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M2 Muscarinic Receptor Activation Impairs Mitotic Progression and Bipolar Mitotic Spindle Formation in Human
Maria Di Bari1, Vanessa Tombolillo1, Francesco Alessandrini1
1Department of Biology and Biotechnologies Charles Darwin, Sapienza University of Rome, 00185 Rome, Italy.
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.
Insights
Arecaidine propargyl ester (APE), an M2 muscarinic acetylcholine receptor agonist, induces aberrant mitosis in glioblastoma cells. This finding supports M2 receptors as a potential therapeutic target for glioblastoma treatment.
Area of Science:
- Neuroscience
- Oncology
- Cell Biology
Background:
- Glioblastoma (GBM) exhibits genetic abnormalities leading to cell cycle deregulation and defective checkpoints.
- Arecaidine propargyl ester (APE), an M2 muscarinic acetylcholine receptor (mAChR) agonist, was previously shown to arrest glioblastoma (GB) cell cycle and reduce survival.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying APE-induced cell cycle arrest in glioblastoma cells.
Main Methods:
- Flow cytometry for cell proliferation analysis.
- Immunocytochemistry and time-lapse microscopy to assess mitosis and mitotic spindles.
- Western blot to evaluate Sirtuin2 and acetylated tubulin expression.
Main Results:
- APE treatment induced M phase arrest, evidenced by increased p-HH3 (ser10) positive cells.
- Significant increases in abnormal mitoses and multipolar mitotic spindle formation were observed post-APE treatment.
- APE treatment modulated SIRT2 and acetylated tubulin expression, impacting mitotic progression.
Conclusions:
- M2 receptor agonism with APE promotes aberrant mitosis in glioblastoma cell lines.
- M2 acetylcholine receptors represent a promising novel therapeutic target for glioblastoma treatment.
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