Molecular and Cellular Biochemistry

, Volume 319, Issue 1–2, pp 61–68 | Cite as

The role of ecto-5′-nucleotidase/CD73 in glioma cell line proliferation

  • Luci Bavaresco
  • Andressa Bernardi
  • Elizandra Braganhol
  • Angélica Regina Cappellari
  • Liliana Rockenbach
  • Patrícia Fernandes Farias
  • Márcia Rosângela Wink
  • Andrés Delgado-Cañedo
  • Ana Maria Oliveira Battastini
Article

Abstract

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 μM APCP, a competitive ecto-5′-NT/CD73 inhibitor, caused a significant reduction of 30% in glioma cell proliferation. In addition, 100 μM 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.

Keywords

Ecto-5′-nucleotidase/CD73 Glioma cells Cell proliferation 

Notes

Acknowledgment

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

References

  1. 1.
    Schwartzbaum JA, Fisher JL, Aldape KD, Wrensch M (2006) Epidemiology and molecular pathology of gliomas. Nat Clin Pract Neurol 2(9):494–501PubMedCrossRefGoogle Scholar
  2. 2.
    Dai C, Holland EC (2001) Glioma models. Biochem Biophys Acta 1551:M19–M27PubMedGoogle Scholar
  3. 3.
    Holland EC (2001) Gliomagenesis: genetic alterations and mouse models. Nat Rev Genet 2:120–129PubMedCrossRefGoogle Scholar
  4. 4.
    Sathornsumetee S, Reardon D, Desjardins A, Quinn J, Vredenburgh JJ, Rich JN (2007) Moleculary targeted therapy for malignant gliomas. Cancer 110(1):13–24. doi: 10.1002/cncr.22741 PubMedCrossRefGoogle Scholar
  5. 5.
    Sanai N, Alvarez-Buylla A, Berger MS (2005) Neural stem cells and the origin of gliomas. N Engl J Med 353:811–822. doi: 10.1056/NEJMra043666 PubMedCrossRefGoogle Scholar
  6. 6.
    Kleihues P, Louis DN, Scheithauer BW, Rorke LB, Reifenberger G, Burger PC et al (2002) The WHO classification of tumors of the nervous system. J Neuropathol Exp Neurol 61(3):215–225PubMedGoogle Scholar
  7. 7.
    Aaronson SA (1991) Growth factors and cancer. Science 254:1146–1153. doi: 10.1126/science.1659742 PubMedCrossRefGoogle Scholar
  8. 8.
    Brendel M, Pollack IF (1999) The p21-Ras signal transduction pathway and growth regulation in human high-grade gliomas. Brain Res Brain Res Rev 29:232–259. doi: 10.1016/S0165-0173(98)00057-5 CrossRefGoogle Scholar
  9. 9.
    Singh SK, Clarke ID, Hide T, Dirks PB (2004) Cancer stem cells in nervous system tumors. Oncogene 23:7267–7273. doi: 10.1038/sj.onc.1207946 PubMedCrossRefGoogle Scholar
  10. 10.
    Kondo T (2006) Brain cancer stem-like cells. Eur J Cancer 42:1237–1242. doi: 10.1016/j.ejca.2006.01.038 PubMedCrossRefGoogle Scholar
  11. 11.
    Zimmermann H (1992) 5′-nucleotidase: molecular structure and functional aspects. Biochem J 285(2):345–365PubMedGoogle Scholar
  12. 12.
    Heymann D, Reddington M, Kreutzberg GW (1984) Subcellular localization of 5′-nucleotidase in rat brain. J Neurochem 43(4):971–978. doi: 10.1111/j.1471-4159.1984.tb12832.x PubMedCrossRefGoogle Scholar
  13. 13.
    Turnay J, Olmo N, Rissi G, Von der Mark K, Lizarbe MA (1989) 5′-nucleotidase activity in cultured cell lines, effect of different assay conditions and correlation with cell proliferation. In Vitro Cell Dev Biol 25(11):1055–1061. doi: 10.1007/BF02624141 PubMedCrossRefGoogle Scholar
  14. 14.
    Navarro JM, Olmo N, Turnay J, López-Conejo MT, Lizarbe MA (1998) Ecto-5′-nucleotidase from a human colon adenocarcinoma cell line. Correlation between enzyme activity and levels in intact cells. Mol Cell Biochem 187:121–131. doi: 10.1023/A:1006808232059 PubMedCrossRefGoogle Scholar
  15. 15.
    Vogel M, Kowalewski HJ, Zimmermenn H, Janetzko A, Margolis RU, Wollny HE (1991) Association of the HNK-1 epitope with 5′-nucleotidase from Torpedo marmorata. Biochem J 278:199–202PubMedGoogle Scholar
