M2 Muscarinic Receptor Activation Impairs Mitotic Progression and Bipolar Mitotic Spindle Formation in Human Glioblastoma Cell Lines

Abstract

Background: Glioblastoma multiforme (GBM) is characterized by several genetic abnormalities, leading to cell cycle deregulation and abnormal mitosis caused by a defective checkpoint. We previously demonstrated that arecaidine propargyl ester (APE), an orthosteric agonist of M2 muscarinic acetylcholine receptors (mAChRs), arrests the cell cycle of glioblastoma (GB) cells, reducing their survival. The aim of this work was to better characterize the molecular mechanisms responsible for this cell cycle arrest. Methods: The arrest of cell proliferation was evaluated by flow cytometry analysis. Using immunocytochemistry and time-lapse analysis, the percentage of abnormal mitosis and aberrant mitotic spindles were assessed in both cell lines. Western blot analysis was used to evaluate the modulation of Sirtuin2 and acetylated tubulin—factors involved in the control of cell cycle progression. Results: APE treatment caused arrest in the M phase, as indicated by the increase in p-HH3 (ser10)-positive cells. By immunocytochemistry, we found a significant increase in abnormal mitoses and multipolar mitotic spindle formation after APE treatment. Time-lapse analysis confirmed that the APE-treated GB cells were unable to correctly complete the mitosis. The modulated expression of SIRT2 and acetylated tubulin in APE-treated cells provides new insights into the mechanisms of altered mitotic progression in both GB cell lines. Conclusions: Our data show that the M2 agonist increases aberrant mitosis in GB cell lines. These results strengthen the idea of considering M2 acetylcholine receptors a novel promising therapeutic target for the glioblastoma treatment.

Analysis of Signal Transduction Pathways Downstream M2 Receptor Activation: Effects on Schwann Cell Migration and Morphology

“…The concentration was established by previous studies demonstrating that 100 µM of APE was able to produce the best effects in terms of inhibition of cell proliferation and upregulation of differentiation factors expression. Moreover, pharmacological binding experiments and M2 knockdown with short interference RNA technique demonstrated that APE selectively binds M2 muscarinic subtype in SCs as well as in other cell models [8][9][10][11][21][22][23][24][25].…”

Editorial to Summarize the Papers Published in the Special Issue “10th Anniversary of Cells—Advances in Cell Cycle”

“…Therefore, most cancer drugs are designed to specifically target mitotic cells. The next two research articles published in this Special Issue focused on the mitotic phase of the cell cycle [ 2 , 3 ].…”

“…The research article by Bari et al, aimed to develop a novel therapeutic agent against Glioblastoma multiforme (GBM) by arresting the cancer cells in mitosis [ 2 ]. GBM is the most malignant and frequent human brain tumor, representing more than 60% of all brain tumors in adults [ 2 ].…”

“…The research article by Bari et al, aimed to develop a novel therapeutic agent against Glioblastoma multiforme (GBM) by arresting the cancer cells in mitosis [ 2 ]. GBM is the most malignant and frequent human brain tumor, representing more than 60% of all brain tumors in adults [ 2 ]. Despite the great advances made in understanding the molecular alterations that occur in Glioblastoma (GB), there is no definitive cure, and mortality is still very high.…”

“…The Italian research team previously led by Dr. Tata showed that the arecaidine propargyl ester (APE), an orthosteric agonist of M2 muscarinic acetylcholine receptors (mAChRs), arrests the cell cycle of glioblastoma (GB) cells and reduces their survival. They showed that M2 agonist treatment causes an arrest in cell cycle progression with an accumulation of cells during the pro-metaphase/metaphase transition, which causes a significant increase in abnormal mitosis and multipolar mitotic spindle formation [ 2 ]. These findings highlight the M2 muscarinic receptor as a new strategic therapeutic target in GBM therapy.…”