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.