J Neurochem. 2026 May;170(5):e70454. doi: 10.1111/jnc.70454.
ABSTRACT
Glioblastoma (GB) is the most malignant form of human brain tumor, characterized by heterogeneous cell populations, including undifferentiated cells defined as GB stem cells (GSCs), responsible for initiating the neoplastic process as well as recurrence. Previous studies demonstrated that the activation of M2 muscarinic acetylcholine receptor (M2 mAChR) by the orthosteric agonist arecaidine propargyl ester (APE) and the dualsteric agonist Iper-N-naphthalimide (N-8-Iper) caused a significant decrease in cell proliferation and survival in both GSCs and GB cell lines. Interestingly, N-8-Iper activates M2 mAChR with higher efficacy and at a lower concentration than APE. The work aimed to better investigate the mechanisms downstream of M2 mAChR activation by both agonists responsible for cytotoxic and pro-apoptotic effects in both U251 cell line and G166 cells (GSCs). To this end, we assessed mitochondrial function by using cell-based assays. Our results demonstrate the ability of N-8-Iper, both at the high (100 μM) and low (25 μM) dose, to induce alteration of mitochondrial morphology and activity, affecting both cellular respiration and ROS production in U251 and G166 cells. Instead, APE causes the same alterations but only in the U251 cell line. Given the relevance of lipid metabolism analysis in the study of cancer, lipid droplets (LDs) were evaluated in the presence or absence of the two M2 agonists. LDs accumulation within the cells was detected after N-8-Iper treatment in both cell lines, whereas APE produced similar effects only in the U251 cell line. No cytotoxic effects and mitochondrial alteration were detected on normal human astrocytes. These results clearly suggest that N-8-Iper has a more potent action on GSCs than APE, thus making this dual-acting agonist a promising muscarinic ligand able to better characterize the inhibitory effects of the M2 muscarinic receptor in glioblastoma cells as well as in other tumor types.
PMID:42141821 | DOI:10.1111/jnc.70454