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

Maria Di Bari,Mario Fiore,Vanessa Tombolillo,Ada Maria Tata,Claudia Guerriero,Giulia Guarguaglini,Francesco Alessandrini,Miriam Sciaccaluga,Francesca Degrassi,Emilia Castigli,Italia Anna Asteriti

doi:10.3390/cells10071727

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.

Highlights

Glioblastoma multiforme (GBM) is the most malignant and frequent human brain tumor, representing more than 60% of all brain tumors in adults [1]
Given the ability of arecaidine propargyl ester (APE) to increase ROS levels and to cause cytotoxic damage, including chromosomal aberrations [17], in the present work, we investigated in depth the cell cycle progression upon M2 receptor activation in these two cell lines
Our previous data indicated that APE induces G2/M arrest in the U251 glioblastoma cell line [16]

Summary

Introduction

Glioblastoma multiforme (GBM) is the most malignant and frequent human brain tumor, representing more than 60% of all brain tumors in adults [1]. Glioblastoma multiforme (GBM) is characterized by several genetic abnormalities, leading to cell cycle deregulation and abnormal mitosis caused by a defective checkpoint. Using immunocytochemistry and time-lapse analysis, the percentage of abnormal mitosis and aberrant mitotic spindles were assessed in both cell lines. 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

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