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.

Purinergic signaling elements are correlated with coagulation players in peripheral blood and leukocyte samples from COVID-19 patients.

For over a year, the coronavirus disease 2019 has been affecting the world population by causing severe tissue injuries and death in infected people. Adenosine triphosphate (ATP) and the nicotinamide adenine dinucleotide (NAD +) are two molecules that are released into the extracellular microenvironment after direct virus infection or cell death caused by hyper inflammation and coagulopathy. Also, these molecules are well known to participate in multiple pathways and have a pivotal role in the purinergic signaling pathway. Thus, using public datasets available on the Gene Expression Omnibus (GEO), we analyzed raw proteomics data acquired using mass spectrometry (the gold standard method) and raw genomics data from COVID-19 patient samples obtained by microarray. The data was analyzed using bioinformatics and statistical methods according to our objectives. Here, we compared the purinergic profile of the total leukocyte population and evaluated the levels of these soluble biomolecules in the blood, and their correlation with coagulation components in COVID-19 patients, in comparison to healthy people or non-COVID-19 patients. The blood metabolite analysis showed a stage-dependent inosine increase in COVID-19 patients, while the nucleotides ATP and ADP had positive correlations with fibrinogen and other coagulation proteins. Also, ATP, ADP, inosine, and hypoxanthine had positive and negative correlations with clinical features. Regarding leukocyte gene expression, COVID-19 patients showed an upregulation of the P2RX1, P2RX4, P2RX5, P2RX7, P2RY1, P2RY12, PANX1, ADORA2B, NLPR3, and F3 genes. Yet, the ectoenzymes of the canonical and non-canonical adenosinergic pathway (ENTPD1 and CD38) are upregulated, suggesting that adenosine is produced by both active adenosinergic pathways. Hence, approaches targeting these biomolecules or their specific purinoreceptors and ectoenzymes may attenuate the high inflammatory state and the coagulopathy seen in COVID-19 patients. KEY MESSAGES : Adenosinergic pathways are modulated on leukocytes from COVID-19 patients. Plasmatic inosine levels are increased in COVID-19 patients. ATP, ADP, AMP, hypoxanthine, and inosine are correlated with coagulation players. The nucleotides and nucleosides are correlated with patients' clinical features. The P2 receptors and ectoenzymes are correlated with Tissue factor in COVID-19.

The implications of the purinergic signaling throughout pregnancy.

Purinergic signaling is a necessary mechanism to trigger or even amplify cell communication. Its ligands, notably adenosine triphosphate (ATP) and adenosine, modulate specific membrane-bound receptors in virtually all human cells. Regardless of the stage of the pregnancy, cellular communication between maternal, placental, and fetal cells is the paramount mechanism to sustain its optimal status. In this review, we describe the crucial role of purinergic signaling on the regulation of the maternal-fetal trophic exchanges, immune control, and endocrine exchanges throughout pregnancy. The nature of the modulation of both ATP and adenosine on the embryo-maternal interface, going through placental invasion until birth delivery depends on the general maternal-fetal health state and consequently on the selective activation of their specific receptors. In addition, an increasing number of studies have been demonstrating the pivotal role of ATP and adenosine in modulating deleterious effects of suboptimal conditions of pregnancy. Here, we discuss the role of purinergic signaling on the balance that coordinates the embryo-maternal exchanges and a promising therapeutic venue in the context of pregnancy disorders.

The adenosinergic pathway in mesenchymal stem cell fate and functions.

