Investigation of co-treatment multi-targeting approaches in breast cancer cell lines.

In 2020, breast cancer (BC) has surpassed lung cancer as the most diagnosed cancer in the world. Tumor microenvironment (TME) plays a critical role in resistance to standard therapies and tumor progression. Two key factors within the TME include adenosine, an immunosuppressive molecule, and glucose, which serves as the primary energy source for tumor cells. In this scenario, inhibiting the purinergic pathway and glucose uptake might be a promising strategy. Therefore, we sought to evaluated different treatment approaches in BC cells (Dapagliflozin, a SGLT2 inhibitor; Paclitaxel, the standard chemotherapy for BC; and ARL67156/APCP, inhibitors of CD39 and CD73, respectively). The expression of some membrane markers relevant to resistance was assessed. BC cell-lines (MCF-7 and MDA-MB-231) were co-treated and cell viability, cell cycle, and annexin/PI assays were performed. Our analysis showed promising results, where the combination of these compounds led to cell death by apoptosis/necrosis and cell cycle arrest. Dapagliflozin showed more impact on early apoptosis, whereas Paclitaxel led to late apoptosis/necrosis as the main mechanism of cell death. Inhibiting purinergic signaling also contributed to reducing cell viability together with the other drugs, suggesting it could have an influence on breast cancer survival mechanisms. Indeed, the overexpression of the NT5E gene in patients with ER+ tumors is strongly associated with reduced overall survival and progression-free interval. However, more studies are needed to fully understand the interactions and mechanism underlying these co-treatment multi-targeting approaches.

Characterization of purinergic signaling in tumor-infiltrating lymphocytes from lower- and high-grade gliomas.

Malignant gliomas are highly heterogeneous glia-derived tumors that present an aggressive and invasive nature, with a dismal prognosis. The multi-dimensional interactions between glioma cells and other tumor microenvironment (TME) non-tumoral components constitute a challenge to finding successful treatment strategies. Several molecules, such as extracellular purines, participate in signaling events and support the immunosuppressive TME of glioma patients. The purinergic signaling and the ectoenzymes network involved in the metabolism of these extracellular nucleotides are still unexplored in the glioma TME, especially in lower-grade gliomas (LGG). Also, differences between IDH-mutant (IDH-Mut) versus wild-type (IDH-WT) gliomas are still unknown in this context. For the first time, to our knowledge, this study characterizes the TME of LGG, high-grade gliomas (HGG) IDH-Mut, and HGG IDH-WT patients regarding purinergic ectoenzymes and P1 receptors, focusing on tumor-infiltrating lymphocytes. Here, we show that ectoenzymes from both canonical and non-canonical pathways are increased in the TME when compared to the peripheral blood. We hypothesize this enhancement supports extracellular adenosine generation, hence increasing TME immunosuppression.

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.

Influence of body composition and cardiorespiratory fitness on plasma HSP72, norepinephrine, insulin, and glucose responses to an acute aerobic exercise bout performed in the fed state.

Being overweight is already considered a metabolic risk factor, which can be overcome by increasing cardiorespiratory fitness (CRF). Acute exercise is known to induce changes in plasma hormones and heat shock proteins release. However, there is a lack of studies investigating the impact of body composition and CRF on these variables following acute aerobic exercise. To assess the influence of body composition and cardiorespiratory fitness on plasma heat shock protein 72 kDa (HSP72), norepinephrine (NE), insulin, and glucose responses to an acute aerobic exercise bout in the fed state. Twenty-four healthy male adults were recruited and allocated into three groups: overweight sedentary (n = 8), normal weight sedentary (n = 8), and normal weight active (n = 8). The volunteers performed an acute moderate exercise session on a treadmill at 70% of VO2 peak. Blood samples were drawn at baseline, immediately post-exercise, and at 1-h post-exercise. The exercise session did not induce changes in HSP72 nor NE but changes in glucose and insulin were affected by body mass index. Also, subjects with elevated CRF maintain reduced NE through exercise. At baseline, the overweight sedentary group showed elevated NE, insulin, and glucose; these last two impacting the HOMA-IR index. Thirty minutes of aerobic exercise at 70% VO2 peak, in the fed state, did not change the levels of plasma NE and HSP72. Elevated body composition seems to impact metabolic profile and increase sympathetic activity. Conversely, subjects with increased cardiorespiratory fitness seem to have attenuated sympathetic activity.

