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Methotrexate up-regulates ecto-5′-nucleotidase/CD73 and reduces the frequency of T lymphocytes in the glioblastoma microenvironment

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Abstract

Glioblastoma multiforme (GBM) is a deadly cancer characterized by a pro-tumoral immune response. T-regulatory (Treg) lymphocytes suppress effector immune cells through cytokine secretion and the adenosinergic system. Ecto-5′-nucleotidase/CD73 plays a crucial role in Treg-mediated immunosuppression in the GBM microenvironment (GME). Methotrexate (MTX) is an immunosuppressive drug that can increase the extracellular concentration of adenosine. In this manuscript, C6 GBM cells were treated with 1.0 μM MTX, and ecto-5′-nucleotidase/CD73 expression and extracellular AMP metabolism were analyzed in vitro. For in vivo studies, rats with implanted GBM were treated for 10 days with MTX-loaded lipid-core nanocapsules (MTX-LNCs, 1 mg/kg/day). The activity of ectonucleotidase and the expression of NTPDase1/CD39 and ecto-5′-nucleotidase/CD73 were measured. The frequencies of T lymphocytes (CD3+CD4+, CD3+CD8+, and CD4+CD25highCD39+) were quantified. In vitro, treatment with MTX increased CD73 expression and activity in C6 cells, which is in agreement with higher levels of extracellular adenosine. In vivo, MTX-LNC treatment increased CD39 expression on CD3+CD8+ lymphocytes. In addition, MTX-LNC treatment up-regulated CD73 expression in tissue isolated from GBM, a finding that is in agreement with the higher activity of this enzyme. More specifically, the treatment increased CD73 expression on CD3+CD4+ and CD3+CD8+ lymphocytes. Treatment with MTX-LNCs decreased the frequencies of T-cytotoxic, T-helper, and Treg lymphocytes in the GME. Although more studies are necessary to better understand the complex cross-talk mediated by supra-physiological concentrations of adenosine in the GME, these studies demonstrate that MTX treatment increases CD73 enzyme expression and AMP hydrolysis, leading to an increase in adenosine production and immunosuppressive capability.

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Acknowledgments

This study was supported by the following Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Proc. 472577/2013-1, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS).

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Authors and Affiliations

  1. Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

    Fabrício Figueiró, Letícia S. Bergamin, Liliana Rockenbach, Franciane B. Mendes, Letícia F. Pettenuzzo & Ana Maria O. Battastini

  2. Departamento de Química Orgânica, Instituto de Química, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

    Catiúscia P. de Oliveira & Adriana R. Pohlmann

  3. Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Av. Ramiro Barcelos, 2600-Anexo, CEP 90035-003, Porto Alegre, RS, Brazil

    Elisa Helena F. Jandrey & Ana Maria O. Battastini

  4. Programa de Pós-Graduação em Medicina: Ciências Médicas, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

    Cesar Eduardo J. Moritz

  5. Centre de Recherche du CHU de Québec, Université Laval, Québec, QC, Canada

    Jean Sévigny

  6. Département de microbiologie-infectiologie et d’immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada

    Jean Sévigny

  7. Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

    Silvia S. Guterres & Adriana R. Pohlmann

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  1. Fabrício Figueiró
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  2. Catiúscia P. de Oliveira
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  3. Letícia S. Bergamin
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  6. Elisa Helena F. Jandrey
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  7. Cesar Eduardo J. Moritz
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  8. Letícia F. Pettenuzzo
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  9. Jean Sévigny
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  10. Silvia S. Guterres
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  11. Adriana R. Pohlmann
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  12. Ana Maria O. Battastini
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Corresponding author

Correspondence to Ana Maria O. Battastini.

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All of the procedures used in the present study followed the “Principles of Laboratory Animal Care” from the National Institutes of Health (NIH) and were approved by the Ethical Committee of the Universidade Federal do Rio Grande do Sul (Protocol # 26389).

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Figueiró, F., de Oliveira, C.P., Bergamin, L.S. et al. Methotrexate up-regulates ecto-5′-nucleotidase/CD73 and reduces the frequency of T lymphocytes in the glioblastoma microenvironment. Purinergic Signalling 12, 303–312 (2016). https://doi.org/10.1007/s11302-016-9505-8

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