Mesenchymal stem cells (MSCs) play an important role in tissue homeostasis and damage repair through their ability to differentiate into cells of different tissues, trophic support, and immunomodulation. These properties made them attractive for clinical applications in regenerative medicine, immune disorders, and cell transplantation. However, despite multiple preclinical and clinical studies demonstrating beneficial effects of MSCs, their native identity and mechanisms of action remain inconclusive. Since its discovery, the CD73/ecto-5'-nucleotidase is known as a classic marker for MSCs, but its role goes far beyond a phenotypic characterization antigen. CD73 contributes to adenosine production, therefore, is an essential component of purinergic signaling, a pathway composed of different nucleotides and nucleosides, which concentrations are finely regulated by the ectoenzymes and receptors. Thus, purinergic signaling controls pathophysiological functions such as proliferation, migration, cell fate, and immune responses. Despite the remarkable progress already achieved in considering adenosinergic pathway as a therapeutic target in different pathologies, its role is not fully explored in the context of the therapeutic functions of MSCs. Therefore, in this review, we provide an overview of the role of CD73 and adenosine-mediated signaling in the functions ascribed to MSCs, such as homing and proliferation, cell differentiation, and immunomodulation. Additionally, we will discuss the pathophysiological role of MSCs, via CD73 and adenosine, in different diseases, as well as in tumor development and progression. A better understanding of the adenosinergic pathway in the regulation of MSCs functions will help to provide improved therapeutic strategies applicable in regenerative medicine.

Development of chitosan, gelatin and liposome film and analysis of its biocompatibility in vitro.

A film of chitosan, gelatin and liposome has been designed for dermatological applications. Several adaptations were required throughout development to facilitate in vitro analysis, physicochemical characterization and biocompatibility evaluation. The final version of the film was characterized by differential scanning calorimetry, evaluation of swelling and scanning electron microscopy. The biocompatibility of the film was assessed by investigating cellular parameters of three types of human cells by direct contact or through films extracts: I) primary culture of adipose-derived mesenchymal stromal cells (ADCSs) and melanoma cell lines were used to test cell adhesion and morphology by direct cell culture on the material; II) ADSCs and immortalized keratinocytes were used in cell viability assay using different films extracts. The film showed physicochemical characteristics that favored cellular input, being suitable for in vitro analysis, which allowed its biocompatible characteristics such as the absence of toxicity to be verified without causing significant morphological changes in ADSCs and melanoma cell line. Altogether, these results suggest that the material has a potential application for drug delivery and promotion of skin tissue repair and is therefore worthwhile for further investigations using preclinical models to cover dermal lesions.

Decitabine limits escape from MEK inhibition in uveal melanoma.

MEK inhibitors (MEKi) demonstrate anti-proliferative activity in patients with metastatic uveal melanoma, but responses are short-lived. In the present study, we evaluated the MEKi trametinib alone and in combination with drugs targeting epigenetic regulators, including DOT1L, EZH2, LSD1, DNA methyltransferases, and histone acetyltransferases. The DNA methyltransferase inhibitor (DNMTi) decitabine effectively enhanced the anti-proliferative activity of trametinib in cell viability, colony formation, and 3D organoid assays. RNA-Seq analysis showed the MEKi-DNMTi combination primarily affected the expression of genes involved in G1 and G2/2M checkpoints, cell survival, chromosome segregation and mitotic spindle. The DNMTi-MEKi combination did not appear to induce a DNA damage response (as measured by γH2AX foci) or senescence (as measured by ß-galactosidase staining) compared to either MEKi or DNMTi alone. Instead, the combination increased expression of the CDK inhibitor p21 and the pro-apoptotic protein BIM. In vivo, the DNMTi-MEKi combination was more effective at suppressing growth of MP41 uveal melanoma xenografts than either drug alone. Our studies indicate that DNMTi may enhance the activity of MEKi in uveal melanoma.

Dipyridamole impairs autophagic flux and exerts antiproliferative activity on prostate cancer cells.

