Istradefylline induces A2A/P2X7 crosstalk expression inducing pro-inflammatory signal, and reduces AKT/mTOR signaling in melanoma-bearing mice.

ATP and adenosine (ADO) are critical players in the context of cancer. In the tumor microenvironment, the signaling dependent on these molecules, and immune cells, is regulated by an enzymatic chain and purinergic receptors called purinome. Primarily, the A2A receptor (A2AR) has a pro-tumor action since it reduces the immune response and favors the growth of malignant melanoma. Therefore, this study aimed to verify the effects of A2AR antagonism with Istradefylline (IST) on the purinergic signaling profile of the melanoma tumor and immunological compartments. We observed reduced tumor growth of melanoma in IST-treated animals. IST inhibited AKT/mTOR pathway, which is involved with tumor growth. In the tumor, spleen, and thymus, the modulation of purinergic enzymes (CD39, CD73, and E-ADA) characterized a pro-inflammatory profile since it favored increased extracellular concentrations of ATP to the detriment of ADO. A2AR inhibition generated a compensatory feedback process with increased A2AR expression at the tumor level. However, there was also an increase in the expression of the P2X7 receptor (P2X7R), which culminated in an increase in pro-inflammatory pathways with the release of IL-1ß and pro-inflammatory cytokines such as IFN-γ and TNF-α. Our data evidence the cross-involvement between expression and action of the A2AR and P2X7R. We suggest that IST is a promising drug for off-label use in cancer since it promotes an anti-tumoral response by producing pro-inflammatory cytokines and blocking of AKT/mTOR tumor growth pathway.

Distinct kinetics for nucleotide hydrolysis in lymphocytes isolated from blood, spleen and cervical lymph nodes: Characterization of ectonucleotidase activity.

Ectonucleotidases are a plasma membrane-bound enzyme that hydrolyses extracellular adenosine triphosphate (eATP) and adenosine diphosphate (eADP) to adenosine monophosphate (AMP). It regulates normal function of lymphocytes, acts as an inflammatory marker and represents a molecular target for new therapeutics. Thus, this study sought to isolate lymphocytes from blood (BL), spleen (SL) and cervical lymph node (CLL), and characterize the eATP and eADP enzymatic hydrolysis in Wistar rats. The hydrolysis of the nucleotides occurred primarily at pH 8.0, 37°C in the presence of Ca2+ or Mg2+ . Chevillard-plot showed the hydrolysis of eATP and eADP at the same active site. The inhibitors of some classical ATDPases did not cause any significant change on enzymatic activity. Inhibitors of E-NTPDase (-1, -2, -3 isoforms) and E-NPP-1 decrease the enzyme activity in all resident lymphocytes. Furthermore, kinetic parameters (Vmax and Km) revealed that SL had significantly (P < .001) higher enzymatic activity when compared to BL and CLL. In conclusion, this study standardized kinetic values for eATP and eADP hydrolysis for resident lymphocytes isolated from BL, SL and CLL.

Astrocaryum aculeatum fruit improves inflammation and redox balance in phytohemagglutinin-stimulated macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Astrocaryum aculeatum G.Mey. (tucumã) is highly consumed by riverside communities in the Amazonian region. These communities have recently been shown to have increased longevity and reduced prevalence of age-related morbidity. Tucumã, which is locally used in their diet and traditional medicine may contribute to these features. AIM OF THE STUDY: To investigate the anti-inflammatory and antioxidant properties of A. aculeatum extract against phytohemagglutinin-induced inflammation in cell cultures. MATERIALS AND METHODS: Cell viability and cytotoxicity assays, gene expression of interleukins IL-1ß, IL-6, IL-10, levels of reactive oxygen species (ROS), nitric oxide (NO) and thiols were employed, as well as the activities of antioxidant enzymes in RAW 264.7 cells stimulated with phytohemagglutinin to mimic inflammation. RESULTS: The extract of A. aculeatum fruit inhibited macrophage proliferation (P < 0.05), arrested the cell cycle in G0/G1 phase (P < 0.001), increased antioxidant defenses (P < 0.01), reduced oxidative stress (P < 0.01), and modulated genes related to the inflammatory response (P < 0.001). CONCLUSION: Our results demonstrate that A. aculeatum fruit has anti-inflammatory and antioxidant capacities. These beneficial effects of tucumã on cells are also likely to be seen in vivo, thereby suggesting that its extract is a suitable therapeutic adjuvant in the prevention or treatment of inflammatory diseases.

