M2 receptor activation inhibits cell cycle progression and survival in human glioblastoma cells.
Bottom Line: Cell growth analysis has demonstrated that the M2 agonist arecaidine strongly decreased cell proliferation in both glioma cell lines and primary cultures.This effect was dose and time dependent.Chemosensitivity assays have, moreover, shown arecaidine and temozolomide similar effects on glioma cell lines, although IC50 value for arecaidine was significantly lower than temozolomide.
Affiliation: Department of Biology and Biotechnologies Charles Darwin, Research Centre of Neurobiology Daniel Bovet, La Sapienza, University of Rome, P.le Aldo Moro, 5-00185 Roma, Italy.Show MeSH
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Mentions: To exclude the ability of arecaidine to activate also other muscarinic receptor subtypes we have cultured U251MG and U87MG lines in 24 well trays for 24 and 48 hrs, in presence of either the M1 antagonist pirenzepine (final concentration 10−6 M) or M3 antagonist 4-DAMP (final concentration 10−8 M) alone or in addition to arecaidine (final concentration 100 μM). The MTT assay was used to evaluate the cell number in the different experimental conditions. As reported in Figure 12 the treatment with M1 and M3 antagonists did not affect cell growth in both glioma cell lines, as indicated by a comparable cell number in control conditions and in antagonists treated cells, at 24 and 48 hrs of treatment. Arecaidine, as previously reported 28 inhibits cell growth in both glioma cell lines and this effect was not counteracted by the presence of M1 and M3 antagonists. Finally, to confirm that the effect mediated by arecaidine was completely dependent on M2 receptor activation and to exclude a cytotoxic effect of the molecule, we have treated glioma cell lines with arecaidine after M2 receptor silencing using a mix of four siRNAs targeting human M2 receptor. Seventy per cent efficiency was obtained in siRNA transfection experiments, as indicated by the inhibition of GAPDH expression, following transfection of Chromo-siRNA GAPDH (20 nM/well) used as positive control (Fig. 13A and B). The use of a mix of four siRNAs (40 nM each/well) specific for CHRM2 reduced by 30–40% M2 expression in U251 cells, as indicated by Western blot analysis performed 72 hrs after transfection (Fig. 13C). Although the transfection caused a decrease in cell number possibly due to lipofectamine toxicity, 24 hrs of treatment with arecaidine after M2 silencing showed the inability of M2 agonist to inhibit cell proliferation in both glioma cell lines (Fig. 13D).
Affiliation: Department of Biology and Biotechnologies Charles Darwin, Research Centre of Neurobiology Daniel Bovet, La Sapienza, University of Rome, P.le Aldo Moro, 5-00185 Roma, Italy.