M2 acetylcholine receptor activation shows anti-proliferative and pro-apoptotic effects in human glioblastoma cells

Introduction. Muscarinic receptors are expressed in several primary and metastatic tumors and appear involved in their growth and propagation. In the present work we have studied the effects of M2 receptor activation on cell growth and survival of human glioblastoma cells. Materials and Methods. Glioma cell lines and primary cultures from biopsies were used for our analysis. FACS analysis was used to evaluate cell proliferation and apoptosis. Real time PCR, immunocytochemistry, western blot and Elisa were also used to analyze the modulated expression of genes and proteins involved in cell cycle arrest and apoptosis. Results. We demonstrated that the activation of M2 receptor by selective agonist arecaidine caused the arrest of cell cycle progression in two glioblastoma cell lines (U251MG and U87MG), with cell accumulation in G1 phase for U87 and in G2/M for U251 cells. Moreover FACS analysis and Elisa for cytoplasmic nucleosomes have demonstrated that arecaidine induced apoptosis in glioblastoma cells. The same effects were also observed in primary cell cultures. Apoptotic cell death was counteracted by the treatment with N-acetyl-L-cysteine, a scavenger of ROS, suggesting that arecaidine induced oxidative stress in glioblastoma cells. In particular in U251 cells, the oxidative stress caused DNA damage, cell cycle arrest in M phase, disregulation of mitotic spindle assembly and misalignment of chromosomes. Conclusion. Our data demonstrate that M2 receptor activation, counteracting glioblastoma cell proliferation and survival may represent a new interesting therapeutic target in glioblastoma therapy. This project was supported by Cofin-Prin 2007 funds