In previous studies, we found that the orthosteric muscarinic agonist arecaidine propargyl ester (APE) (100 μM) induced a decreased cell proliferation and severe apoptosis in glioblastoma cancer stem cells (GSCs). In this report, we have investigated the effects mediated by hybrid (orthosteric/allosteric) muscarinic agonists P-6-Iper and N-8-Iper on GSCs survival. At variance with APE, the agonist N-8-Iper inhibited cell growth in a dose dependent manner and also impaired cell survival at low doses. The inhibitory effects of the N-8-Iper action appear to be mediated by M2 receptor activation, since they were strongly reduced by co-administration of the selective M2 receptor antagonist methoctramine as well as upon M2 receptor silencing. Moreover, analysis of the expression of phosphorylated histone H2AX (γ-H2AX) indicated that the treatment with N-8-Iper produced a decreased cell survival by induction of DNA damage. The ability of N-8-Iper to produce a cytotoxic effect and apoptosis at low doses indicates that this muscarinic agonist is a suitable probe in a putative therapeutic intervention on glioblastoma through M2 receptor activation.

在以前的研究中，我们发现正构毒蕈碱激动剂槟榔碱炔丙基酯（APE）（100μM）在胶质母细胞瘤癌干细胞（GSC）中诱导了细胞增殖减少和严重凋亡。在本报告中，我们研究了杂种（正构/变构）毒蕈碱激动剂P-6-Iper和N-8-Iper对GSC存活的介导作用。与APE不同，激动剂N-8-Iper以剂量依赖性方式抑制细胞生长，并且在低剂量时也损害细胞存活。N-8-Iper作用的抑制作用似乎是由M2受体激活介导的，因为选择性M2受体拮抗剂甲辛胺的共同给药以及M2受体沉默会大大降低它们的抑制作用。而且，磷酸化组蛋白H2AX（γ-H2AX）表达的分析表明，用N-8-Iper处理会诱导DNA损伤，从而降低细胞存活率。N-8-Iper在低剂量时产生细胞毒性作用和凋亡的能力表明，该毒蕈碱激动剂是通过M2受体激活对胶质母细胞瘤进行假定治疗的合适探针。