Dottorato in BIOLOGIA CELLULARE E DELLO SVILUPPO

Titolo della tesi: Effects mediated by new dualsteric agonist for M2 muscarinic receptors in glioblastoma cancer stem cells

Glioblastoma (GBM) is the most malignant human brain tumor characterized by heterogeneous cell populations, including undifferentiated cells defined GBM Stem cells (GSCs), responsible for the beginning of neoplastic process and recurrence formation. Previous studies demonstrated how the activation of M2 muscarinic receptor (M2 mAChR) 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 GBM cell lines. Interestingly N-8-Iper is capable to activate M2 mAChR with higher affinity and at a lower concentration than APE. The aim of this first part of my PhD project was to better investigate the mechanisms downstream of M2 mAChR activation by both agonists responsible of the cytotoxic and pro-apoptotic effects both in U251 cell line and in GSCs (G166 cells). To this end, we assessed mitochondrial function, by cell-based assays and by measurement of oxygen consumption. Our results demonstrate the ability of N-8-Iper, at the high dose (100 μM) and the first effective dose (25 μM), to induce oxidative stress, alteration of mitochondrial morphology and activity, resulting in altered cellular respiration in both the U251 cell line and G166 cells. APE causes the same alterations but only in U251 cell line. Given the importance of lipid metabolism analysis in the study of cancer, lipid droplets, cellular neutral lipid content and lipid metabolic enzymes expression were evaluated in the presence or absence of M2 agonists; the data reported demonstrated lipid accumulation within the cells after treatment with M2 agonists mediated by N-8-Iper in both cell lines. APE produces similar effects but only in U251 cell line. In recent years, the role of autophagy in promoting cell death has emerged. In this part of the project, I have evaluated whether, downstream the cytotoxic processes observed following M2 mAChR activation, the autophagy process was induced. The data obtained suggest that activation of M2 mAChR by N-8-Iper activates autophagy in U251 cell line and GB7 cells (GSCs) as suggested by the modulation of LC3B-II expression analyzed by western blot analysis and transfection of LC3B-EGFP construct. The autophagy induction by M2 mAChRs is dependent on its ability to downregulate the PI3K/AKT/mTORC1 pathway and upregulate pAMPK expression, pathways classically involved in the regulation of autophagy process. In contrast, APE seems to have the same effect only on the U251 stable cell line, but not in GB7 cells. Following autophagy, the expression of caspases 9 and 3, which are involved in the apoptotic process, was assessed. After APE treatment, upregulation of pro-apoptotic proteins was observed only in U251 cells, while N-8-Iper is able to activate apoptosis in both cell lines, demonstrating the greater efficacy of the dualsteric agonist over the orthosteric one. Finally, the demonstration that M2 agonists produced no toxic effect on normal human astrocytes confirms that the severe effects produced by orthosteric and dualosteric agonists are specific only to tumor cells.