Potentiation of temozolomide antitumor effect by purine receptor ligands able to restrain the in vitro growth of human glioblastoma stem cells

Glioblastoma multiforme (GBM), the most common and aggressive brain tumor in humans, comprises a population of stem-like cells (GSCs) that are currently investigated as potential target for GBM therapy. Here, we used GSCs isolated from three different GBM surgical specimens to examine the antitumor activity of purines. Cultured GSCs expressed either metabotropic adenosine P1 and ATP P2Y receptors or ionotropic P2X₇ receptors. GSC exposure for 48 h to 10–150 μM ATP, P2R ligand, or to ADPβS or MRS2365, P2Y₁R agonists, enhanced cell expansion. This effect was counteracted by the PY₁R antagonist MRS2500. In contrast, 48-h treatment with higher doses of ATP or UTP, which binds to P2Y_2/4R, or 2′(3′)-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), P2X₇R agonist, decreased GSC proliferation. Such a reduction was due to apoptotic or necrotic cell death but mostly to growth arrest. Accordingly, cell regrowth and secondary neurosphere formation were observed 2 weeks after the end of treatment. Suramin, nonselective P2R antagonist, MRS1220 or AZ11645373, selective A₃R or P2X₇R antagonists, respectively, counteracted ATP antiproliferative effects. AZ11645373 also abolished the inhibitory effect of Bz-ATP low doses on GSC growth. These findings provide important clues on the anticancer potential of ligands for A₃R, P2Y₁R, and P2X₇R, which are involved in the GSC growth control. Interestingly, ATP and BzATP potentiated the cytotoxicity of temozolomide (TMZ), currently used for GBM therapy, enabling it to cause a greater and long-lasting inhibitory effect on GSC duplication when readded to cells previously treated with purine nucleotides plus TMZ. These are the first findings identifying purine nucleotides as able to enhance TMZ antitumor efficacy and might have an immediate translational impact.