Autophagy is a cellular bulk degradation process used as an alternative source of energy and metabolites and implicated in various diseases. Inefficient autophagy in nutrient-deprived cancer cells would be beneficial for cancer therapy making its modulation valuable as a therapeutic strategy for cancer treatment, especially in combination with chemotherapy. Dipyridamole (DIP) is a vasodilator and antithrombotic drug. Its major effects involve the block of nucleoside uptake and phosphodiestesase inhibition, leading to increased levels of intracellular cAMP. Here we report that DIP increases autophagic markers due to autophagic flux blockage, resembling autophagosome maturation and/or closure impairment. Treatment with DIP results in an increased number of autophagosomes and autolysosomes and impairs degradation of SQSTM1/p62. As blockage of autophagic flux decreases the recycling of cellular components, DIP reduced the intracellular ATP levels in cancer cells. Autophagic flux blockage was neither through inhibition of lysosome function nor blockage of nucleoside uptake, but could be prevented by treatment with a PKA inhibitor, suggesting that autophagic flux failure mediated by DIP results from increased intracellular levels of cAMP. Treatment with DIP presented antiproliferative effects in vitro alone and in combination with chemotherapy drugs. Collectively, these data demonstrate that DIP can impair autophagic degradation, by preventing the normal autophagosome maturation, and might be useful in combination anticancer therapy.

Physical exercise and human adipose-derived mesenchymal stem cells ameliorate motor disturbances in a male rat model of Parkinson's disease.

Parkinson's disease (PD) is a disabling and highly costly neurodegenerative condition with worldwide prevalence. Despite advances in treatments that slow progression and minimize locomotor impairments, its clinical management is still a challenge. Previous preclinical studies, using mesenchymal stem cell (MSC) transplantation and isolated physical exercise (EX), reported beneficial results for treatment of PD. Therefore, this experimental randomized study aimed to elucidate the therapeutic potential of combined therapy using adipose-derived human MSCs (ADSCs) grafted into the striatum in conjunction with aerobic treadmill training, specifically in terms of locomotor performance in a unilateral PD rat model induced by 6-hydroxydopamine (6-OHDA). Forty-one male Wistar rats were categorized into five groups in accordance with the type of treatment to which they were subjected (Sham, 6-OHDA - injury, 6-OHDA + exercise, 6-OHDA + cells, and 6-OHDA + combined). Subsequently, dopaminergic depletion was assessed by the methylphenidate challenge and the specified therapeutic intervention was conducted in each group. The foot fault task was performed at the end of the experiment to serve as an assessment of motor skills. The results showed that despite disturbances in motor balance and coordination, locomotor dysfunction was ameliorated in all treatment categories in comparison to the injury group (sign test, p < 0.001, effect size: 0.71). The exercise alone and combined groups were the categories that exhibited the best recovery in terms of movement performance (p < 0.001). Overall, this study confirms that exercise is a powerful option to improve motor function and a promising adjuvant intervention for stem cell transplantation in the treatment of PD motor symptoms. OPEN PRACTICES: This article has been awarded Open Data. All materials and data are publicly accessible at https://figshare.com/s/18a543c101a17a1d5560. Learn more about the Open Practices badges from the Center for Open Science: https://osf.io/tvyxz/wiki.

EMT-like process in glioblastomas and reactive astrocytes.

The concept of the epithelial-to-mesenchymal transition (EMT) in epithelial cells has accelerated our understanding about cancer spreading. Fortunately, much of this information has been able to be extrapolated to non-epithelial cancers, such as glioblastoma (GBM). Interestingly, reactive astrocytes, which are present in the tumor edge in association with glioma cells, might also undergo EMT-like under stimuli of GBM cells. As result, the positive feedback of reactive astrocytes and glioma cells, could act to promote cancer progression making use of the dynamism of the mesenchymal phenotype. Nonetheless, the interpretation of data regarding EMT-like in GBM and astrocytes requires prudence, mainly because the program of EMT in epithelial tumors may not necessarily be the same as those in gliomas and astrocytes.

Comparison of Human Denuded Amniotic Membrane and Porcine Small Intestine Submucosa as Scaffolds for Limbal Mesenchymal Stem Cells.

