traditional chemo- and immunotherapy.

New insights into cytotoxic mechanisms of bozepinib against glioblastoma.

Glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults and the current treatments only have a modest effect on patient survival. Recent studies show that bozepinib (BZP), a purine derivative, has potential applications in cancer treatment. The aim of this study was to evaluate the effect of BZP against GBM cells, specially concerning the purinergic system. Thus, GBM cells (C6 and U138 cell lines) were treated with BZP and cell viability, cell cycle, and annexin/PI assays, and active caspase-3 measurements were carried out. Besides, the effect of BZP over the purinergic system was also evaluated in silico and in vitro. Finally, we evaluate the action of BZP against important markers related to cancer progression, such as Akt, NF-κB, and CD133. We demonstrate here that BZP reduces GBM cell viability (IC50 = 5.7 ± 0.3 µM and 12.7 ± 1.5 µM, in C6 and U138 cells, respectively), inducing cell death through caspase-dependent apoptosis, autophagosome formation, activation of NF-κB, without any change in cell cycle progression or on the Akt pathway. Also, BZP modulates the purinergic system, inducing an increase in CD39 enzyme expression and activity, while inhibiting CD73 activity and adenosine formation, without altering CD73 enzyme expression. Curiously, one cycle of treatment resulted in enrichment of GBM cells expressing NF-κB and CD133+, suggesting resistant cells selection. However, after another treatment round, the resistant cells were eliminated. Altogether, BZP presented in vitro anti-glioma activity, encouraging further in vivo studies in order to better understand its mechanism of action.

Influence of NSAIDs and methotrexate on CD73 expression and glioma cell growth.

Glioblastoma (GBM) is the most malignant and deadly brain tumor. GBM cells overexpress the CD73 enzyme, which controls the level of extracellular adenosine, an immunosuppressive molecule. Studies have shown that some nonsteroidal anti-inflammatory drugs (NSAIDs) and methotrexate (MTX) have antiproliferative and modulatory effects on CD73 in vitro and in vivo. However, it remains unclear whether the antiproliferative effects of MTX and NSAIDS in GBM cells are mediated by increases in CD73 expression and adenosine formation. The aim of this study was to evaluate the effect of the NSAIDs, naproxen, piroxicam, meloxicam, ibuprofen, sodium diclofenac, acetylsalicylic acid, nimesulide, and ketoprofen on CD73 expression in GBM and mononuclear cells. In addition, we sought to understand whether the effects of MTX may be mediated by CD73 expression and activity. Cell viability and CD73 expression were evaluated in C6 and mononuclear cells after exposure to NSAIDs. For analysis of the mechanism of action of MTX, GBM cells were treated with APCP (CD73 inhibitor), dipyridamole (inhibitor of adenosine uptake), ABT-702 (adenosine kinase enzyme inhibitor), or caffeine (P1 adenosine receptor antagonist), before treatment with MTX and AMP, in the presence or not of mononuclear cells. In summary, only MTX increased the expression of CD73 in GBM cells decreasing cells viability by mechanisms independent of the adenosinergic system. Further studies are needed to understand the role of MTX in the GBM microenvironment.

Characterization and antiproliferative activity of glioma-derived extracellular vesicles.

Aim: To characterize a method to isolate glioma-derived extracellular vesicles (GEVs) and understand their role in immune system modulation and glioma progression. Materials & methods: GEVs were isolated by differential centrifugation from C6 cell supernatant and characterized by size and expression of CD9, HSP70, CD39 and CD73. The glioma model was performed by injecting C6 glioma cells into the right striatum of Wistar rats in the following groups: controls (C6 cells alone), coinjection (C6 cells + GEVs) and GEVs by intranasal administration followed by immune cells, tumor size and cells proliferation analyses. Results: GEVs presented uniform size (175 nm), expressed CD9, HSP70, CD39, CD73 and produced adenosine. In vivo, we observed a reduction in tumor size, in cell proliferation (Ki-67) and in a regulatory cell marker (FoxP3). Conclusion: GEVs, administered before or at tumor challenge, have antiproliferative properties and reduce regulatory cells in the glioma microenvironment.