  16. 16.
    Airas L, Hellman J, Salmi M, Bono P, Purune SmithDJ, Jalkanen S (1995) CD73 is involved in lymphocyte binding to the endothelium: characterization of lymphocyte-vascular adhesion protein 2 identifies it as CD73. J Exp Med 182:1603–1608. doi: 10.1084/jem.182.5.1603 PubMedCrossRefGoogle Scholar
  17. 17.
    Sadej R, Spychala J, Skladanowski C (2006) Expression of ecto-5′-nucleotidase (eN, CD73) in cell lines from various stages of human melanoma. Melanoma Res 16:213–222. doi: 10.1097/01.cmr.0000215030.69823.11 PubMedCrossRefGoogle Scholar
  18. 18.
    Wang L, Zhou X, Zhou T, Ma D, Chen S, Zhi X et al (2007) Ecto-5′-nucleotidase promotes invasion, migration and adhesion of human breast cancer cells. J Cancer Res Clin Oncol 134(3):365–372. doi: 10.1007/s00432-007-0292-z PubMedCrossRefGoogle Scholar
  19. 19.
    Ujházy P, Berleth ES, Pietkiewicz JM, Kitano H, Skaar JR, Ehrke MJ et al (1996) Evidence for the involvement of ecto-5′-nucleotidase (CD73) in drug resistance. Int J Cancer 68:493–500. doi:10.1002/(SICI)1097-0215(19961115)68:4<493::AID-IJC15>3.0.CO;2-6PubMedCrossRefGoogle Scholar
  20. 20.
    Spychala J (2000) Tumor-promoting functions of adenosine. Pharmacol Ther 87:161–173. doi: 10.1016/S0163-7258(00)00053-X PubMedCrossRefGoogle Scholar
  21. 21.
    Eroglu A, Cambolat O, Demirci S, Kocaoglu H, Eryavuz Y, Akgul H (1997) Activities of adenosine deaminase and 5′-nucleotidase in cancerous and noncancerous human colorectal tissues. Med Oncol 17:319–324. doi: 10.1007/BF02782198 CrossRefGoogle Scholar
  22. 22.
    Jacobson KA, Hoffmann C, Cattabeni F, Abbracchio MP (1999) Adenosine-induced cell death: evidence for receptor-mediated signalling. Apoptosis 4:197–211. doi: 10.1023/A:1009666707307 PubMedCrossRefGoogle Scholar
  23. 23.
    Resta R, Thompson LF (1997) T cell signalling through CD73. Cell Signal 9:131–139. doi: 10.1016/S0898-6568(96)00132-5 PubMedCrossRefGoogle Scholar
  24. 24.
    Merighi S, Mirandola P, Varani K, Gessi S, Leung E, Baraldi PG et al (2003) A glance at adenosine receptors: novel target for antitumor therapy. Pharmacol Ther 100:31–48. doi: 10.1016/S0163-7258(03)00084-6 PubMedCrossRefGoogle Scholar
  25. 25.
    Morrone FB, Jacques-Silva MC, Horn AP, Bernardi A, Schwartsmann G, Rodnight R et al (2003) Extracellular nucleotides and nucleosides induce proliferation and increase nucleoside transport in human glioma cell lines. J Neurooncol 64:211–218. doi: 10.1023/A:1025699932270 PubMedCrossRefGoogle Scholar
  26. 26.
    Merighi S, Mirandola P, Milani D, Varani K, Gessi S, Klotz KN et al (2002) Adenosine receptors as mediators of both cell proliferation and cell death of cultured human melanoma cells. J Invest Dermatol 119:923–933. doi: 10.1046/j.1523-1747.2002.00111.x PubMedCrossRefGoogle Scholar
  27. 27.
    Wink MR, Lenz G, Braganhol E, Tamajusuku ASK, Schwartsmann G, Sarkis JJF et al (2003) Altered extracellular ATP, ADP and AMP catabolism in glioma cell lines. Cancer Lett 198:211–218. doi: 10.1016/S0304-3835(03)00308-2 PubMedCrossRefGoogle Scholar
  28. 28.
    Chan KM, Delfert D, Junger KD (1986) A direct colorimetric assay for Ca2 + stimulated ATPase activity. Anal Biochem 157:375–380. doi: 10.1016/0003-2697(86)90640-8 PubMedCrossRefGoogle Scholar
  29. 29.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. doi: 10.1016/0003-2697(76)90527-3 PubMedCrossRefGoogle Scholar
  30. 30.
    Fenoglio C, Necchi D, Civallero M, Ceroni M, Nano R (1997) Cytochemical demonstration of nitric oxide synthase and 5′nucleotidase in human glioblastoma. Anticancer Res 17:2507–2511PubMedGoogle Scholar