Blinding corneal scarring is usually treated with allogeneic graft tissue. Nevertheless, the global shortage of donors leaves millions of patients in need of therapy. Traditional tissue engineering strategies involves the combination of cells, growth factors, and scaffolds that can supply cellular biological components allowing to restore the tissue function. The mesenchymal stem cells found in the limbal stroma (L-MSCs) have a self-renewal potential for multilineage differentiation. Thus, in this work we compared the potential of human amniotic membrane (hAM) and porcine small intestine submucosa (SIS) as scaffolds for L-MSCs, aiming at potential applications in corneal regeneration. For that, L-MSCs were seeded on hAM and SIS and we analyzed their viability, actin cytoskeleton, nuclei morphology, cell density, adhesion and surface markers. Our results showed that cells adhered and integrated into both membranes with a high cell density, an important characteristic for cell therapy. However, due to its transparency, the hAM allowed a better observation of L-MSCs. In addition, the analysis of surface markers expression on L-MSCs after two weeks showed a slight increase in the percentages of negative markers for MSCs grown on SIS membrane. Thus, considering a long-term culture, the hAM was considered better in maintaining the MSCs phenotype. Regarding the function as scaffolds, SIS was as efficient as the amniotic membrane, considering that these two types of biological matrices maintained the cell viability, actin cytoskeleton, nuclei morphology and mesenchymal phenotype, without causing cell death. Therefore, our data in vitro provides evidence for future pre-clinical studies were these membranes can be used as a support to transport mesenchymal stem cells to the injured area, creating a kind of temporary curative, allowing the release of bioactive molecules, such as cytokines and growth factors and then promoting the tissue regeneration, both in human and veterinary medicine.

Cervical cancer stem-like cells: systematic review and identification of reference genes for gene expression.

Cervical cancer is the fourth most common cancer affecting women worldwide. Among many factors, the presence of cancer stem cells, a subpopulation of cells inside the tumor, has been associated with a worse prognosis. Considering the importance of gene expression studies to understand the biology of cervical cancer stem cells (CCSC), this work identifies stable reference genes for cervical cancer cell lines SiHa, HeLa, and ME180 as well as their respective cancer stem-like cells. A literature review was performed to identify validated reference genes currently used to normalize RT-qPCR data in cervical cancer cell lines. Then, cell lines were cultured in regular monolayer or in a condition that favors tumor sphere formation. RT-qPCR was performed using five reference genes: ACTB, B2M, GAPDH, HPRT1, and TBP. Stability was assessed to validate the selected genes as suitable reference genes. The evaluation validated B2M, GAPDH, HPRT1, and TBP in these experimental conditions. Among them, GAPDH and TBP presented the lowest variability according to the analysis by Normfinder, Bestkeeper, and ΔCq methods, being therefore the most adequate genes to normalize the combination of all samples. These results suggest that B2M, GAPDH, HPRT1, and TBP are suitable reference genes to normalize RT-qPCR data of established cervical cancer cell lines SiHa, HeLa, and ME180 as well as their derived cancer stem-like cells. Indeed, GAPDH and TBP seem to be the most convenient choices for studying gene expression in these cells in monolayers or spheres.

Extracellular Nucleotide Hydrolysis in Dermal and Limbal Mesenchymal Stem Cells: A Source of Adenosine Production.

Human Limbal (L-MSCs) and Dermal Mesenchymal Stem Cell (D-MSCs) possess many properties that increase their therapeutic potential in ophthalmology and dermatology. It is known that purinergic signaling plays a role in many aspects of mesenchymal stem cells physiology. They release and respond to purinergic ligands, altering proliferation, migration, differentiation, and apoptosis. Therefore, more information on these processes would be crucial for establishing future clinical applications using their differentiation potential, but without undesirable side effects. This study evaluated and compared the expression of ecto-nucleotidases, the enzymatic activity of degradation of extracellular nucleotides and the metabolism of extracellular ATP in D-MSCs and L-MSCs, isolated from discard tissues of human skin and sclerocorneal rims. The D-MSCs and L-MSCs showed a differentiation potential into osteogenic, adipogenic, and chondrogenic lineages and the expression of markers CD105+ , CD44+ , CD14- , CD34- , CD45- , as expected. Both cells hydrolyzed low levels of extracellular ATP and high levels of AMP, leading to adenosine accumulation that can regulate inflammation and tissue repair. These cells expressed mRNA for ENTPD1, 2, 3, 5 and 6, and CD73 that corresponded to the observed enzymatic activities. Thus, considering the degradation of ATP and adenosine production, limbal MSCs are very similar to dermal MSCs, indicating that from the aspect of extracellular nucleotide metabolism L-MSCs are very similar to the characterized D-MSCs. J. Cell. Biochem. 118: 2430-2442, 2017. © 2017 Wiley Periodicals, Inc.