Imatinib mesylate affects extracellular ATP catabolism and expression of NTPDases in a chronic myeloid leukemia cell line.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, characterized by the occurrence of the t(9;22)(q34;q11) translocation. First-line therapy for CML consists of treatment with imatinib mesylate, which selectively inhibits the BCR-ABL protein by competing for its ATP-binding site. Adenine nucleotide signaling is modulated by the ectonucleotidases and this pathway is related to tumorigenic processes. Considering the relationship between ATP and cancer, we aimed to evaluate the influence of imatinib mesylate on the expressions and functions of the NTPDase and ecto-5'-nucleotidase (CD73) enzymes in imatinib-sensitive and -resistant K-562 cell lines. mRNA analysis showed that K-562 cells express all ENTPDs and NT5E. However, when treated with imatinib mesylate for 24 h, the expression of ENTPD1, -2, -3 and -5 increased, leading to a higher nucleotides hydrolysis rate. HPLC analysis identified increased ATP degradation in cells after 24 h of treatment, with consequent ADP and AMP formation, corroborating the increase in gene and protein expression of ectonucleotidases as observed in previous results. On the other hand, we observed that imatinib-resistant K-562 cells presented a decrease in nucleotide hydrolysis and expressions of ENTPD1 and -5. These results suggest an involvement of imatinib in modulating ectonucleotidases in CML that will need further investigation. Since these ectonucleotidases have important catalytic activities in the tumor microenvironment, their modulation in CML cells may represent an important therapeutic approach to regulate levels of extracellular adenine nucleotides.

Development and characterization of CD73-siRNA-loaded nanoemulsion: effect on C6 glioma cells and primary astrocytes.

Introduction: Glioblastoma (GB) is the most common malignant brain tumor and is characterized by high invasiveness, poor prognosis, and limited therapeutic options. Silencing gene expression, through the use of small interfering RNA (siRNA), has been proposed as an alternative to conventional cancer therapy. Here, we evaluated the potential of CD73 as a new therapeutic target, since it is overexpressed in solid tumors and has emerged as a promising target to control GB progression.Methods: A cationic nanoemulsion (NE) as an intravenous siRNA-CD73 delivery system was developed and its effect on C6 glioma cell viability was determined.Results: The nanostructured system was effective in complexing oligonucleotides for delivery to target cells. In addition, we observed that the NE-siRNA-CD73 complex was effective in reducing CD73 protein levels and AMPase activity, which were related to decreased C6 glioma cell viability.Conclusions: These findings indicate the potential of siRNA-CD73-loaded cationic NE as a therapeutic alternative for glioma treatment.

Characterization of soluble CD39 (SolCD39/NTPDase1) from PiggyBac nonviral system as a tool to control the nucleotides level.

Extracellular ATP (eATP) and its metabolites have emerged as key modulators of different diseases and comprise a complex pathway called purinergic signaling. An increased number of tools have been developed to study the role of nucleotides and nucleosides in cell proliferation and migration, influence on the immune system and tumor progression. These tools include receptor agonists/antagonists, engineered ectonucleotidases, interference RNAs and ectonucleotidase inhibitors that allow the control and quantification of nucleotide levels. NTPDase1 (also called apyrase, ecto-ATPase and CD39) is one of the main enzymes responsible for the hydrolysis of eATP, and purified enzymes, such as apyrase purified from potato, or engineered as soluble CD39 (SolCD39), have been widely used in in vitro and in vivo experiments. However, the commercial apyrase had its effects recently questioned and SolCD39 exhibits limitations, such as short half-life and need of high doses to reach the expected enzymatic activity. Therefore, this study investigated a non-viral method to improve the overexpression of SolCD39 and evaluated its impact on other enzymes of the purinergic system. Our data demonstrated that PiggyBac transposon system proved to be a fast and efficient method to generate cells stably expressing SolCD39, producing high amounts of the enzyme from a limited number of cells and with high hydrolytic activity. In addition, the soluble form of NTPDase1/CD39 did not alter the expression or catalytic activity of other enzymes from the purinergic system. Altogether, these findings set the groundwork for prospective studies on the function and therapeutic role of eATP and its metabolites in physiological and pathological conditions.

