M2 muscarinic receptor agonists interfere with the cell proliferation and survival of human glioblastoma cancer stem cells

Glioblastomas are the most aggressive brain tumors in humans. They show a marked cell heterogeneity; in particular glioblastoma stem cells (GCSs), a multipotent cell subpopulation capable of self-renewals, have been identified in these tumors. Considering that these cells show high resistance to chemiotherapy, the identification of drugs able to inhibit GCS proliferation and survival is considered of great clinical relevance. The involvement of muscarinic receptors in cancer has been reported. Recently we have demonstrated that the activation of M2 muscarinic receptors, through the agonist arecaidine, arrests cell proliferation and induces apoptosis in primary and stable glioblastoma cell lines. Following previous data, we have investigated the effect produced by M2 stimulation on GCS proliferation. The expression of M2 receptors was analyzed in several GCS cell lines derived from human biopsies. Considering the M2 receptor levels, we have selected two cell lines (GB7 and GB8). In both cell lines the treatment with the M2 agonist arecaidine decreases cell proliferation; in particular in GB7 cells the decrease of cell proliferation is time and dose dependent. Moreover co-treatment of GB7 cells with M1 and M3 antagonists (pirenzepine and 4-DAMP respectively) has indicated that the decreased cell proliferation–arecaidine induced was probably dependent on M2 activation. FACS analysis has also shown that in GB7 cells, arecaidine inhibits cell cycle progression. Conversely in GB8 cells, arecaidine causes a decreased cell survival. The analysis of EGFR and Notch expression has clearly indicated that at least in GB7 cells the decrease of EGFR expression may be responsible on the decreased cell proliferation induced by arecaidine. Considering the previous data, the use of a more selective agonist for M2 receptor may have a strategic relevance for the glioblastoma therapy. As the difficulty of finding ligands with high selectivity, the exploitation of allosteric sites on G protein-coupled receptors (GPCRs) opens up a new range of possibilities in the research of selective orthosteric ligands in particular for GPCRs as mAChRs, which are characterized by highly conserved orthosteric sites among different receptor subtypes. Actually we are testing the effects produced by a new generation of dualsteric ligands for M2 receptors on glioblastoma cell proliferation both in stable cell lines and in the GCSs.