The Epithelial-to-Mesenchymal Transition-Like Process in Glioblastoma: An Updated Systematic Review and In Silico Investigation.

Glioblastoma multiforme (GBM) is the most aggressive form of brain cancer due to its highly invasive nature that impedes the surgical removal of all tumor cells, making relapse inevitable. However, the mechanisms used by glioma cells to invade the surrounding tissue are still unclear. In this context, epithelial-to-mesenchymal transition (EMT) has emerged as a key regulator of this invasive state and although the real relevance of this program in malignant glioma is still controversial, it has been strongly associated with GBM malignancy. EMT is a very complex process regulated by several families of transcriptional factors through many signaling pathways that form a network that allows cancer cells to acquire invasive properties and penetrate the neighboring stroma, resulting in the formation of an advantageous microenvironment for cancer progression and metastasis. In this systematic review, we focus on the molecular mechanisms of EMT including EMT-factors, drug resistance, miRNA, and new therapeutic strategies. In addition, we address controversial questions about mesenchymal shift in GBMs with a bioinformatics analysis to show that in terms of epithelial and mesenchymal phenotype, the majority of GBMs samples analyzed have a profile more mesenchymal than epithelial. If induced, this phenotype can be shifted toward an even more mesenchymal phenotype in an EMT-like process in glioma cells. A better understanding of the molecular regulation of the EMT during tumor spreading will help to provide potential therapeutic interventions to target this program when treating GBM.

Modulation of Autophagy by Calcium Signalosome in Human Disease.

Autophagy is a catabolic process that is largely regulated by extracellular and intracellular signaling pathways that are central to cellular metabolism and growth. Mounting evidence has shown that ion channels and transporters are important for basal autophagy functioning and influence autophagy to handle stressful situations. Besides its role in cell proliferation and apoptosis, intracellular Ca(2+) is widely recognized as a key regulator of autophagy, acting through the modulation of pathways such as the mechanistic target of rapamycin complex 1, calcium/calmodulin-dependent protein kinase kinase 2, and protein kinase C. Proper spatiotemporal Ca(2+) availability, coupled with a controlled ionic flow among the extracellular milieu, storage compartments, and the cytosol, is critical in determining the role played by Ca(2+) on autophagy and on cell fate. The crosstalk between Ca(2+) and autophagy has a central role in cellular homeostasis and survival during several physiologic and pathologic conditions. Here we review the main findings concerning the mechanisms and roles of Ca(2+)-modulated autophagy, focusing on human disorders ranging from cancer to neurologic diseases and immunity. By identifying mechanisms, players, and pathways that either induce or suppress autophagy, new promising approaches for preventing and treating human disorders emerge, including those based on the modulation of Ca(2+)-mediated autophagy.

Conditioned Medium from Adipose-Derived Stem Cells (ADSCs) Promotes Epithelial-to-Mesenchymal-Like Transition (EMT-Like) in Glioma Cells In vitro.