CD73 Downregulation Decreases In Vitro and In Vivo Glioblastoma Growth.

Glioblastoma is the worst and most common primary brain tumor. Here, we demonstrated the role of CD73, an enzyme responsible for adenosine (ADO) production, in glioblastoma progression. ADO increased glioma cell viability via A1 receptor sensitization. CD73 downregulation decreased glioma cell migration and invasion by reducing metalloproteinase-2 and vimentin expression and reduced cell proliferation by 40%, which was related to necrosis and sub-G1 phase blockage of cell cycle. Those effects also involved the stimulation of Akt/NF-kB pathways. Additionally, CD73 knockdown or enzyme inhibition potentiated temozolomide cytotoxic effect on glioma cells by decreasing the IC50 value and sensitizing cells to a non-cytotoxic drug concentration. CD73 inhibition also decreased in vivo rat glioblastoma progression. Delivery of siRNA-CD73 or APCP reduced tumor size by 45 and 40%, respectively, when compared with control. This effect was followed by a parallel 95% reduction of ADO levels in cerebrospinal fluid, indicating the role of extracellular ADO in in vivo glioma growth. Treatment did not induce systemic damage or mortality. Altogether, we conclude that CD73 is an interesting target for glioblastoma treatment and its inhibition may provide new opportunities to improve the treatment of brain tumors. Graphical Abstract ᅟ.

Physical exercise prevents alterations in purinergic system and oxidative status in lipopolysaccharide-induced sepsis in rats.

Sepsis is a generalized infection that involves alterations in inflammatory parameters, oxidant status, and purinergic signaling in many tissues. Physical exercise has emerged as a tool to prevent this disease because of its anti-inflammatory and antioxidant properties. Thus, in this study, we investigated the effects of physical exercise on preventing alterations in purinergic system components, oxidative stress, and inflammatory parameters in lipopolysaccharide (LPS)-induced sepsis in rats. Male Wistar rats were divided into four groups: control, exercise (EX), LPS, and EX+LPS. The resisted physical exercise was performed for 12 weeks on a ladder with 1 m height. After 72 hours of the last exercise session, the animals received 2.5 mg/kg of LPS for induction of sepsis, and after 24 hours, lungs and blood samples were collected for analysis. The results showed that the exercise protocol used was able to prevent, in septic animals: (1) the increase in body temperature; (2) the increase of lipid peroxidation and reactive species levels in the lung, (3) the increase in adenosine triphosphate levels in serum; (4) the change in the activity of the enzymes ectonucleotidases in lymphocytes, partially; (5) the change in the density of purinergic enzymes and receptors in the lung, and (6) the increase of IL-6 and IL-1ß gene expression. Our results revealed the involvement of purinergic signaling and oxidative damage in the mechanisms by which exercise prevents sepsis aggravations. Therefore, the regular practice of physical exercise is encouraged as a better way to prepare the body against sepsis complications.

Ecto-5'-nucleotidase/CD73 contributes to the radiosensitivity of T24 human bladder cancer cell line.

