Glioblastoma multiforme is the most frequent malignant brain tumor. A key feature of malignant gliomas is their cellular heterogeneity. In particular, the presence of an undifferentiated cell population defined Glioblastoma Stem cells (GSCs) has been reported. GSCs are considered the main cell population responsible for the beginning of neoplastic process and recurrence formation. Several studies report how muscarinic receptors (mAChRs) are involved in the regulation of cell cycle, chemotaxis of brain tumors. For years, our research group has been studying the antiproliferative effects of M2 mAChR activation in different tumors, such as glioblastoma, neuroblastoma and breast cancer [1¿5]. Our previous data have showed that the activation of M2 mAChR by orthosteric agonist Arecaidine Propargyl Ester (APE) caused a significant decrease of cell proliferation and survival in GSCs [2]. In our lab we have also characterized the N-8-Iper (N8), a dualsteric agonist able to activate M2 mAChR with higher affinity than APE. N8 is able to induce a pro-apoptotic and cytotoxic effect already at low doses [3]. These results suggest that N8 may be a promising therapeutic drug for the treatment of glioblastoma. M2 mAChR belongs to the class of G-protein-coupled receptors (GPCRs). After being activated, GPCR signaling is mediated by G-proteins and is followed by their rapid desensitization, i.e. the rapid return to the basal level of GPCR. This process is mainly orchestrated by GPCR kinases and ß-arrestins [6]. In recent years, a new role of ß-arrestins has emerged. These proteins, upon activation of a GPCR, are in turn activated by directing several signaling pathways of particular interest for the study of tumor progression, such as cell proliferation, cell survival and migration [7,8]. Based on this evidence, the aim of this project is to study the signaling pathways downstream of the M2 mAChR activated by APE or N8, focusing on the possible role of ß-arrestins in tumor migration.
For the first time, ACh muscarinic receptors were characterized in human Glioblastoma stem cells. In order to find a new cancer treatment and increase the knowledge on the role of M2 mAChR in brain tumors, it appears relevant to better characterize the effects produced by this receptor in cancer stem cells. GSCs play a key role in tumor formation and progression. These cells show high levels of resistance to chemo- and radio-therapy, as they have very efficient mechanisms of DNA repair and up-regulation of drug efflux pumps. These represent the main obstacle to the pharmacological treatment and lead to a poor prognosis for the patient [23]. Therefore, the identification of new molecules able to counteract GSCs survival represents the greatest challenge in the field of glioblastoma cancer therapy.
M2 agonist APE is capable to arrest in glioblastoma cell lines and GSCs cell cycle progression with a significant decrease of cells in S phase. M2 mAChR activation reduces cell survival, inducing a dramatic apoptosis in both cell lines. N8, a new dualsteric agonist, is able to activate M2 mAChR at lower doses than APE, reducing possible side effects. This makes it a more promising molecule than APE for potential glioblastoma treatment. The invasiveness of GSCs after treatment with the two M2 agonists has not yet been investigated. From recent findings on the role of ß-arrestins in tumor invasiveness and progression [18,19,22], we believe that was relevant to understand whether this protein family is involved in the signaling pathways downstream of the M2 mAChR in glioma cells. The identification of a potential new drugs able to impair the GSC survival, proliferation and migration, may have a strategic relevance in clinical treatment of GBM.
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