Mesenchymal stem cells (MSCs) have recently been described to home to brain tumors and to integrate into the tumor-associated stroma. Understanding the communication between cancer cells and MSCs has become fundamental to determine whether MSC-tumor interactions should be exploited as a vehicle for therapeutic agents or considered a target for intervention. Therefore, we investigated whether conditioned medium from adipose-derived stem cells (ADSCs-CM) modulate glioma tumor cells by analyzing several cell biology processes in vitro. C6 rat glioma cells were treated with ADSCs-CM, and cell proliferation, cell cycle, cell viability, cell morphology, adhesion, migration, and expression of epithelial-mesenchymal transition (EMT)-related surface markers were analyzed. ADSCs-CM did not alter cell viability, cell cycle, and growth rate of C6 glioma cells but increased their migratory capacity. Moreover, C6 cells treated with ADSC-CM showed reduced adhesion and underwent changes in cell morphology. Up-regulation of EMT-associated markers (vimentin, MMP2, and NRAS) was also observed following treatment with ADSC-CM. Our findings demonstrate that the paracrine factors released by ADSCs are able to modulate glioma cell biology. Therefore, ADSC-tumor cell interactions in a tumor microenvironment must be considered in the design of clinical application of stem cell therapy. Graphical Abstract Factors released by adipose-derived stem cells (ADSCs) may modulate the biology of C6 glioma cells. When C6 cells are exposed to a conditioned medium from adipose-derived stem cells (ADSCs-CM), some of these cells can undergo an EMT-like process and trans-differentiate into cells with a more mesenchymal phenotype, characterized by enhanced expression of EMT-related surface markers, reduced cell adhesion capacity, increased migratory capacity, as well as changes in cell and nuclei morphology.

Mesenchymal stem cells from different murine tissues have differential capacity to metabolize extracellular nucleotides.

Mesenchymal stem cells (MSCs) have shown a great potential for cell-based therapy and many different therapeutic purposes. Despite the recent advances in the knowledge of MSCs biology, their biochemical and molecular properties are still poorly defined. Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) and ecto-5'-nucleotidase (eNT/CD73) are widely expressed enzymes that hydrolyze extracellular nucleotides, generating an important cellular signaling cascade. Currently, studies have evidenced the relationship between the purinergic system and the development, maintenance, and differentiation of stem cells. The objective of this study is to identify the NTPDases and eNT/CD73 and compare the levels of nucleotide hydrolysis on MSCs isolated from different murine tissues (bone marrow, lung, vena cava, kidney, pancreas, spleen, skin, and adipose tissue). MSCs from all tissues investigated expressed the ectoenzymes at different levels. In MSCs from pancreas and adipose tissue, the hydrolysis of triphosphonucleosides was significantly higher when compared to the other cells. The diphosphonucleosides were hydrolyzed at a higher rate by MSC from pancreas when compared to MSC from other tissues. The differential nucleotide hydrolysis activity and enzyme expression in these cells suggests that MSCs play different roles in regulating the purinergic system in these tissues. Overall MSCs are an attractive adult-derived cell population for therapies, however, the fact that ecto-nucleotide metabolism can affect the microenvironment, modulating important events, such as immune response, makes the assessment of this metabolism an important part of the characterization of MSCs to be applied therapeutically.

Ectonucleotidase expression profile and activity in human cervical cancer cell lines.

Cervical cancer is the third most frequent cancer in women worldwide. Adenine nucleotide signaling is modulated by the ectonucleotidases that act in sequence, forming an enzymatic cascade. Considering the relationship between the purinergic signaling and cancer, we studied the E-NTPDases, ecto-5'-nucleotidase, and E-NPPs in human cervical cancer cell lines and keratinocytes. We evaluated the expression profiles of these enzymes using RT-PCR and quantitative real-time PCR analysis. The activities of these enzymes were examined using ATP, ADP, AMP, and p-nitrophenyl-5'-thymidine monophosphate (p-Nph-5'-TMP) as substrate, in a colorimetric assay. The extracellular adenine nucleotide hydrolysis was estimated by HPLC analysis. The hydrolysis of all substrates exhibited a linear pattern and these activities were cation-dependent. An interesting difference in the degradation rate was observed between cervical cancer cell lines SiHa, HeLa, and C33A and normal imortalized keratinocytes, HaCaT cells. The mRNA of ecto-5'-nucleotidase, E-NTPDases 5 and 6 were detectable in all cell lines, and the dominant gene expressed was the Entpd 5 enzyme, in SiHa cell line (HPV16 positive). In accordance with this result, a higher hydrolysis activity for UDP and GDP nucleotides was observed in the supernatant of the SiHa cells. Both normal and cancer cells presented activity and mRNAs of members of the NPP family. Considering that these enzymes exert an important catalytic activity, controlling purinergic nucleotide concentrations in tumors, the presence of ectonucleotidases in cervical cancer cells can be important to regulate the levels of extracellular adenine nucleotides, limiting their effects.

