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Purinergic Signalling

, Volume 8, Issue 1, pp 39–48 | Cite as

Extracellular ATP reduces tumor sphere growth and cancer stem cell population in glioblastoma cells

  • Pítia Flores Ledur
  • Emilly Schlee Villodre
  • Romela Paulus
  • Lavinia Almeida Cruz
  • Débora Gazzana Flores
  • Guido Lenz
Original Article

Abstract

Glioblastoma is the most aggressive tumor in the CNS and is characterized by having a cancer stem cell (CSC) subpopulation essential for tumor survival. The purinergic system plays an important role in glioma growth, since adenosine triphosphate (ATP) can induce proliferation of glioma cells, and alteration in extracellular ATP degradation by the use of exogenous nucleotidases dramatically alters the size of gliomas in rats. The aim of this work was to characterize the effect of the purinergic system on glioma CSCs. Human U87 glioma cultures presented tumor spheres that express the markers of glioma cancer stem cells CD133, Oct-4, and Nanog. Messenger RNA of several purinergic receptors were differently expressed in spheres when compared to a cell monolayer not containing spheres. Treatment of human gliomas U87 or U343 as well as rat C6 gliomas with 100 μM of ATP reduced the number of tumor spheres when grown in neural stem cell medium supplemented with epidermal growth factor and basic fibroblast growth factor. Moreover, ATP caused a decline in the number of spheres observed in culture in a dose-dependent manner. ATP also reduces the expression of Nanog, as determined by flow cytometry, as well as CD133 and Oct-4, as analyzed by flow cytometry and RT-PCR in U87 cells. The differential expression of purinergic receptor in tumor spheres when compared to adherent cells and the effect of ATP in reducing tumor spheres suggest that the purinergic system affects CSC biology and that ATP may be a potential agonist for differentiation therapy.

Keywords

Glioma Cancer stem cells CD133 Purinergic system ATP 

Abbreviations

CSC

Cancer stem cell

Oct-4

Octamer-4

GLAST

Glutamate–aspartate transporter

CAMKII

Calcium-calmodulin-dependent kinase II

NMDA

N-methyl-d-aspartate

FGF

Fibroblast growth factor

EGF

Epidermal growth factor

Notes

Acknowledgments

The present research was supported by grants from Brazilian funding agencies FAPERGS (Procoredes III 06/1376.0 and Pronex 10/0044-3) and CNPq (420036/2005-9). PL, ESV, DGF. and GL are recipient of CNPq fellowships.

Conflict of interest

We declare that we have no conflict of interest.

Supplementary material

11302_2011_9252_MOESM1_ESM.pdf (981 kb)
Supplementary Figure 1 Presence of CD133 marker in spheres versus monolayer and in control versus ATP treatments. Spheres and monolayer cells were stained with anti-CD133 antibody and analyzed by flow cytometry (a). CD133 analysis by flow cytometry of cells grown for 7 days in the presence or absence of ATP in soft agar plates (spheres) or adherent plates in the same conditions as used in the sphere formation assay with FBS medium (representative of three independent experiments) (b, c) (PDF 980kb)
11302_2011_9252_MOESM2_ESM.pdf (2.2 mb)
Supplementary Figure 2 Titration of ATP effect on sphere formation. Proportion of spheres formed in control and in ATP treatment at concentrations of 1, 10, and 100 μM, in U87 and U343 cells grown on NSC medium (a). Bars represent SEM (n = 4). t test values: *p < 0.05 and **p < 0.001. Representative figures of U87 cells treated with the indicated concentrations of ATP for 7 days (b). Bar size = 100 μm (PDF 2242kb)
11302_2011_9252_MOESM3_ESM.doc (48 kb)
Supplementary Table 1 Primers used in RT-PCR and annealing temperature (T) (DOC 48 kb)
11302_2011_9252_MOESM4_ESM.doc (38 kb)
Supplementary Table 2 mRNA expression of purinergic receptors in different gliomas (DOC 38 kb)

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Pítia Flores Ledur
    • 1
  • Emilly Schlee Villodre
    • 1
  • Romela Paulus
    • 1
  • Lavinia Almeida Cruz
    • 1
  • Débora Gazzana Flores
    • 2
  • Guido Lenz
    • 1
    • 3
  1. 1.Biophysics Department, IBFederal University of Rio Grande do Sul (UFRGS)Porto AlegreBrazil
  2. 2.Cancer Research LaboratoryFederal University of Rio Grande do Sul (UFRGS)Porto AlegreBrazil
  3. 3.Center of BiotechnologyFederal University of Rio Grande do Sul (UFRGS)Porto AlegreBrazil

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