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Abstract Background Extracellular ATP–AMP–adenosine metabolism plays a pivotal role in modulating tumor immune responses. Previous studies have shown that the conversion of ATP to AMP is primarily catalysed by Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), a widely studied ATPase, which is expressed in tumor-associated immune cells. However, the function of ATPases derived from tumor cells themselves remains poorly understood. The purpose of this study was to investigate the role of colon cancer cell–derived ATPases in the development and progression of colon cancer. Methods Bioinformatic and tissue microarray analyses were performed to investigate the expression of ATPase family members in colon cancer. An ATP hydrolysis assay, high-performance liquid chromatography (HPLC), and CCK8 and colony formation assays were used to determine the effects of ENTPD2 on the biological functions of colon cancer cells. Flow cytometric and RNA-seq analyses were used to explore the function of CD8+ T cells. Immunoelectron microscopy and western blotting were used to evaluate the expression of ENTPD2 in exosomes. Double-labelling immunofluorescence and western blotting were used to examine the expression of ENTPD2 in serum exosomes and colon cancer tissues. Results We found that ENTPD2, rather than the well-known ATPase CD39, is highly expressed in cancer cells and is significantly positively associated with poor patient prognosis in patients with colon cancer. The overexpression of ENTPD2 in cancer cells augmented tumor progression in immunocompetent mice by inhibiting the function of CD8+ T cells. Moreover, ENTPD2 is localized primarily within exosomes. On the one hand, exosomal ENTPD2 reduces extracellular ATP levels, thereby inhibiting P2X7R-mediated NFATc1 nuclear transcription; on the other hand, it facilitates the increased conversion of ATP to adenosine, hence promoting adenosine-A2AR pathway activity. In patients with colon cancer, the serum level of exosomal ENTPD2 is positively associated with advanced TNM stage and high tumor invasion depth. Moreover, the level of ENTPD2 in the serum exosomes of colon cancer patients is positively correlated with the ENTPD2 expression level in paired colon cancer tissues, and the ENTPD2 level in both serum exosomes and tissues is significantly negatively correlated with the ENTPD2 expression level in tumor-infiltrating CD8+ T cells. Conclusion Our study suggests that exosomal ENTPD2, originated from colon cancer cells, contributes to the immunosuppressive microenvironment by promoting ATP–adenosine metabolism. These findings highlight the importance of exosome-derived hydrolytic enzymes as independent entities in shaping the tumor immune microenvironment.

Background: Extracellular ATP–AMP–adenosine metabolism plays a pivotal role in modulating tumor immune responses. Previous studies have shown that the conversion of ATP to AMP is primarily catalysed by Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), a widely studied ATPase, which is expressed in tumor-associated immune cells. However, the function of ATPases derived from tumor cells themselves remains poorly understood. The purpose of this study was to investigate the role of colon cancer cell–derived ATPases in the development and progression of colon cancer. Methods: Bioinformatic and tissue microarray analyses were performed to investigate the expression of ATPase family members in colon cancer. An ATP hydrolysis assay, high-performance liquid chromatography (HPLC), and CCK8 and colony formation assays were used to determine the effects of ENTPD2 on the biological functions of colon cancer cells. Flow cytometric and RNA-seq analyses were used to explore the function of CD8+ T cells. Immunoelectron microscopy and western blotting were used to evaluate the expression of ENTPD2 in exosomes. Double-labelling immunofluorescence and western blotting were used to examine the expression of ENTPD2 in serum exosomes and colon cancer tissues. Results: We found that ENTPD2, rather than the well-known ATPase CD39, is highly expressed in cancer cells and is significantly positively associated with poor patient prognosis in patients with colon cancer. The overexpression of ENTPD2 in cancer cells augmented tumor progression in immunocompetent mice by inhibiting the function of CD8+ T cells. Moreover, ENTPD2 is localized primarily within exosomes. On the one hand, exosomal ENTPD2 reduces extracellular ATP levels, thereby inhibiting P2X7R-mediated NFATc1 nuclear transcription; on the other hand, it facilitates the increased conversion of ATP to adenosine, hence promoting adenosine-A2AR pathway activity. In patients with colon cancer, the serum level of exosomal ENTPD2 is positively associated with advanced TNM stage and high tumor invasion depth. Moreover, the level of ENTPD2 in the serum exosomes of colon cancer patients is positively correlated with the ENTPD2 expression level in paired colon cancer tissues, and the ENTPD2 level in both serum exosomes and tissues is significantly negatively correlated with the ENTPD2 expression level in tumor-infiltrating CD8+ T cells. Conclusion: Our study suggests that exosomal ENTPD2, originated from colon cancer cells, contributes to the immunosuppressive microenvironment by promoting ATP–adenosine metabolism. These findings highlight the importance of exosome-derived hydrolytic enzymes as independent entities in shaping the tumor immune microenvironment. [ABSTRACT FROM AUTHOR]

Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) family members, which hydrolyse extracellular ATP and ADP and ecto-5′-nucleotidase, which hydrolyses AMP, have been characterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10% serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1% serum). The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi) released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.

Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5′-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E- NTPDase and ecto-5′-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5′-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final accumulation of the nucleoside. The transcript levels of the five TvNTPDases gene sequences were analyzed by qRT-PCR and the highest gene expressions were found for TvNTPDase 2 and 4 . The extracellular guanosine uptake was observed as 13C GTP nucleotide into parasite DNA and it was lower than that observed for adenosine, labeled as 13C ATP. These findings indicate the T. vaginalis preference for adenosine uptake and the accumulation of guanosine in the extracellular milieu, corroborating with HPLC data. Our data demonstrate, for the first time, the cascade of guanine nucleotides in T. vaginalis and open possibilities on the study of guanine-related purines other than the classical intracellular activity of G proteins for signal transduction. [ABSTRACT FROM AUTHOR]

Abstract: Human health is under constant threat of a wide variety of dangers, both self and nonself. The immune system is occupied with protecting the host against such dangers in order to preserve human health. For that purpose, the immune system is equipped with a diverse array of both cellular and non-cellular effectors that are in continuous communication with each other. The naturally occurring nucleotide adenosine 5′-triphosphate (ATP) and its metabolite adenosine (Ado) probably constitute an intrinsic part of this extensive immunological network through purinergic signaling by their cognate receptors, which are widely expressed throughout the body. This review provides a thorough overview of the effects of ATP and Ado on major immune cell types. The overwhelming evidence indicates that ATP and Ado are important endogenous signaling molecules in immunity and inflammation. Although the role of ATP and Ado during the course of inflammatory and immune responses in vivo appears to be extremely complex, we propose that their immunological role is both interdependent and multifaceted, meaning that the nature of their effects may shift from immunostimulatory to immunoregulatory or vice versa depending on extracellular concentrations as well as on expression patterns of purinergic receptors and ecto-enzymes. Purinergic signaling thus contributes to the fine-tuning of inflammatory and immune responses in such a way that the danger to the host is eliminated efficiently with minimal damage to healthy tissues. [Copyright &y& Elsevier]

We report here that induction of ectoATPase by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is cell-type specific and not a generalized response to aryl hydrocarbon (Ah) receptor activation. TCDD increased [14C]-ATP and -ADP metabolism in two mouse hepatoma lines, Hepa1c1c7 and Hepa1-6 cells, but not in human hepatoma HepG2 or HuH-7 cells, human umbilical vein endothelial cells (HUVEC), chick hepatoma (LMH) cells, or chick primary hepatocytes or cardiac myocytes, even though all of those cell types were Ah receptor-responsive, as evidenced by cytochrome P4501A induction. To determine whether the differences in ectonucleotidase responsiveness to TCDD might be related to differences in cell-type ectonucleotidase expression, ATP and ADP metabolite patterns, the products of several classes of ectonucleotidases including ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), ectophosphodiesterase/pyrophosphatases (E-NPP enzymes) and ectoalkaline phosphatase activities were examined. Those patterns, together with results of enzyme assays, Western blotting, or semiquantitative RT–PCR show that NTPDase2 is the main ectonucleotidase for murine and human hepatoma cells, NTPDase3 for chick hepatocytes and LMH cells, and an E-NPP enzyme for chick cardiac myocytes. Evidence for NTPDase2 expression was lacking in all cells except the mouse and human hepatoma cells. TCDD increased expression of the NTPDase2 gene but only in the mouse and not in the human hepatoma cells. TCDD did not increase NTPDase3, NTPDase1, E-NPP, or alkaline phosphatase in any of the cell types examined. The failure of TCDD to increase ATP metabolism in HUVEC, chick LMH cells, hepatocytes, and cardiac myocytes can be attributed to their lack of NTPDase2 expression, while the increase in ATP metabolism by TCDD in the mouse but not the human hepatoma cells can be explained by differences in TCDD effects on mouse and human hepatoma NTPDase2 gene expression. In addition to characterizing effects of TCDD on ectonucleotidases, these studies reveal major differences in the complements of ectonucleotidases present in different cell types. It is likely that such differences are important for cell-specific susceptibility to extracellular nucleotide toxicity and responses to purinergic signaling. [Copyright &y& Elsevier]

Adenosine (ADO) is a retaliatory metabolite that is expressed in conditions of injury or stress. During these conditions ATP is released at the extracellular level and is metabolized to adenosine. For this reason, adenosine is defined as a "danger signal" for cells and organs, in addition to its important role as homeostatic regulator. Its physiological functions are mediated through interaction with four specific transmembrane receptors called ADORA1, ADORA2A, ADORA2B and ADORA3. In the lungs of mice and humans all four adenosine receptors are expressed with different roles, having pro- and anti-inflammatory roles, determining bronchoconstriction and regulating lung inflammation and airway remodeling. Adenosine receptors can also promote differentiation of lung fibroblasts into myofibroblasts, typical of the fibrotic event. This last function suggests a potential involvement of adenosine in the fibrotic lung disease processes, which are characterized by different degrees of inflammation and fibrosis. Idiopathic pulmonary fibrosis (IPF) is the pathology with the highest degree of fibrosis and is of unknown etiology and burdened by lack of effective treatments in humans., (Copyright © 2013 Elsevier Ltd. All rights reserved.)