Background: Although autophagy is a pro-survival process of tumor cells, it can stimulate cell death in particular conditions and when differently regulated by specific signals. We previously demonstrated that the selective stimulation of the M2 muscarinic receptor subtype (mAChR) negatively controls cell proliferation and survival and causes oxidative stress and cytotoxic and genotoxic effects in both GBM cell lines and GBM stem cells (GSCs). In this work, we have evaluated whether autophagy was induced as a downstream mechanism of the observed cytotoxic processes induced by M2 mAChR activation by the orthosteric agonist APE or the dualsteric agonist N8-Iper (N8). Methods: To assess the activation of autophagy, we analyzed the expression of LC3B using Western blot analysis and in LC3B-EGFP transfected cell lines. Apoptosis was assessed by measuring the protein expression of Caspases 3 and 9. Results: Our data indicate that activation of M2 mAChR by N8 promotes autophagy in both U251 and GB7 cell lines as suggested by the LC3B-II expression level and analysis of the transfected cells by fluorescence microscopy. Autophagy induction by M2 mAChRs is regulated by the decreased activity of the PI3K/AKT/mTORC1 pathway and upregulated by pAMPK expression. Downstream of autophagy activation, an increase in apoptosis was also observed in both cell lines after treatment with the two M2 agonists. Conclusions: N8 treatment causes autophagy via pAMPK upregulation, followed by apoptosis in both investigated cell lines. In contrast, the absence of autophagy in APE-treated GSC cells seems to indicate that cell death could be triggered by mechanisms alternative to those observed for N8.
背景：尽管自噬是肿瘤细胞的一种促生存过程，但在特定条件下以及受不同信号调控时，它也可能刺激细胞死亡。我们先前的研究表明，选择性刺激M2毒蕈碱受体亚型（mAChR）会负向调控细胞增殖与存活，并在胶质母细胞瘤（GBM）细胞系及GBM干细胞（GSCs）中引发氧化应激、细胞毒性和遗传毒性效应。本研究旨在评估自噬是否作为M2 mAChR激活所诱导细胞毒性过程的下游机制被触发，其中M2 mAChR的激活通过正构激动剂APE或双位点激动剂N8-Iper（N8）实现。 方法：为评估自噬激活情况，我们通过蛋白质印迹分析及LC3B-EGFP转染细胞系检测LC3B的表达水平。通过检测Caspase 3和Caspase 9的蛋白表达来评估细胞凋亡。 结果：数据显示，N8激活M2 mAChR可促进U251和GB7细胞系的自噬，这通过LC3B-II表达水平及荧光显微镜下对转染细胞的分析得以证实。M2 mAChR诱导的自噬受PI3K/AKT/mTORC1通路活性下调的调控，并受pAMPK表达上调的促进。在自噬激活的下游，两种M2激动剂处理后的两种细胞系均观察到细胞凋亡的增加。 结论：N8处理通过上调pAMPK引发自噬，随后在所研究的两种细胞系中诱导细胞凋亡。相比之下，APE处理的GSC细胞中未出现自噬，这可能表明其细胞死亡由不同于N8的机制触发。
肿瘤(癌症)患者之家