Selective agonists for M2 muscarinic acetylcholine receptors inhibit cell proliferation and survival in human glioblastoma cells: possible effects on drug resistance

Introduction. The involvement of muscarinic acetylcholine receptors (mAChRs) in cancer has been largely documented. Recently, we have demonstrated that activation of M2AChRs arrests cell proliferation and induces apoptosis in glioblastoma (GB) cell lines and in glioblastoma cancer stem cells (GSC). In the present work, we compared the effects mediated by the preferential activation of M2AChRs, through the M2 agonist arecaidine propargyl ester (APE) and the dualsteric agonist N8-Iper, both in GB cell lines and in GSC, analyzing their antiproliferative and pro-apoptotic effects and their potential use in combined therapy with conventional drugs (i.e., temozolomide). Material and Methods. U251 established cell lines and GSC (GB7 cell line) obtained from human biopsy were used as cell models. MTT assay, trypan blue staining and FACS analysis were used to evaluate cell viability and cell death. The M2 silencing (by siRNA) and pharmacological competition were used to confirm the ability of M2 agonists to selectively bind this receptor subtype. Transcript levels for multidrug efflux pumps (e.g., ATP binding cassette, ABC) were analyzed by Real-Time PCR and Western Blot analysis. Results. In GB7 cells, treatment with the N8-Iper decreased cell proliferation in a time and dose dependent manner as previously demonstrated after APE treatments. FACS analysis has also shown that in GB7 cells N8-Iper caused a significant increase of cell death. The pharmacological competition with M1 and M3 antagonists (pirenzepine and 4-DAMP, respectively) and the M2 antagonist methoctramine indicated that the effects of N8-Iper were dependent on selective activation of the M2AChR subtype. The ability of APE and N-8-Iper to activate M2 receptor populations was also confirmed by CHRM2 siRNA transfection. Moreover, APE and N-8-Iper decreased the mRNA levels and the protein expression of the ABC-G2 pump. Finally, the combined treatment with an M2 agonist and temozolomide indicated a synergic effect of the two drugs in decreasing GB cell survival. Conclusions. Our findings suggest that M2AChR agonists represent a new putative therapeutic tool in glioblastoma treatment. Moreover, the ability of M2 agonists to decrease the ABC-G2 drug efflux pumps expression in GSC cells, coupled with their synergic effect in combination with temozolomide, are indicative of their relevant role in reducing the GB chemoresistance.