LaSOM 335, active against bladder cancer cells, interferes with Let-60 (hRas) and reduces CD73 expression/activity.

Bladder cancer is the fourth most common malignancy in men. It can present along the entire continuum of severity, from mild to well-differentiated disease to extremely malignant tumors with low survival rates. Human RAS genes are the most frequently mutated oncogenes in human cancers, and the critical role of aberrant Ras protein function in carcinogenesis is well established. Therefore, considerable efforts have been devoted to the development of anti-Ras inhibitors for cancer treatment. This study presents the biphenyl dihydropyrimidinone LaSOM 335 with high activity against T24 bladder cancer cells (IC50 = 10.73 ± 0.53 µM) and selectivity of cytotoxicity for this cancer cell line compared to two non-cancer cell lines investigated. Furthermore, we also show that this compound reduced vulvar development in the mutant let-60 gene of Caenorhabditis elegans. Let-60 is a homolog of the mammalian Ras gene. In addition, we observed that LaSOM 335 inhibits the enzymatic activity of CD73 and decreases CD73 expression. Possibly, this expression decrease is due to downstream EGFR signaling via the Ras-Raf-ERK pathway, that directly regulates CD73 expression via ERK1/2. Evidence suggests that non-immunomodulating functions of CD73 play an equally important role for cancer cell survival, progression, and migration. Regarding we also notice that LaSOM 335 was safe in the in vivo model of C. elegans. The set of these findings makes this biphenyl dihydropyrimidinone a promising candidate for further investigations in the bladder cancer field.

Targeting ecto-5'-nucleotidase: A comprehensive review into small molecule inhibitors and expression modulators.

The purinergic signaling has drawn attention from academia and more recently from pharmaceutical industries as a potential therapeutic route for cancer treatment, since ATP may act as chemotactic agent and possess in vitro antineoplastic activity. On the other way, adenosine, produced in extracellular medium by ecto-5'-NT, acts as immunosuppressor and is related to neoangiogenesis, vasculogenesis and evasion to the immune system. Consequently, inhibitors of ecto-5'-NT may prevent tumor progression, reducing adenosine concentrations, preventing escape from the host's immune system and slowing cancer's growth. This review aims to highlight important biochemical and structural features of ecto-5'NT, highlight its expression profile in normal and cancer cell lines detailing compounds which may act as expression regulators and to review the several classes of ecto-5'NT inhibitors developed in the past 12 years, in order to build a general structure-activity relationship model to guide further compound design.

Impairment of adenosine signaling disrupts early embryo development: unveiling the underlying mechanisms.

Purinergic signaling has been implicated in many biological functions, including development. In this study, we investigate the functions of extracellular adenosine and adenosine receptors using a mouse embryonic stem cell (ESC) line and morula stages isolated from mouse embryos. Feeder-free mouse ESC was investigated in the absence and presence of the leukemia inhibitory factor (LIF), configuring undifferentiated cells and cells undergoing spontaneous differentiation. High alkaline phosphatase (ALPL) and low CD73 levels resulting in low adenosine (eADO) levels were characteristic for pluripotent cells in the presence of the LIF, while LIF deprivation resulted in augmented adenosine levels and reduced pluripotency marker expression, which indicated differentiation. Tracing ESC proliferation by BrdU labeling revealed that the inhibition of ALPL by levamisole resulted in a decrease in proliferation due to less eADO accumulation. Furthermore, caffeine and levamisole treatment, inhibiting adenosine receptor and eADO accumulation, respectively, reduced ESC migration, similar to that observed in the absence of the LIF. Pharmacological approaches of selective adenosine receptor subtype inhibition triggered specific adenosine receptor activities, thus triggering calcium or MAP kinase pathways leading to differentiation. In line with the in vitro data, mouse embryos at the morula stage were sensitive to treatments with A1 and A3 receptor antagonists, leading to the conclusion that A1 receptor and A3 receptor inhibition impairs proliferation and self-renewal and triggers inappropriate differentiation, respectively. The findings herein define the functions of eADO signaling in early development with implications for developmental disorders, in which adenosine receptors or ectonucleotidase dysfunctions are involved, and which could lead to malformations and miscarriages, due to exposure to caffeine.

