Combined treatments with the dualsteric agonist N-8-Iper plus chemotherapy drugs affect drug resistance in glioblastoma cancer stem cells

Glioblastoma multiforme (GBM), the most aggressive brain tumor in humans, is characterized by heterogeneous cell populations, including Glioblastoma Stem Cells (GSCs) that share some similarities with Neural Stem Cells. Nowadays, one of the greatest challenges in the treatment of GBM is multidrug resistance (MDR), a complex and multifactorial phenomenon involving a variety of mechanisms that make tumor cells resistant to different therapeutic treatments. In GBM, MDR has been observed especially in GSCs with respect to non-stem-like GBM cells. Our previous studies demonstrated that the selective stimulation of M2 muscarinic receptor subtypes by the orthosteric agonist Arecaidine Propargyl Ester (APE) decreased cell proliferation and survival, causing an arrest of the cell cycle both in GBM cell lines and in GSCs. In addition to the orthosteric binding site, muscarinic receptors also have allosteric binding sites. The different muscarinic subtypes show a high degree of sequence homology in the orthosteric binding site, which makes orthosteric ligands not always selective. They often lead to therapeutic ineffectiveness or numerous side effects due to the high dose required. For this reason, a new approach in drug design has been developed; the new synthetic compounds are called 'dualsteric ligands'. These molecules are hybrid derivates, containing the substructures of both orthosteric and allosteric ligands bridges by a spacer chain.N-8-Iper, is the most effective dualsteric agonist for the M2 receptor tested in our lab. Our study demonstrated that N-8-Iper was able to reduce GSC proliferation in the comparable manner of APE, but N-8-Iper showed a significant ability to reduce cell proliferation also at lower doses. The aim of the present study was to analyze the ability of the M2 dualsteric agonist N-8-Iper to counteract GSCs drug resistance. By using sub-toxic concentrations of N8-Iperand of the single chemotherapy drugs, we found that only M2 agonist low doses combined to doxorubicin or cisplatin or temozolomide, are significantly able to counteract cell growth in different GSC cell lines (GB7 and G166lines). The analysis of ABC efflux pumps expression levels following exposure to high and low doses of N-8-Iper, enlightened the ability of M2 receptors to significantly driving their downregulation. Our data demonstrated the ability of this novel M2 agonist to counteract drug resistance in GSCs at least partially through downregulating ABC drug efflux pumps expression. The combined effects of low doses of conventional chemotherapy drugs and M2 agonists may thus represent a novel promising pharmacological approach to impair the GSCs-drug resistance in GBM therapy.