Rotenone, nicardipine, Fluo-4 AM, the CASP3 assay kit, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin were obtained from Gibco-BRL (Grand Island, NY, USA). The in situ cell death detection terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) kit was obtained from Roche Diagnostics (Indianapolis, IN, USA). Anti-JNK, anti-phospho-JNK (Thr 183 and Tyr 185), anti-p38 MAPK, anti-phospho-p38 MAPK (Thr 180 and Tyr 182), anti-cleaved CASP9, anti-cleaved CASP3, anti-cleaved poly (ADP-ribose) polymerase-1 (PARP) and anti-β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-B-cell lymphoma protein 2 (Bcl2) and anti-Bcl2-associated X protein (BAX) antibodies and horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

SH-SY5Y cells were obtained from American Type Culture Company (Rockville, MD, USA). Cells were grown in DMEM supplemented with 10% FBS and 100 U/ml penicillin/streptomycin. Cultures were maintained in a humidified incubator at 37°C in an atmosphere of 5% CO₂ and 95% air. Cell culture medium was changed every 2 days.

Rotenone was freshly prepared in dimethyl sulfoxide (DMSO) prior to each experiment. Nicardipine was freshly prepared in saline and was added 4 h prior to rotenone addition to the cultures.

Cell viability was determined by MTT assay. For detecting cytotoxicity of rotenone on SH-SY5Y cells, cells were seeded in triplicate at a concentration of 2×10⁵ cells/ml on a 96-well plate. Cells were exposed to rotenone (0.1, 1, 5, 10 and 20 μM) containing 0.1% (v/v) DMSO or vehicle for 24 h. In the experiment for detecting the effect of nicardipine, cells were pretreated with nicardipine or saline for 4 h prior to rotenone treatment with concentrations of 0.1, 0.5, 1 and 5 μM. Following this, cells were exposed to rotenone (10 μM) containing 0.1% (v/v) DMSO or vehicle for 24 h. MTT (0.5 mg/ml) was added to each group and the cells were incubated for 4 h. Then, cells were incubated for an additional 1 h in the solution in which MTT was dissolved. Viability was read with an absorbance microplate reader (Molecular Devices, Toronto, ON, Canada) at a test wavelength of 595 nm with a reference wavelength of 690 nm. Optical density (OD) was calculated as the difference between the reference and test wavelength. Percent viability was calculated as (OD_drug/OD_control) × 100.

SH-SY5Y cells (1×10⁵ cells/ml) were cultured on four-chamber slides (Nalge Nunc International, Naperville, IL, USA). Following pretreatment with nicardipine (5 μM, 4 h) or saline, cells were incubated with rotenone (10 μM) or vehicle for 24 h. Then, cells were fixed in methanol and incubated in 1 μg/ml DAPI solution for 30 min in the dark. The stained cells were observed with a fluorescence microscope (Carl Zeiss, Oberköchen, Germany).

TUNEL assays were performed according to the manufacturer's instructions. SH-SY5Y cells (1×10⁵ cells/ml) pretreated with 5 μM nicardipine (4 h) or saline were exposed to 10 μM rotenone or vehicle for 24 h and then fixed in acetic acid at −20°C. Fixed cells were incubated with the TUNEL reaction mixture (terminal deoxynucleotidyl transferase and nucleotide) for 1 h at 37°C, followed by the addition of peroxidase-conjugated detection antibody. DNA fragments were stained using 3,3′-diaminobenzidine as the substrate for the peroxidase.

Cells were pretreated with nicardipine (5 μM) for 4 h, incubated with 3 μM Fluo-4 AM (dissolved in DMSO) in serum-free growth medium at 37°C for 45 min in a CO₂ incubator and then washed with calcium-free Hank's balanced salt solution (Gibco-BRL). Ca²⁺ fluorescence was assessed at intervals of 10 sec in an absorption spectrum (488 nm) and an emission wavelength (515 nm) using laser scanning confocal microscope (Leica Lasertechnik, Heidelberg GmbH, Wetzlar, Germany). Baseline [Ca²⁺]_i was observed for 100 sec and 10 μM rotenone was added and changes in [Ca²⁺]_i were measured. Results are expressed as relative fluorescence intensity (21).

Cells were lysed in RIPA buffer containing protease inhibitors. The protein content was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein (80 μg) were separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane (Schleicher & Schuell, Postfach, Germany). Following blocking with 5% skimmed milk, membranes were probed with rabbit anti-JNK, anti-phospho-JNK, anti-p38, anti-phospho-p38 MAPK, anti-cleaved CASP9, anti-cleaved CASP3, anti-cleaved PARP or mouse anti-β-actin antibodies overnight at 4°C. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG were used as the secondary antibodies. Band detection was performed using the Enhanced Chemiluminescence detection system (Amersham Biosciences, Uppsala, Sweden). Results of western blot analysis were quantified using ImageJ image analysis software (v1.4; National Institutes of Health, Bethesda, MD, USA).