Lymphocytes from B-acute lymphoblastic leukemia patients present differential regulation of the adenosinergic axis depending on risk stratification.

PURPOSE: Although risk-stratified chemotherapy regimens improve B-cell acute lymphoblastic leukemia (B-ALL) clinical outcome, relapse occurs in a significant number of cases. The identification of new therapeutic targets as well as prognostic and diagnostic biomarkers can improve B-ALL patients' clinical outcomes. Purinergic signaling is an important pathway in cancer progression, however the expression of ectonucleotidases and their impact on immune cells in B-ALL lacks exploration. We aimed to analyze the expression of ectonucleotidases in B-ALL patients' lymphocyte subpopulations. METHODS: Peripheral blood samples from 15 patients diagnosed with B-ALL were analyzed. Flow cytometry was used to analyze cellularity, expression level of CD38, CD39, and CD73, and frequency of [Formula: see text], and [Formula: see text] in lymphocyte subpopulations. Plasma was used for cytokines (by CBA kit) and adenine nucleosides/nucleotides detection (by HPLC). RESULTS: Comparing B-ALL patients to health donors, we observed an increase of CD4 + and CD8 + T-cells. In addition, a decrease in CD38 expression in B and Treg subpopulations and an increase in CD39+ CD73+ frequency in Breg and CD8+ T-cells. Analyzing cytokines and adenine nucleosides/nucleotides, we found a decrease in TNF, IL-1ß, and ADO concentrations, together with an increase in AMP in B-ALL patients' plasma. CONCLUSION: As immunomodulators, the expression of ectonucleotidases might be associated with activation states, as well as the abundance of different cellular subsets. We observed a pro-tumor immunity expression profile in B-ALL patients at diagnosis, being associated with cell exhaustion and immune evasion in B-ALL.

Dysfunctional purinergic signaling correlates with disease severity in COVID-19 patients.

Ectonucleotidases modulate inflammatory responses by balancing extracellular ATP and adenosine (ADO) and might be involved in COVID-19 immunopathogenesis. Here, we explored the contribution of extracellular nucleotide metabolism to COVID-19 severity in mild and severe cases of the disease. We verified that the gene expression of ectonucleotidases is reduced in the whole blood of patients with COVID-19 and is negatively correlated to levels of CRP, an inflammatory marker of disease severity. In line with these findings, COVID-19 patients present higher ATP levels in plasma and reduced levels of ADO when compared to healthy controls. Cell type-specific analysis revealed higher frequencies of CD39+ T cells in severely ill patients, while CD4+ and CD8+ expressing CD73 are reduced in this same group. The frequency of B cells CD39+CD73+ is also decreased during acute COVID-19. Interestingly, B cells from COVID-19 patients showed a reduced capacity to hydrolyze ATP into ADP and ADO. Furthermore, impaired expression of ADO receptors and a compromised activation of its signaling pathway is observed in COVID-19 patients. The presence of ADO in vitro, however, suppressed inflammatory responses triggered in patients' cells. In summary, our findings support the idea that alterations in the metabolism of extracellular purines contribute to immune dysregulation during COVID-19, possibly favoring disease severity, and suggest that ADO may be a therapeutic approach for the disease.

Effects of physical exercise on the functionality of human nucleotidases: A systematic review.

