Selective agonists for M2 muscarinic receptors inhibit cell proliferation and survival in human glioblastoma cells: Possible implications in drug resistance

Introduction. The involvement of muscarinic receptors in cancer has been largely documented. Recently, we have demonstrated that the activation of M2 muscarinic receptors, by the agonist Arecaidine Propargyl Ester (APE), arrests cell proliferation and induces apoptosis in glioblastoma (GB) cell lines. In the present work, we investigated the effects mediated by M2 receptors in glioblastoma cancer stem cells (GSC), an undifferentiated GB subpopulation characterized by high chemioresistance. Material and Methods. GB7 and GB8 cell lines obtained from human biopsies were cultured in Euromed-N supplemented with N2, B27, EGF and FGF. MTT assay and trypan blue staining were used to evaluate cell viability and cell death, respectively. By means of M2 silencing (by siRNA) and pharmacological competition we confirmed the ability of M2 agonists to selectively bind this receptor subtype. Transcript levels for muscarinic receptors and multidrug efflux pumps (e.g. ATP binding cassette, ABC) were analyzed by RT-PCR analysis. Results. Our experiments were performed with the M2 agonist APE, the muscarinic orthosteric superagonist Iperoxo and its related dualsteric agonists P-6-Iper and N-8- Iper. In GB7, treatment with the M2 agonist APE (100 μM) decreased cell proliferation in a time and dose dependent manner. In GB8 APE induced cell death. Cell proliferation and survival of U251 and U87 cell lines and GSC cells were unaffected by treatment with Iperoxo and P-6-Iper. Conversely, N-8-Iper decreased cell proliferation in a time and dose dependent manner. Also in GB7 cells, N-8-Iper inhibited cell growth and survival also at lower concentration (12.5 μM). The co-treatment of GB cells with M2 agonists (APE or N-8-iper) and different muscarinic antagonists confirmed that the decreased agonist-induced cell proliferation and survival were dependent on selective activation of M2 receptor. Similarly, the silencing of M2 receptor abolished the M2- mediated agonists effects. Moreover, APE and N-8-Iper decreased, particularly in GB7 cells, the mRNA levels for the ABC drug efflux pumps (C1 and G2). Conclusions. Our data suggest that M2 receptor agonists represent a new relevant tools to investigate glioblastoma-related mechanisms. Furthermore, the ability of M2 agonists to decrease the drug efflux pumps expression, in particular in GSC cells, suggests that they may have a role in reducing the GSC chemoresistance, and make them more responsive to conventional drugs (e.g. temozolomide).