TY - JOUR
T1 - Cytotoxic and genotoxic effects mediated by M2 muscarinic receptor activation in human glioblastoma cells
AU - Di Bari, Maria
AU - Tombolillo, Vanessa
AU - Conte, Claudia
AU - Castigli, Emila
AU - Sciaccaluga, Miriam
AU - Iorio, Egidio
AU - Carpinelli, Giulia
AU - Ricordy, Ruggero
AU - Fiore, Mario
AU - Degrassi, Francesca
AU - Tata, Ada Maria
PY - 2015/11/1
Y1 - 2015/11/1
N2 - Glioblastomas are the most common brain tumors in humans. Previously, we demonstrated that the muscarinic receptor agonist, arecaidine propargyl ester, via M2 receptors, inhibits cell proliferation in a time and dose-dependent manner and induces a severe apoptosis in human U251 and U87 glioblastoma cell lines. In order to clarify the mechanisms causing apoptosis after arecaidine treatment, we analyzed the ability of arecaidine to induce oxidative stress. By dichloro-dihydro-fluorescein diacetate (DCFDA) staining, we demonstrated that arecaidine increased the intracellular ROS levels. ROS accumulation was completely counteracted by the ROS scavenger, N-acetyl-l-cysteine (NAC). Apoptotic cell death appeared directly correlated to ROS production since NAC was able to counteract this effect. Although there was an up-regulation of some detoxifying enzyme expression such as superoxide dismutase (MnSOD) and sirtuin-1 (SIRT1), the cytotoxic effect caused by arecaidine treatment caused DNA damage, as demonstrated by the increase of histone γ-H2AX positive cells, and chromosomal aberrations. These effects were mediated by M2 receptor activation; in fact after silencing of M2 receptors by siRNA, the increase of γ-H2AX positive cells was abolished. In conclusion, in addition to a cytostatic effect previously described, in the present study we have better characterized the mechanisms causing the cytotoxic effects and the apoptotic cell death in glioblastoma cells after M2 receptor activation. These data allow to consider this receptor a new interesting therapeutic tool for the glioblastoma treatment.
AB - Glioblastomas are the most common brain tumors in humans. Previously, we demonstrated that the muscarinic receptor agonist, arecaidine propargyl ester, via M2 receptors, inhibits cell proliferation in a time and dose-dependent manner and induces a severe apoptosis in human U251 and U87 glioblastoma cell lines. In order to clarify the mechanisms causing apoptosis after arecaidine treatment, we analyzed the ability of arecaidine to induce oxidative stress. By dichloro-dihydro-fluorescein diacetate (DCFDA) staining, we demonstrated that arecaidine increased the intracellular ROS levels. ROS accumulation was completely counteracted by the ROS scavenger, N-acetyl-l-cysteine (NAC). Apoptotic cell death appeared directly correlated to ROS production since NAC was able to counteract this effect. Although there was an up-regulation of some detoxifying enzyme expression such as superoxide dismutase (MnSOD) and sirtuin-1 (SIRT1), the cytotoxic effect caused by arecaidine treatment caused DNA damage, as demonstrated by the increase of histone γ-H2AX positive cells, and chromosomal aberrations. These effects were mediated by M2 receptor activation; in fact after silencing of M2 receptors by siRNA, the increase of γ-H2AX positive cells was abolished. In conclusion, in addition to a cytostatic effect previously described, in the present study we have better characterized the mechanisms causing the cytotoxic effects and the apoptotic cell death in glioblastoma cells after M2 receptor activation. These data allow to consider this receptor a new interesting therapeutic tool for the glioblastoma treatment.
KW - Acetylcholine
KW - Apoptosis
KW - DNA damage
KW - M2 receptors
KW - ROS
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U2 - 10.1016/j.neuint.2015.09.008
DO - 10.1016/j.neuint.2015.09.008
M3 - Article
C2 - 26455407
AN - SCOPUS:84946550913
VL - 90
SP - 261
EP - 270
JO - Neurochemistry International
JF - Neurochemistry International
SN - 0197-0186
ER -