Nucleotidases contribute to the regulation of inflammation, coagulation, and cardiovascular activity. Exercise promotes biological adaptations, but its effects on nucleotidase activities and expression are unclear. The objective of this study was to review systematically the effects of exercise on nucleotidase functionality in healthy and unhealthy subjects. The MEDLINE, EMBASE, Cochrane Library, and Web of Science databases were searched to identify, randomized clinical trials, non-randomized clinical trials, uncontrolled clinical trials, quasi-experimental, pre-, and post-interventional studies that evaluated the effects of exercise on nucleotidases in humans, and was not limited by language and date. Two independent reviewers performed the study selection, data extraction, and assessment of risk of bias. Of the 203 articles identified, 12 were included in this review. Eight studies reported that acute exercise, in healthy and unhealthy subjects, elevated the activities or expression of nucleotidases. Four studies evaluated the effects of chronic training on nucleotidase activities in the platelets and lymphocytes of patients with metabolic syndrome, chronic kidney disease, and hypertension and found a decrease in nucleotidase activities in these conditions. Acute and chronic exercise was able to modify the blood plasma and serum levels of nucleotides and nucleosides. Our results suggest that short- and long-term exercise modulate nucleotidase functionality. As such, purinergic signaling may represent a novel molecular adaptation in inflammatory, thrombotic, and vascular responses to exercise.

High P2X6 receptor expression in human bladder cancer predicts good survival prognosis.

As alterations in purinergic signaling have been observed in bladder diseases, we aimed to assess the potential prognostic role of purinergic receptors in bladder cancer in a translational approach based on clinical databases and in vitro data. The prognostic role of purinergic receptors in the survival of patients with bladder cancer and the expression profile of the altered P2 receptors in normal and in tumor samples were determined using The Cancer Genome Atlas databank. In T24 and RT4 human bladder cancer cell lines, the P2 purinergic receptors were characterized by RT-PCR and RT-qPCR analysis including radiotherapy exposure as treatment. The cell number and the cumulative population doubling were also assessed. The expression profile of P2X6 receptor in the cancer pathological stage and in the nodal metastasis status was in agreement with Kaplan-Meier analysis, indicating that high expression of this receptor was related to an increased survival rate in patients with bladder cancer. Of all the P2 receptors expressed on T24 cell line, P2X6 presented high expression after radiotherapy, while it was not altered in RT4 cells. In addition, irradiation promoted a decrease of T24 cell number, but did not change the cell number of RT4 after the same time and radiation dose. Along 7 days after irradiation exposure, both cells regrew. However, while P2X6 receptor was downregulated in T24 cells, it was upregulated in RT4 cells. Our findings indicated that high P2X6 receptor expression induced by radiation in T24 cell line may predict a good survival prognostic factor.

NT5E gene and CD38 protein as potential prognostic biomarkers for childhood B-acute lymphoblastic leukemia.

The risk stratification of B-acute lymphoblastic leukemia (B-ALL) is based on clinical and biological factors. However, B-ALL has significant biological and clinical heterogeneity and 50% of B-ALL patients do not have defined prognostic markers. In this sense, the identification of new prognostic biomarkers is necessary. Considering different cohorts of childhood B-ALL patients, gene (DPP4/CD38/ENTPD1/NT5E) and protein (CD38/CD39/CD73) expressions of ectonucleotidases were analyzed in silico and ex vivo and the association with prognosis was established. In univariate analyses, expression of NT5E was significantly associated with worse progression-free survival (PFS) in bone marrow (BM) samples. In multivariate analyses, Kaplan-Meier analysis, and log-rank test, higher NT5E expression predicted unfavorable PFS in BM samples. Considering minimal residual disease (MRD), higher levels of cellularity were associated with the high NT5E expression at day 8 of induction therapy. In addition, we observed that white blood cells (WBC) of childhood B-ALL patients had more CD38 compared to the same cell population of healthy donors (HD). In fact, MRD > 0.1% patients had higher CD38 protein expression on WBC in comparison to HD. Noteworthy, we observed higher CD38 expression on WBC than blasts in MRD > 0.1% patients. We suggest that NT5E gene and CD38 protein expression, of the ectonucleotidases family, could provide interesting prognostic biomarkers for childhood B-ALL.

