Alteration of the mitochondrial activity and lipidic metabolism caused by the selective stimulation of M2 muscarinic receptors in human glioblastoma cells

Background and aims: Glioblastoma is the most malignant human brain tumor characterized by heterogeneous cell populations, including undifferentiated cells defined Glioblastoma Stem cells (GSCs), responsible for the beginning of neoplastic process and recurrence formation. Previous studies demonstrated how the activation of M2 muscarinic receptor by orthosteric agonist Arecaidine Propargyl Ester (APE) and dualsteric agonist N-8-Iper caused a significant decrease of cell proliferation and survival both in GSCs and in glioblastoma cell lines. Interestingly N8-Iper is capable to activate M2 receptor with higher affinity and at a lower concentration than APE. The aim of this work was to investigate the mechanisms downstream of M2 receptor activation by both agonists responsible of the cytotoxic and pro-apoptotic effects both in U251 cell line and in GSCs. Methods: mitochondrial functionality was evaluated by MITO ID assay, TMRE staining and oxygen consumption measurement by oxygraph. Lipid homeostasis was analyzed by Oil Red O staining, TLC and WB analysis. Autophagy was analyzed by WB analysis and LC3-GFP construct transfection. Results: our results demonstrate the ability of the M2 agonists to induce oxidative stress, alteration of the mitochondrial morphology and activity with consequent alteration of cellular respiration, lipid homeostasis and lipid droplets formation. M2 agonists also induce autophagy, as demonstrated in U251 cells. Conclusions: these results suggest that the selective activation of M2 receptor in particular by N-8-Iper may be a promising therapeutic strategy for the glioblastoma treatment, reducing the possible side effects that may be caused by the high doses of the orthosteric agonist.