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Glioblastoma multiforme (GBM) is the most malignant brain tumor. Hypoxic condition is a predominant feature of the GBM contributing to tumor growth and resistance to conventional therapies. Hence, the identification of drugs able to impair GBM malignancy and aggressiveness is considered of great clinical relevance. Previously, we demonstrated that the activation of M2 muscarinic receptors, through the agonist arecaidine propargyl ester (Ape), arrests cell proliferation in GBM cancer stem cells (GSCs). In the present work, we have characterized the response of GSCs to hypoxic condition showing an upregulation of hypoxia-inducible factors and factors involved in the regulation of GSCs survival and proliferation. Ape treatment in hypoxic conditions is however able to inhibit cell cycle progression, causing a significant increase of aberrant mitosis with consequent decreased cell survival. Additionally, qRT-PCR analysis suggest that Ape downregulates the expression of stemness markers and miR-210 levels, one of the main regulators of the responses to hypoxic condition in different tumor types. Our data demonstrate that Ape impairs the GSCs proliferation and survival also in hypoxic condition, negatively modulating the adaptive response of GSCs to hypoxia.

In previous studies, we found that the orthosteric muscarinic agonist arecaidine propargyl ester (APE) (100 μM) induced a decreased cell proliferation and severe apoptosis in glioblastoma cancer stem cells (GSCs). In this report, we have investigated the effects mediated by hybrid (orthosteric/allosteric) muscarinic agonists P-6-Iper and N-8-Iper on GSCs survival. At variance with APE, the 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 action appear 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. Moreover, analysis of the expression of phosphorylated histone H2AX (γ-H2AX) indicated that the treatment with N-8-Iper produced a decreased cell survival by induction of DNA damage. The ability of N-8-Iper to produce a cytotoxic effect and apoptosis at low doses indicates that this muscarinic agonist is a suitable probe in a putative therapeutic intervention on glioblastoma through M2 receptor activation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The involvement of muscarinic receptors in cancer has been reported. Recently we have demonstrated that the activation of M2 muscarinic receptors, through arecaidine propargyl ester, arrests cell proliferation and induces apoptosis in primary and established glioblastoma cell lines. Considering the inability of conventional drugs to completely counteract the growth of glioblastoma cancer stem cells (GSCs), we have investigated the effect produced by arecaidine on GSC growth and survival. The expression of M2 receptors has been analyzed in GSC cell lines derived from human biopsies. Based on the M2 receptor expression levels, we have selected two gliolastoma cell lines (GB7 and GB8). In both cell lines the treatment with arecaidine decreased GCS cell growth. GB7 cells exhibited a time- and dose-dependent decrease of cell proliferation. Moreover arecaidine caused a reduced cell survival in particular in GB8 cell line. These effects appear to be mediated by M2 receptor activation as suggested by pharmacological experiments performed in the presence of M1 and M3 preferring antagonists (pirenzepine and 4-DAMP respectively) and M2/M4 antagonist methoctramine. M2 receptor silencing by siRNA has further confirmed that the inhibition of cell growth arecaidine-induced was mediated by the M2 receptor activation. These results suggest that the M2 receptors may represent a new interesting therapeutic tool to counteract glioblastoma cancer stem cell growth and survival., (Copyright © 2015 Elsevier B.V. All rights reserved.)