Development of bozepinib-loaded nanocapsules for nose-to-brain delivery: preclinical evaluation in glioblastoma.

Aim: To develop and characterize bozepinib-loaded lipid-core nanocapsules (BZP-LNC+) as a potential treatment for glioblastoma (GBM). Methods: Characterization of nanocapsules was performed by diameter, polydispersity index, Zeta potential, pH and encapsulation efficiency. GBM cell viability, cell cycle and Annexin/PI were evaluated after BZP-LNC+ treatment. Synergism between BZP-LNC+ and temozolomide (TMZ) was performed by CompuSyn software and confirmed in vitro and in vivo. Results: BZP-LNC+ showed adequate particle sizes, positive Zeta potential, narrow size distribution and high encapsulation efficiency. BZP-LNC+ reduces GBM growth by inducing apoptosis. BZP-LNC+ and TMZ showed synergistic effect in vitro and reduced the in vivo glioma growth by approximately 81%. Conclusion: The present study provides proof-of-principle insights for the combination of these drugs for GBM treatment.

Nanoformulation Shows Cytotoxicity against Glioblastoma Cell Lines and Antiangiogenic Activity in Chicken Chorioallantoic Membrane.

Glioblastoma (GB) is a histological and genetically heterogeneous brain tumor that is highly proliferative and vascularized. The prognosis is poor with currently available treatment. In this study, we evaluated the cytotoxicity and antiangiogenic activity of doxorubicin-loaded-chitosan-coated-arginylglycylaspartic acid-functionalized-poly(ε-caprolactone)-alpha bisabolol-LNC (AB-DOX-LNC-L-C-RGD). The nanoformulation was prepared by self-assembling followed by interfacial reactions, physicochemically characterized and evaluated in vitro against GB cell lines (U87MG and U138MG) and in vivo using the chicken chorioallantoic membrane assay (CAM). Spherical shape nanocapsules had a hydrodynamic mean diameter of 138 nm, zeta potential of +13.4 mV, doxorubicin encapsulation of 65%, and RGD conjugation of 92%. After 24 h of treatment (U87MG and U138MG), the median inhibition concentrations (IC50) were 520 and 490 nmol L-1 doxorubicin-equivalent concentrations, respectively. The treatment induced antiproliferative activity with S-phase cell-cycle arrest and apoptosis in the GB cells. Furthermore, after 48 h of exposure, evaluation of antiangiogenic activity (CAM) showed that the relative vessel growth following treatment with the nanocapsules was 5.4 times lower than that with the control treatment. The results support the therapeutic potential of the nanoformulation against GB and, thereby, pave the way for future preclinical studies.

EGFRvIII peptide nanocapsules and bevacizumab nanocapsules: a nose-to-brain multitarget approach against glioblastoma.

Aim: To evaluate the antitumor efficacy of bevacizumab-functionalized nanocapsules in a rat glioblastoma model after the pretreatment with nanocapsules functionalized with a peptide-specific to the epidermal growth factor receptor variant III. Materials & methods: Nanocapsules were prepared, physicochemical characterized and intranasally administered to rats. Parameters such as tumor size, histopathological characteristics and infiltration of CD8+ T lymphocytes were evaluated. Results: The strategy of treatment resulted in a reduction of 87% in the tumor size compared with the control group and a higher infiltration of CD8+ T lymphocytes in tumoral tissue. Conclusion: The block of two different molecular targets using nose-to-brain delivery represents a new and promising approach against glioblastoma.

Post-weaning social isolation impairs purinergic signaling in rat brain.