PURPOSE: Trimodal therapy is a reasonable bladder-preserving option to radical cystectomy. However, many tumors are radioresistive. In this sense, the identification of new prognostic and predictive biomarkers that allow the selection of patients with better responses to radiation therapy would improve outcomes. With the aim of using ecto-5'-nucleotidase/CD73 as a predictive biomarker, the role of this enzyme in the context of radiotherapy in T24 human bladder cancer cell line was investigated. METHODS: T24 cell line was exposure to a single dose of radiation (4 Gray) and trypan blue assay (pharmacological assays of viability/cumulative population doubling), flow cytometry (cell cycle/cell death/active caspase-3/ecto-5'-nucleotidase/CD73 protein staining), DAPI staining (nuclear morphometric assay), RT-PCR and real-time PCR, malachite green method (ectonucleotidase enzymatic assay), and HPLC (analysis of AMP metabolism) were carried out. T24 cell line in which ecto-5'-nucleotidase/CD73 has been completely silenced (5'KO) was also used. RESULTS: The exposure of T24 cell line to a single dose (4 Gray) of radiation-induced cell death and triggered a transitory increase in ecto-5'-nucleotidase/CD73 expression, increased ectonucleotidase activity, and led to adenosine and inosine accumulation in the extracellular medium. Pharmacological inhibition or knocking out ecto-5'-nucleotidase/CD73 rescued cells' proliferative capacity, reducing their sensitivity to radiation. CONCLUSION: Our findings show that the induction of ecto-5'-nucleotidase/CD73 by radiation contributes to the radiosensitivity of T24 cell line.

Methotrexate up-regulates ecto-5'-nucleotidase/CD73 and reduces the frequency of T lymphocytes in the glioblastoma microenvironment.

Glioblastoma multiforme (GBM) is a deadly cancer characterized by a pro-tumoral immune response. T-regulatory (Treg) lymphocytes suppress effector immune cells through cytokine secretion and the adenosinergic system. Ecto-5'-nucleotidase/CD73 plays a crucial role in Treg-mediated immunosuppression in the GBM microenvironment (GME). Methotrexate (MTX) is an immunosuppressive drug that can increase the extracellular concentration of adenosine. In this manuscript, C6 GBM cells were treated with 1.0 µM MTX, and ecto-5'-nucleotidase/CD73 expression and extracellular AMP metabolism were analyzed in vitro. For in vivo studies, rats with implanted GBM were treated for 10 days with MTX-loaded lipid-core nanocapsules (MTX-LNCs, 1 mg/kg/day). The activity of ectonucleotidase and the expression of NTPDase1/CD39 and ecto-5'-nucleotidase/CD73 were measured. The frequencies of T lymphocytes (CD3(+)CD4(+), CD3(+)CD8(+), and CD4(+)CD25(high)CD39(+)) were quantified. In vitro, treatment with MTX increased CD73 expression and activity in C6 cells, which is in agreement with higher levels of extracellular adenosine. In vivo, MTX-LNC treatment increased CD39 expression on CD3(+)CD8(+) lymphocytes. In addition, MTX-LNC treatment up-regulated CD73 expression in tissue isolated from GBM, a finding that is in agreement with the higher activity of this enzyme. More specifically, the treatment increased CD73 expression on CD3(+)CD4(+) and CD3(+)CD8(+) lymphocytes. Treatment with MTX-LNCs decreased the frequencies of T-cytotoxic, T-helper, and Treg lymphocytes in the GME. Although more studies are necessary to better understand the complex cross-talk mediated by supra-physiological concentrations of adenosine in the GME, these studies demonstrate that MTX treatment increases CD73 enzyme expression and AMP hydrolysis, leading to an increase in adenosine production and immunosuppressive capability.

Ecto-5'-Nucleotidase Overexpression Reduces Tumor Growth in a Xenograph Medulloblastoma Model.