  31. 31.
    Ludwing HC, Rausch S, Schallock K, Markakis E (1999) Expression of CD73 (ecto-5′-nucleotidase) in 165 glioblastomas by immunohistochemistry and electronmicroscopic histochemistry. Anticancer Res 19(3A):1747–1752Google Scholar
  32. 32.
    Morrone FB, Oliveira DL, Gamermann PW, Stella J, Wofchuk S, Wink MR et al (2006) Involvement of extracellular ATP on the glioblastoma growth in a rat glioma model. BMC Cancer 6:226–236. doi: 10.1186/1471-2407-6-226 PubMedCrossRefGoogle Scholar
  33. 33.
    Zhou P, Zhi X, Zhou T, Chen S, Li X, Wang L et al (2007) Overexpression of Ecto-5′-nucleotidase (CD73) promotes T-47D human breast cancer cells invasion and adhesion to extracellular matrix. Cancer Biol Ther 6(3):426–431PubMedCrossRefGoogle Scholar
  34. 34.
    Sadej R, Spychala J, Skladanowski AC (2006) Expression of ecto-5′-nucleotidase (eN, CD73) in cell lines from various stages of human melanoma. Melanoma Res 16(3):213–222. doi: 10.1097/01.cmr.0000215030.69823.11 PubMedCrossRefGoogle Scholar
  35. 35.
    Zhi X, Chen S, Zhou P, Shao Z, Wang L, Ou Z et al (2007) RNA interference of ecto-5′-nucleotidase (CD73) inhibits human breast cancer cell growth and invasion. Clin Exp Metastasis 24(6):439–448. doi: 10.1007/s10585-007-9081-y PubMedCrossRefGoogle Scholar
  36. 36.
    Ohana G, Bar-Yehuda S, Barer F, Fishman P (2001) Differential effect of adenosine on tumor and normal cell growth: focus on the A3 adenosine receptor. J Cell Physiol 186:19–23. doi:10.1002/1097-4652(200101)186:1<19::AID-JCP1011>3.0.CO;2-3PubMedCrossRefGoogle Scholar
  37. 37.
    Hugo F, Mazurek S, Zander U, Eigenbrodt E (1992) In vitro effect of extracellular AMP on MCF-7 breast cancer cells: inhibition of glycolysis and cell proliferation. J Cell Physiol 153(3):539–549. doi: 10.1002/jcp.1041530315 PubMedCrossRefGoogle Scholar
  38. 38.
    Mazurek S, Michel A, Eigenbrodt E (1997) Effect of extracellular AMP on cell proliferation and metabolism of breast cancer cell lines with high and low glycolytic rates. J Biol Chem 272(8):4941–4952. doi: 10.1074/jbc.272.8.4941 PubMedCrossRefGoogle Scholar
  39. 39.
    Ohkudo S, Nagata K, Nakahata N (2007) Adenosine uptake-dependent C6 cell growth inhibition. Eur J Pharmacol 577(1–3):35–43. doi: 10.1016/j.ejphar.2007.08.025 CrossRefGoogle Scholar
  40. 40.
    Morrone FB, Horn AP, Stella J, Spiller F, Sarkis JJ, Salbego CG et al (2005) Increased resistance of glioma cell lines to extracellular ATP cytotoxicity. J Neurooncol 71(2):135–140. doi: 10.1007/s11060-004-1383-1 PubMedCrossRefGoogle Scholar
  41. 41.
    Braganhol E, Tamajusuku AS, Bernardi A, Wink MR, Battastini AM (2007) Ecto-5′-nucleotidase/CD73 inhibition by quercetin in the human U138MG glioma cell line. Biochim Biophys Acta 1770(9):1352–1359PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2008

Authors and Affiliations

  • Luci Bavaresco
    • 1
  • Andressa Bernardi
    • 1
  • Elizandra Braganhol
    • 1
  • Angélica Regina Cappellari
    • 1
  • Liliana Rockenbach
    • 1
  • Patrícia Fernandes Farias
    • 1
  • Márcia Rosângela Wink
    • 2
  • Andrés Delgado-Cañedo
    • 3
  • Ana Maria Oliveira Battastini
    • 1
  1. 1.Departamento de Bioquímica, Instituto de Ciências Básicas da SaúdeUniversidade Federal do Rio Grande do SulPorto AlegreBrazil
  2. 2.Departamento de Bioquímica, Ciências FisiológicasFundação Faculdade Federal de Ciências Médicas de Porto AlegrePorto AlegreBrazil
  3. 3.Laboratório de Cardiologia Molecular e CelularInstituto de Cardiologia de Porto AlegrePorto AlegreBrazil

Personalised recommendations