Activation of M2 muscarinic acetylcholine receptors by orthosteric and dualsteric agonists in glioblastoma cancer stem cells: effects on cell proliferation and drug resistance

The involvement of muscarinic receptors in cancer has been largely documented. While M3 muscarinic receptors results to promote tumor growth and progression, M2 subtype negatively modulates cell growth and survival in different tumor types. Our previous studies demonstrated that M2 orthosteric agonist Arecaidine Propargyl Ester (APE), arrested cell proliferation and induced apoptosis in glioblastoma cell lines and in glioblastoma cancer stem cells (GSC), an undifferentiated GB subpopulation characterized by high chemioresistance. Studies on glioblastoma stable cell lines have demonstrated that low doses of M2 agonist APE were able to counteract the drug resistance for conventional drugs such as doxorubicin (Doxo) and Temozolomide, negatively modulating the drug efflux pump AGCG2. Similarly, in GSCs, the hybrid (orthosteric/allosteric) muscarinic agonist N-8-Iper inhibited cell growth in a dose dependent manner and also impaired cell survival at low doses. The inhibitory effects of the N-8-Iper appeared to be mediated by M2 receptor activation, since they were strongly reduced by co-administration of the selective M2 receptor antagonist methoctramine as well as upon M2 receptor silencing. The dualsteric agonist N8-Iper also decreased the expression of ABC drug efflux pumps (C1 and G2) both as transcript and as protein. Moreover the co-treatment of M2 agonists with low doses of Doxo (6.2 microM) significantly affected cell growth compared with the treatment with Doxo alone. Our data suggest that M2 receptor agonists, decreasing the drug efflux pumps expression, may impair the cancer stem cell chemoresistance, making the tumor cells more responsive to low doses of conventional drugs and reducing the side effects induced by chemotherapy.