ABSTRACT
Title
Effect of the stimulation of ATP P2 receptors on the proliferation of human brain cancer stem cells
Authors
A. Lannutti1,2*, I. D’Alimonte1,2*, E. Nargi1,2*, E. Torelli1, N. Petragnani1, L. Ricci-Vitiani3, R. De Maria3, F. Caciagli1, R. Ciccarelli1,2.
1Dept. of Biomedical Sciences and 2Stem TeCh group, University of Chieti; 3Dept. of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità (ISS), Rome. Italy
*Equally contributed to the study
1Dept. of Biomedical Sciences and 2Stem TeCh group, University of Chieti; 3Dept. of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità (ISS), Rome. Italy
*Equally contributed to the study
Abstract
Malignant brain tumors, like other types of cancer, may arise from the transformation of a restrict population of stem cells, named brain cancer stem cells. Since they show an abnormal degree of self-renewal and high resistance to chemotherapy, there is urgent need to explore the cytotoxic potential of new compounds able to impair the survival of these cells. Purine and pyrimidine analogues, used as anti-leukemic agents and experimental cytotoxic compounds to treat solid tumors, are known to act as antimetabolites able to interfere with a large number of intracellular targets to induce cytotoxicity (1). More recent in vitro and in vivo studies on different cancer types, including brain tumors, are now demonstrating the antitumor activity of purines as linked to the stimulation of specific purinoceptors (2). Based on this evidence, we started to investigate the potential antitumoral effects of some purine compounds (natural and synthetic ligands) acting at extracellular levels as signaling molecules in stem cells named glioblastoma-initiating cells (GICs), deriving from different bioptic samples (provided by ISS) of human glioblastoma multiforme, grade IV, the most aggressive glial tumor. At first, we evaluated the expression of known purinoceptors belonging to the families of adenosine P1 (A1, A2A, A2B, A3) and ATP P2 receptors (either metabotropic P2Y or ionotropic P2X subtypes) in cultured GICs. The most part of purinoceptors were present on these cells, with a major expression of the A3 subtype for adenosine and P2Y1, P2Y2, P2Y4, P2Y13 and P2X7 subtypes for ATP. Then, we observed the effects caused by the stimulation of some of the above mentioned receptors on GIC growth, performed 24 h after cell seeding on Matrigel. The exposure of undifferentiated GICs to ATP, the natural ligand of all P2 receptors, for two consecutive days caused a dose-dependent increase (10-100 µM) or decrease (250-1000 µM) of cell survival measured three days after the beginning of the pharmacological treatment. Cell proliferation was significantly diminished also by GIC exposure for two days to 500-1000 µM UTP, that more selectively bind P2Y2/4 receptors, or for only one day to 500-1000 µM benzoyl-ATP (Bz-ATP), agonist of P2X7 receptors. Interestingly, when cell proliferation was evaluated again four days after the end of the pharmacological treatment with purine compounds, a renewal of cell duplication was found only following ATP addition in the range from 100 to 500 µM. On the contrary, cell exposure for two consecutive days to ADPβS (10-200 µM), a rather selective agonist for P2Y1/11/13 receptors, dose-dependently stimulated GIC proliferation and this effect was evident even four days after the pharmacological treatment suspension. The effect of ATP, UTP and ADPβS was counteracted by the non selective P2 receptor antagonist, suramin; that induced by Bz-ATP was antagonized by the specific P2X7 receptor antagonist, oxidized-ATP. We are currently evaluating whether the same purinergic agonists are also able to affect GIC differentiation towards an astrocytic phenotype, the molecular mechanisms underlying these effects and the eventual synergistic action between purinergic ligands (able to inhibit proliferation and/or to favor differentiation) with cytotoxic antitumoral agents currently used for brain cancer chemotherapy. At present, our results indicate that the stimulation of some ATP P2 receptors may exert an important antitumoral effect on cells originating malignant brain tumors such as GICs, thus revealing further aspects of the activity of purinoceptor ligands and hopefully contributing to open a new exciting avenue of investigation.
1. Galmarini CM et al. (2008) Curr. Med. Chem.15: 1072-82.
2. White N. & Burnstock G. (2006) TiPS 27: 211-17.