BACKGROUND: Ecto-5'-nucleotidase/CD73 (ecto-5'-NT) participates in extracellular ATP catabolism by converting adenosine monophosphate (AMP) into adenosine. This enzyme affects the progression and invasiveness of different tumors. Furthermore, the expression of ecto-5'-NT has also been suggested as a favorable prognostic marker, attributing to this enzyme contradictory functions in cancer. Medulloblastoma (MB) is the most common brain tumor of the cerebellum and affects mainly children. MATERIALS AND METHODS: The effects of ecto-5'-NT overexpression on human MB tumor growth were studied in an in vivo model. Balb/c immunodeficient (nude) 6 to 14-week-old mice were used for dorsal subcutaneous xenograph tumor implant. Tumor development was evaluated by pathophysiological analysis. In addition, the expression patterns of adenosine receptors were verified. RESULTS: The human MB cell line D283, transfected with ecto-5'-NT (D283hCD73), revealed reduced tumor growth compared to the original cell line transfected with an empty vector. D283hCD73 generated tumors with a reduced proliferative index, lower vascularization, the presence of differentiated cells and increased active caspase-3 expression. Prominent A1 adenosine receptor expression rates were detected in MB cells overexpressing ecto-5'-NT. CONCLUSION: This work suggests that ecto-5'-NT promotes reduced tumor growth to reduce cell proliferation and vascularization, promote higher differentiation rates and initiate apoptosis, supposedly by accumulating adenosine, which then acts through A1 adenosine receptors. Therefore, ecto-5'-NT might be considered an important prognostic marker, being associated with good prognosis and used as a potential target for therapy.

Involvement of purinergic system in the release of cytokines by macrophages exposed to glioma-conditioned medium.

Macrophages are involved in cancer progression. M1 macrophages have an antitumor effect, whereas M2 phenotype are associated with tumor growth. The progression of gliomas involves the participation of an inflammatory microenvironment. Adenosine triphosphate (ATP) can act as pro-inflammatory signal, whereas adenosine has opposite properties. The biological effects of extracellular nucleotides/nucleosides mediated by purinergic receptors are controlled by ectonucleotidases. In the present work, we evaluated whether glioma-conditioned medium (GL-CM) modulates macrophage differentiation and the participation of ATP and adenosine in the release of pro-and anti-inflammatory cytokines by these cells. The results show that macrophages exposed to GL-CM were modulated to an M2-like phenotype. HPLC analysis of GL-CM demonstrated the presence of significant amounts of ATP and its metabolites. Macrophages exposed to GL-CM presented decreased ATP and AMP hydrolysis and increased IL-10 and MCP-1 secretion, effects that were diminished by P1 or P2 antagonists. GL-CM did not alter the release of IL-6 by macrophages, although treatment with ATP promoted an increase in the release of IL-6, which was prevented by a P2X7 antagonist. In summary, we found that A2A and P2X7 activation is necessary for IL-10, MCP-1, and IL-6 release by macrophages exposed to GL-CM, which, in turn, modulates the macrophages to M2-phenotype. The present study establishes a relationship between M2-like polarization, cytokine release and purinergic receptor activation in macrophages exposed to GL-CM. Therefore, the data presented herein contributes to advancing in the field of cancer-related inflammation and point specific purinergic receptors as targets for modulation of the phenotype of glioma-associated macrophages.

Nucleotide receptors control IL-8/CXCL8 and MCP-1/CCL2 secretions as well as proliferation in human glioma cells.

Glioma cells release cytokines to stimulate inflammation that facilitates cell proliferation. Here, we show that Lipopolysaccharide (LPS) treatment could induce glioma cells to proliferate and this process was dependent on nucleotide receptor activation as well as interleukin-8 (IL-8/CXCL8) secretion. We observed that extracellular nucleotides controlled IL-8/CXCL8 and monocyte chemoattractant protein 1 (MCP-1/CCL2) release by U251MG and U87MG human glioma cell lines via P2X7 and P2Y6 receptor activation. The LPS-induced release of these cytokines was also modulated by purinergic receptor activation since IL-8 and MCP-1 release was decreased by the nucleotide scavenger apyrase as well as by the pharmacological P2Y6 receptor antagonists suramin and MRS2578. In agreement with these observations, the knockdown of P2Y6 expression decreased LPS-induced IL-8 release as well as the spontaneous release of IL-8 and MCP-1, suggesting an endogenous basal release of nucleotides. Moreover, high millimolar concentrations of ATP increased IL-8 and MCP-1 release by the glioma cells stimulated with suboptimal LPS concentration which were blocked by P2X7 and P2Y6 antagonists. Altogether, these data suggest that extracellular nucleotides control glioma growth via P2 receptor-dependent IL-8 and MCP-1 secretions.