背景:尽管自噬是肿瘤细胞的促存活过程,它可以在特定条件下刺激细胞死亡,并且当受到特定信号的不同调节时。我们先前证明,M2毒蕈碱受体亚型(mAChR)的选择性刺激对细胞增殖和存活产生负面影响,并在GBM细胞系和GBM干细胞(GSC)中引起氧化应激和细胞毒性和遗传毒性作用。在这项工作中,我们已经评估了自噬是否被诱导为观察到的细胞毒性过程的下游机制,所述细胞毒性过程由正构激动剂APE或双构激动剂N8-Iper(N8)的M2mAChR激活诱导.
方法:为了评估自噬的激活,我们使用Western印迹分析和LC3B-EGFP转染的细胞系分析了LC3B的表达。通过测量胱天蛋白酶3和9的蛋白表达来评估细胞凋亡。
结果:我们的数据表明,N8对M2mAChR的激活促进了U251和GB7细胞系中的自噬,如LC3B-II表达水平和通过荧光显微镜分析转染细胞所表明的。M2mAChRs的自噬诱导受PI3K/AKT/mTORC1途径活性降低调节,并受pAMPK表达上调。自噬激活的下游,在用两种M2激动剂处理后,在两种细胞系中也观察到细胞凋亡的增加。
结论:N8治疗通过pAMPK上调引起自噬,随后在两个研究细胞系中进行细胞凋亡。相比之下,APE处理的GSC细胞中不存在自噬,这似乎表明细胞死亡可能是由N8观察到的替代机制触发的.
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.