Overexpression of NTPDase2 in gliomas promotes systemic inflammation and pulmonary injury.

Gliomas are the most common and devastating type of primary brain tumor. Many non-neoplastic cells, including immune cells, comprise the tumor microenvironment where they create a milieu that appears to dictate cancer development. ATP and the phosphohydrolytic products ADP and adenosine by activating P2 and P1 receptors may participate in these interactions among malignant and immune cells. Purinergic receptor-mediated cell communication is closely regulated by ectonucleotidases, such as by members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, which hydrolyze extracellular nucleotides. We have shown that gliomas, unlike astrocytes, exhibit low NTPDase activity. Furthermore, ATP induces glioma cell proliferation and the co-administration of apyrase decreases progression of injected cells in vivo. We have previously shown that NTPDase2 reconstitution dramatically increases tumor growth in vivo. Here we evaluated whether NTPDase2 reconstitution to gliomas modulates systemic inflammatory responses. We observed that NTPDase2 overexpression modulated pro-inflammatory cytokine production and platelet reactivity. Additionally, pathological alterations in the lungs were observed in rats bearing these tumors. Our results suggest that disruption of purinergic signaling via ADP accumulation creates an inflammatory state that may promote tumor spread and dictate clinical progression.

Ecto-5'-nucleotidase/CD73 knockdown increases cell migration and mRNA level of collagen I in a hepatic stellate cell line.

Ecto-5'-nucleotidase (eNT/CD73, E.C.3.1.3.5) is a glycosyl phosphatidylinositol (GPI)-linked cell-surface protein with several functions, including the local generation of adenosine from AMP, with the consequent activation of adenosine receptors and the salvaging of extracellular nucleotides. It also apparently functions independently of this activity, e.g., in the mediation of cell-cell adhesion. Liver fibrosis can be considered as a dynamic and integrated cellular response to chronic liver injury and the activation of hepatic stellate cells (HSCs) plays a role in the fibrogenic process. eNT/CD73 and adenosine are reported to play an important role in hepatic fibrosis in murine models. Knockdown of eNT/CD73 leads to an increase in mRNA expression of tissue non-specific alkaline phosphatase (TNALP), another AMP-degrading enzyme and thus no alteration is seen in the total ecto-AMPase activity of the cell. eNT/CD73 knockdown also leads to changes in the expression of collagen I and a clear alteration of cell migration. We suggest that eNT/CD73 protein expression controls cell migration and collagen expression in a mechanism independent of changes in nucleotide metabolism.

The nucleotide hydrolysis is altered in blood serum of streptozotocin-induced diabetic rats.

Ectonucleotidases and the nucleotide metabolism have been implicated as important regulators of various tissue functions in diabetes disease. Here we evaluated the ectonucleotidase activities and the profile of extracellular ATP metabolism in blood serum of streptozotocin (STZ)-induced diabetic rats. We observed a raise in ATP, ADP, AMP, and 5'-TMP hydrolysis in blood serum after 30 days of diabetes induction, when compared with the citrate group. However, in serum of rats treated 6 days with insulin, the hydrolysis returned to the control levels. Extracellular ATP metabolism estimated by HPLC analysis showed a rapid hydrolysis of extracellular ATP by diabetic animals, leading to the formation of high levels of adenosine when compared with citrate and insulin groups. Since in diabetes the vascular disease is frequently present, the alterations observed are important, because these enzymes control the nucleotides/nucleosides ratio in the circulation and thus the events related to haemostasis.