Malignant gliomas are the most common and devastating primary tumors in the brain and, despite treatment, patients with these tumors have a poor prognosis. The participation of ecto-5'-NT/CD73 per se as a proliferative factor, being involved in the control of cell growth, differentiation, invasion, migration and metastasis processes has been previously proposed. In the present study, we evaluated the activity and functions of ecto-5'-NT/CD73 during the proliferation process of rat C6 and human U138MG glioma cell lines. Increasing confluences and culture times led to an increase in ecto-5'-NT/CD73 activity in both C6 and U138MG glioma cells. RT-PCR analysis and flow cytometry analysis showed a significant increase in ecto-5'-NT/CD73 mRNA and protein levels, respectively, comparing confluent with sub-confluent cultures in human U138MG glioma cells. Ecto-5'-nucleotidase/CD73 may regulate the extracellular adenosine 5'-monophosphate (AMP) and adenosine levels. Treatment with 1 microM APCP, a competitive ecto-5'-NT/CD73 inhibitor, caused a significant reduction of 30% in glioma cell proliferation. In addition, 100 microM adenosine increases cell proliferation by 36%, and the treatment with adenosine plus NBTI and dipyridamole, produced an additional and significant increase of on cell proliferation. The inhibitory effect on cell proliferation caused by APCP was reverted by co-treatment with NBTI and dipyridamole. AMP (1 mM and 3 mM) decreased U138MG glioma cell proliferation by 29% and 42%, respectively. Taken together, these results suggest the participation of ecto-5'-NT/CD73 in cell proliferation and that this process is dependent upon the enzyme's production of adenosine, a proliferative factor, and removal of AMP, a toxic molecule for gliomas.
Medulloblastoma (MB) is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5′NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy) and inosine accumulation (ONS76). On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5′NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5′NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5′NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.
Nucleotidases participate in the regulation of physiological and pathological events, such as inflammation and coagulation. Exercise promotes distinct adaptations, and can influence purinergic signaling. In the present study, we investigated soluble nucleotidase activities in the blood serum of sedentary young male adults at pre- and post-acute moderate aerobic exercise. In addition, we evaluated how this kind of exercise could influence adenine nucleotide concentrations in the blood serum. Sedentary individuals were submitted to moderate aerobic exercise on a treadmill; blood samples were collected pre- and post-exercise, and serum was separated for analysis. Results showed increases in ATP, ADP, and AMP hydrolysis post-exercise, compared to pre-exercise values. The ecto-nucleotide pyrophosphatase/phosphodiesterase was also evaluated, showing an increased activity post-exercise, compared to pre-exercise. Purine levels were analyzed by HPLC in the blood serum, pre- and post-exercise. Decreased levels of ATP and ADP were found post-exercise, in contrast with pre-exercise values. Conversely, post-exercise levels of adenosine and inosine increased compared to pre-exercise levels. Our results indicate an influence of acute exercise on ATP metabolism, modifying enzymatic behavior to promote a protective biological environment.
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