The role of Ca2+ on the DADS-induced apoptosis in mouse-rat hybrid retina ganglion cells (N18)

Abstract

Diallyl disulfide (DADS), a component of garlic, has been shown to induce growth inhibition and apoptosis in human cancer cell types. The present studies were designed to investigate the effects of DADS on mouse-rat hybrid retina ganglion cells (N18) to better understand its effect on apoptosis and apoptosis-related genes in vitro. Cell viability, cell cycle analysis, reactive oxygen species (ROS), Ca2+ production, mitochondria membrane potential, apoptosis induction, associated gene expression and caspases-3 activity were examined by flow cytometric assay and/or Western blot. After 24-h treatment with DADS, a dose- and time-dependent decrease in the viability of N18 cells was observed and the approximate IC50 was 27.6 microM. The decreased percentage of viable cells are associated with the production of ROS then followed by the production of Ca2+ which is induced by DADS. DADS induced apoptosis in N18 cells via the activation of caspase-3. DADS increased the protein levels of p53, cytochrome c and phosphated JNK within 24 h of treatment and it decreased the levels of Bcl-2 and those factors may have led to the mitochondria depolarization of N18 cells. DADS induced apoptosis were accompanied by increased levels of Ca2+ and decreased mitochondrial membrane potential which then led to release the cytochrome c, cleavage of pro-caspase-3. Deleted levels of Ca2+ by BAPTA-AM 10 microM (intracellular calcium chelator) then led to decrease DADS-induced apoptosis. Inhibition of caspase-3 activation by inhibitor (z-VAD-fmk) completely blocked DADS-induced apoptosis on N18 cells. The results indicated that oxidative stress modulates cell proliferation and Ca2+ modulates the cell death induced by DADS.