Co-Nanoencapsulation of Vitamin D3 and Curcumin Regulates Inflammation and Purine Metabolism in a Model of Arthritis.

We analyzed the effects of a nanoencapsulated association of curcumin and vitamin D3 on purine metabolism enzymes in neutrophils, lymphocytes, and platelets in a model of adjuvant-induced arthritis (AIA) in rats. Following AIA induction, the animals were treated for 15 days with free and nanoencapsulated curcumin (4 mg/kg), nanocapsules of vitamin D3 (VD3) (15.84 IU/day), a nanoencapsulated combination of curcumin and VD3, vehicle, or blank nanocapsules. The animals were euthanized, and blood was collected to evaluate the activities of E-NTPDase, adenosine deaminase (ADA), and myeloperoxidase (MPO), as well as reactive oxygen species (ROS) levels and biochemical parameters. Also, the liver and kidney were collected for histological analysis. The changes in the activities of purinergic enzymes indicated that inflammation was significantly reverted by vitamin D3 and curcumin co-nanoencapsulation treatments in the arthritic rats. The reduction of inflammation was confirmed by the reduction in the signs and symptoms of AIA, as well as in MPO activity by all formulations. The treatments with nanocapsules reverted histological alterations in the kidney. Serum parameters were not altered by the induction or treatments. Our results suggest that co-nanoencapsulation of vitamin D3 and curcumin is an efficient alternative adjuvant treatment for rheumatoid arthritis as it reverts the changes in the purine metabolism and reduces arthritis-associated inflammation.

Characterization of E-NTPDase (EC 3.6.1.5) activity in hepatic lymphocytes: A different activity profile from peripheral lymphocytes.

The activity of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5) was characterized in hepatic lymphocytes (HL) of rats. For this purpose, a specific method for the isolation of lymphocytes from hepatic tissue was developed. Subsequently, E-NTPDase activity of rat HL was compared with that of rat peripheral lymphocytes. The HL showed high cell count and viability. Also, the characterization test revealed that the optimal E-NTPDase activities were attained at 37°C and pH 8.0 in the presence of Ca2+ . In addition, in the presence of specific E-NTPDase inhibitors (20mM sodium azide and 0.3mM suramin), there were significant inhibitions in nucleotide hydrolysis. However, there was no significant change in adenosine triphosphate (ATP) or adenosine diphosphate (ADP) hydrolysis in the presence of inhibitors of other E-ATPase (0.1mM Ouabain, 0.5mM orthovanadate, and 1mM, 5mM, and 10mM sodium azide). Furthermore, the kinetic behavior of the enzyme in HL showed apparent Km of 134.90 ± 0.03µM and 214.40 ± 0.06µM as well as Vmax of 345.0 ± 28.32 and 242.0 ± 27.55 Æžmol Pi/min/mg of protein for ATP and ADP, respectively. The Chevillard plot revealed that ATP and ADP were hydrolyzed at the same active site of the enzyme. Our results suggest that the degradation of extracellular nucleotides in HL may have been primarily accomplished by E-NTPDase. The higher E-NTPDase activity observed in HL may be attributed to the important physiological functions of ATP and ADP in HL. SIGNIFICANCE OF THE STUDY: Extracellular purine nucleotides are able to interact with specific receptors and trigger a number of important physiological functions in cells. This interaction is controlled by ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), enzyme that present their catalytic site at the extracellular space and degrades nucleotides. This purinergic signaling has important functions in peripheral lymphocytes and may represent an important new therapeutic target for the treatment of immunological diseases. However, there is dearth of information on the involvement of E-NTPDase in liver lymphocytes. The liver is an important organ, which performs both metabolic and toxicological roles in living organism, and hepatic lymphocytes may play crucial action in the regulation of immune responses in the liver tissue. Furthermore, various chronic diseases such as cirrhosis may be treated with novel pharmacotherapy by targeting the modulation of hepatic lymphocytes. Thus, the significance of this study is to evaluate the activity of E-NTPDase in liver lymphocyte and compare its activity with the peripheral lymphocytes.