Early life stressors, such as social isolation (SI), can disrupt brain development contributing to behavioral and neurochemical alterations in adulthood. Purinergic receptors and ectonucleotidases are key regulators of brain development in embryonic and postnatal periods, and they are involved in several psychiatric disorders, including schizophrenia. The extracellular ATP drives purinergic signaling by activating P2X and P2Y receptors and it is hydrolyzed by ectonucleotidases in adenosine, which activates P1 receptors. The purpose of this study was to investigate if SI, a rodent model used to replicate abnormal behavior relevant to schizophrenia, impacts purinergic signaling. Male Wistar rats were reared from weaning in group-housed or SI conditions for 8 weeks. SI rats exhibited impairment in prepulse inhibition and social interaction. SI presented increased ADP levels in cerebrospinal fluid and ADP hydrolysis in the hippocampus and striatum synaptosomes. Purinergic receptor expressions were upregulated in the prefrontal cortex and downregulated in the hippocampus and striatum. A2A receptors were differentially expressed in SI prefrontal cortex and the striatum, suggesting distinct roles in these brain structures. SI also presented decreased ADP, adenosine, and guanosine levels in the cerebrospinal fluid in response to D-amphetamine. Like patients with schizophrenia, uric acid levels were prominently increased in SI rats after D-amphetamine challenge. We suggest that the SI-induced deficits in prepulse inhibition might be related to the SI-induced changes in purinergic signaling. We provide new evidence that purinergic signaling is markedly affected in a rat model relevant to schizophrenia, pointing out the importance of purinergic system in psychiatry conditions.

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.

Acute moderate-intensity aerobic exercise promotes purinergic and inflammatory responses in sedentary, overweight and physically active subjects.

NEW FINDING: What is the central question of this study? How does moderate-intensity aerobic exercise affect the behaviour of purinergic enzymes in sedentary, overweight and physically active subjects? What is the relationship between purinergic and inflammatory responses triggered by exercise? What is the main finding and its importance? Moderate-intensity aerobic exercise modifies the activity of purinergic enzymes and the levels of nucleotides and nucleosides. These results are similar in subjects with different biological characteristics. 5'-Nucleotidase activity and adenosine levels are associated with inflammatory responses. This study suggests that a purinergic pathway is related to the inflammatory responses triggered by exercise. ABSTRACT: Purinergic signalling is a mechanism of extracellular communication that modulates events related to exercise, such as inflammation and coagulation. Herein, we evaluated the effects of acute moderate-intensity exercise on the activities of purinergic enzymes and plasma levels of adenine nucleotides in individuals with distinct metabolic characteristics. We analysed the relationship between purinergic parameters, inflammatory responses and cardiometabolic markers. Twenty-four healthy males were assigned to three groups: normal weight sedentary (n = 8), overweight sedentary (n = 8) and normal weight physically active (n = 8). The volunteers performed an acute session of moderate-intensity aerobic exercise on a treadmill at 70% of V̇O2peak ; blood samples were drawn at baseline, immediately post-exercise and at 1 h post-exercise. Immediately post-exercise, all subjects showed increases in ATP, ADP, AMP and p-nitrophenyl thymidine 5'-monophosphate hydrolysis, while AMP hydrolysis remained increased at 1 h after exercise. High-performance liquid chromatography analysis demonstrated lower levels of ATP and ADP at post- and 1 h post-exercise in all groups. Conversely, adenosine and inosine levels increased at post-exercise, but only adenosine remained augmented at 1 h after exercise in all groups. With regard to inflammatory responses, the exercise protocol increased tumour necrosis factor α (TNF-α) and interleukin 8 (IL-8) concentrations in all subjects, but only TNF-α remained elevated at 1 h after exercise. Significant correlations were found between the activity of 5'-nucleotidase, adenosine levels, V̇O2peak , triglyceride, TNF-α and IL-8 levels. Our findings suggest a purinergic signalling pathway that participates, at least partially, in the inflammatory responses triggered by acute moderate-intensity exercise. The response of soluble nucleotidases to acute moderate exercise appears to be similar between subjects of different biological profiles.