CASP3 activity was measured using an assay kit according to the manufacturer's instructions. SH-SY5Y cells (2×10⁵ cells/ml) were lysed following treatment with 5 μM nicardipine and 10 μM rotenone for 24 h. The CASP3 substrate (Ac-DVED-p-NA) was added to cell lysates and the mixtures were incubated overnight in a humidified environment at 37°C. Control lysates were preincubated with the CASP3 inhibitor Ac-DEVD-CHO to determine on-specific background substrate breakdown. The concentration of p-NA released from the CASP3 substrate was measured using an absorbance microplate reader (Molecular Devices) as absorbance values at 405 nm and calculated from a calibration curve of p-NA standards.

Data are presented as mean ± SEM from three independent experiments. All experiments were performed at least three times independently. Data were analyzed by one-way ANOVA, followed by Tukey's HSD post hoc test, using the SPSS software (v13.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

SH-SY5Y cells were treated with rotenone of various concentrations (0.1–20 μM) for 24 h. Rotenone revealed significant cytotoxic effects in a dose-dependent manner in SH-SY5Y cells (Fig. 1A; P<0.05 vs. non-treated cells). The viability of SH-SY5Y cells exposed to rotenone at concentrations of 10 and 20 μM was 58.3±3.7 and 33.7±3.3% of the control value, respectively.

To examine the effect of nicardipine on rotenone-induced cytotoxicity, cells were treated with various concentrations of nicardipine with or without rotenone (10 μM). As demonstrated in Fig. 1B, nicardipine pretreatment (4 h prior to rotenone treatment) increased cell viability in rotenone-treated cells in a dose-dependent manner. The viability of SH-SY5Y cells was 61.8±4.1, 69.9±4.5, 80.5±1.3 and 91.8±1.6% at concentrations of 0.1, 0.5, 1 and 5 μM nicardipine, respectively. Further experiments were performed using 5 μM nicardipine to determine the precise effects of nicardipine.

Apoptosis of rotenone-treated cells was determined by DAPI staining and TUNEL assay. As revealed in Fig. 1C (upper panel), 5 μM nicardipine protected against shrinkage of SH-SY5Y cells treated with 10 μM rotenone for 24 h. DAPI staining demonstrated nuclear condensation, DNA fragmentation and perinuclear apoptotic bodies upon treatment of rotenone, whereas nicardipine pretreatment inhibited these apoptotic features (Fig. 1C, middle panel). TUNEL assay also revealed DNA strand breaks, indicative of rotenone-induced apoptosis. By contrast, nicardipine prevented the induction of apoptosis by rotenone (Fig. 1C, lower panel).

Increased [Ca²⁺]_i has been postulated to be associated with rotenone-induced cell death in previous studies (7,8). As demonstrated in Fig. 2, [Ca²⁺]_i was rapidly increased by treatment with 10 μM rotenone and increased [Ca²⁺]_i was sustained to the end of the experiment. By contrast, nicardipine (5 μM) prevented rotenone-induced elevation of [Ca²⁺]_i. Nicardipine did not affect [Ca²⁺]_i in rotenone-untreated cells.

The effect of nicardipine on the phosphorylation of JNK and p38 MAPK in SH-SY5Y cells treated with rotenone was investigated. Cells were treated with 10 μM rotenone for up to 120 min. Fig. 3A indictates that rotenone induced phosphorylation of JNK in a time-dependent manner. Phosphorylation of p38 MAPK was also transiently upregulated following exposure to rotenone, with peak expression observed at 10 min. The effect of nicardipine on phosphorylation of JNK and p38 MAPK was examined in cells exposed to rotenone for 120 and 10 min, respectively. In nicardipine (5 μM)-pretreated cells, the phosphorylation of JNK and p38 MAPK triggered by rotenone was abrogated (Fig. 3B and C).

The effect of nicardipine on Bcl2 family proteins, including Bcl2 and BAX, was analyzed in SH-SY5Y cells exposed to rotenone through western blot analysis. Rotenone (10 μM, 6–24 h) decreased the expression of the anti-apoptotic protein Bcl2 and increased the expression of pro-apoptotic protein BAX in a time-dependent manner (Fig. 4A). As demonstrated in Fig. 4B and C, 5 μM nicardipine pretreatment prevented the reduction of Bcl2 and the elevation of BAX induced by rotenone (10 μM, 24 h).

The effect of nicardipine on CASP9 and 3 and PARP was assessed in rotenone-treated SH-SY5Y cells. Rotenone (10 μM, 6–24 h) increased cleavage of CASP9 and 3 and PARP in a time-dependent manner (Fig. 5A). As revealed in Fig. 5B and C, rotenone-induced cleavage of CASP9 and 3 and PARP was prevented by pretreatment with 5 μM nicardipine.

In addition, the effect of nicardipine on CASP3 enzyme activity, the primary executioner of apoptosis, was analyzed in SH-SY5Y cells treated with 10 μM rotenone for 24 h. The activity was measured by hydrolysis of the peptide substrate, Ac-DEVD-p-NA. Nicardipine pretreatment significantly attenuated the cleavage of Ac-DEVD-p-NA elevated by rotenone (Fig. 6).