Protective Effect of Cilnidipine on Oxidative Stress-Injured Neuronally-Differentiated PC12 Cells

Abstract

Background & Objectives: The effects of cilnidipine, a calcium channel blocker, on the viability of neuronal cells and cell signals, including phosphatidylinositol 3-kinase (PI3K)/Akt, glycogen synthase kinase-3 (GSK-3), cytochrome c, caspase-3, and poly(ADP-ribose) polymerase (PARP), were investigated in PC12 cells neuronally-differentiated by nerve growth factor

Method: To evaluate the toxicity of cilnidipine itself, nPC12 cells were treated with several concentrations of cilnidipine, and 3,(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and trypan blue stain were performed. Free radical level, membrane lipid peroxidation, intracellular signaling proteins were evaluated.

Results: The viability was not affected by low concentration of cilnidipine, up to 20 μM, but, it was decreased at higher than this concentration. The levels of free radicals and membrane lipid peroxidation were significantly increased in nPC12 cells when treated with more than 50 μM cilnidipine, and treatment of PC12 cells with 100 μM cilnidipine killed the cells by inhibiting PI3K/Akt and by promoting activation of GSK-3 and caspase-3, release of cytochrome c, and cleavage of PARP. To evaluate the protective effects of low concentration of cilnidipine on oxidative stress-injured nPC12 cells, the viability of the cells (pretreated with cilnidipine for 2 hours vs. not pretreated) was evaluated 24 hours after exposure to 100 μM H2O2 for 30 min; Compared to the cells treated with only 100 μM H2O2, pretreatment of the cells with 20 μM cilnidipine before exposure to 100 μM H2O2 increased the viability and induced activation of PI3K and Akt, inactivation of GSK-3, and inhibition of cytochrome c release, caspase-3 activation, and PARP cleavage.

Conclusion: These results indicate that low concentration of cilnidipine has neuroprotective effects by activating PI3K/Akt and by inhibiting GSK-3 activation, cytochrome c release, caspase-3 activation, and PARP cleavage, whereas high concentration is rather cytotoxic. Therefore, some specific optimum concentration of cilnidipine may be a new potential therapeutic strategy for oxidative stress injured in vitro model of neurodegenerative diseases.