The reagents and chemicals such as DMEM medium, MTT, dimethyl sulfoxide (DMSO), penicillinG, streptomycin, sodium bicarbonate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and sodium pyruvate were obtained from Sigma-Aldrich; Merck Millipore, (Darmstadt, Germany). Fetal bovine serum (FBS) was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and Annex in V-FITC/PI Apoptosis Detection kit (cat. no. 556570) from BD Biosciences (San Jose, CA, USA).

N2a neuroblastoma cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and strictly grown as per the manufacturer's protocol. Dulbecco's modified Eagle's medium was supplemented with 10% fetal bovine serum (FBS), penicillin G (70 mg/l), streptomycin (100 mg/l) and NaHCO₃ (3.7 g/l). Cells were maintained at 37°C in a humidified CO₂ incubator with 95% humidity and 5% CO₂.

The effect of etomidate on cell viability was investigated using an MTT assay. Briefly, in a 96-well plate, the cells were seeded at a density of 2 ×10⁶ cells/well and allowed to grow for 24 h. After 24 h, the cells were treated with increasing concentrations of etomidate and were maintained for an additional 48 h. The 2.5 mg/ml MTT was added 4 h prior to the termination of experiment. After 4 h, the media was removed and formazan crystals were dissolved by adding 150 µl DMSO per well. The absorbance was quantified using the Synergy MX microplate reader at 570 nm.

Cells at the density of 2×10⁶ were seeded in 60 mm dish and maintained for 24 h after the 0, 1, 5 and 10 µM etomidate treatment was administered for 48 h. Rhodamine-123 was added to the cells at 37°C for 30 min prior to the termination of the experiment. Finally, the cells were collected in respective tubes, centrifuged for 5 min at 400 × g at 37°C and then washed three times with PBS. In flow tubes, the samples were finally transferred and resuspended in 500 µl PBS. The inverted fluorescent microscope was used to visualize the mitochondrial depolarization level, the mitochondrial depolarization level was quantitatively analyzed by imaging software Image J (version 1.49; National Institutes of Health, Bethesda, MD, USA).

In a 6-well plate, N2a cells were seeded at 2.0×10⁶ cells/well cultured for 24 h followed by treatment with 0, 1, 5 and 10 µM etomidate for 48 h at 37°C. Subsequently the cells were trypsinized and replated in a 6-well plate with 500 cells/well. The cells were subsequently cultured for 21 days at 37°C. During termination of the experiment, the cells were washed 3 times with PBS and were subsequently fixed for 10–12 min in 4% paraformaldehyde. Crystal violet (0.06%) was used to stain (30–60 min at 37°C) the live cells and the number of colonies (>50 cells/colony) was counted under an inverted microscope using a camera (Olympus Corporation, Tokyo, Japan).

In order to determine the effect of etomidate on the generation of ROS, N2a cells at a cell count of 2.0×10⁶ were seeded in 6-well plates and maintained for 24 h. Subsequently, the cells were treated with 0.5, 1, 5 and 10 µM etomidate for 48 h. Finally, cells were collected in tubes, washed three times with PBS and then resuspended in 500 µl PBS to which 10 µM DCFH-DA was added and cells were incubated for 30 min in dark at 37°C. ROS-induced green fluorescence of DCF-DA was imaged using 488-nm laser excitation. The laser power was set to 1–3%. This power setting allowed complete discrimination of DCF fluorescence and the autofluorescence originating from the oxidized form of mitochondrial flavoproteins. The 515–530 nm emission range was used to monitor an increase in dichlorofluorescein, the oxidized product of DCF-DA.

Cells (1×10⁶) were seeded in 50-ml dishes and incubated for 24 h at 37°C. Subsequently etomidate at different concentration was added directly to the dishes and incubated for additional 24, 48 and 72 h. Cells were collected, washed with PBS and resuspended in PBS. Apoptotic cell death was identified by double supravital staining with recombinant FITC-conjugated Annexin V and PI, using the Annexin V-FITC Apoptosis Detection kit (BD Biosciences) according to the manufacturer's protocol. Flow cytometry analysis was performed immediately following supravital staining. Data acquisition and analysis were performed in a Becton-Dickinson FACS Calibur flow cytometer using Cell Quest software (version 5.1; BD Biosciences).

In the 60 mm dishes, the N2a cells (at a density 2×10⁶) were seeded and maintained for 24 h after which they were treated with 0.5, 1, 5 and 10 µM etomidate for 48 h. Cells were trypsinized and lysed using a radioimmunoprecipitation assay buffer (Abcam, Cambridge, MA, USA). Bradford method (Thermo Fisher Scientific, Inc.) was used for protein quantification, after which proteins were separated (10 µg/sample) by 12% SDS-PAGE and were then transferred at 100 V for 2 h onto a PVDF membrane. Blocking of the membrane was performed with 5% skimmed milk for 1 h to avoid non-specific binding of the antibodies. Membranes were incubated with primary antibodies: Caspase-3 (procaspase-3 and active caspase-3; cat. no. 9665; 1:1,000), caspase-9 (procaspase-9 and active caspase-9; cat. no. 9502; 1:1,000), poly ADP-ribose polymerase (PARP; cleaved PARP p89; cat. no. 9542; 1:1,000), β-actin (cat. no. 4967; 1:1,000) all from Cell Signaling Technology, Inc., (Beverly, MA, USA) at 4°C for 12–14 h after which membrane was washed twice with TBST for 5 min each. The secondary antibody, horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG; cat. no. K0211589; 1:3,000; KOMA Biotech, Seoul, South Korea) was added at room temperature for 1 h. The blots were analyzed with enhanced chemiluminescence detection system (GE Healthcare, Chicago, IL, USA) and visualized using a LAS 4000 (GE Healthcare, Chicago, IL, USA) imaging system and images were processed using ImageJ software (version 1.49; National Institutes of Health).

All experiments were performed in triplicate and data were presented as the mean ± standard error of the mean. One-way analysis of variance or the independent samples Kruskal-Wallis H test, as appropriate was used to compare differences among normally distributed variables. For statistically significant differences, post hoc pairwise comparisons were performed by using Tukey's honestly significant difference test or Dunn's test. P<0.05 was considered to indicate a statistically significant difference.

N2a cells were first treated with a single concentration of etomidate (20 µM) for 48 h and the cell viability was reduced up to 80% in 48 h (data not shown). For the IC₅₀ value determination, the cells were treated with increasing concentrations of etomidate for 48 h in a 96-well plate. The cell viability was reduced with increased etomidate concentration (0.5, 1, 5 and 10 µM). These findings demonstrated that N2a cells responded to etomidate in a dose-dependent manner. The IC₅₀ was determined as 5 µM (Fig. 1). These findings collectively suggested that etomidate reduced the viability of N2a neuroblastoma cells.

When N2a cells were treated with different concentrations of etomidate, the clonogenic potential of the cells reduced with the increase in etomidate concentration. This led to an inhibition of the colony formation potential of N2a cells in a dose-dependent manner as presented in Fig. 2.

In order to confirm that etomidate induces apoptosis in N2a cells, Annexin V/PI assay was performed. The current study determined that etomidate increased the apoptotic population in a dose-dependent manner as presented in in Fig. 3. These finding sclearly indicated that etomidate induced concentration dependent apoptosis in N2a cells.

As a fore mentioned etomidate induced apoptosis in N2a cells; therefore, the current study investigated the effect of etomidate on the generation of reactive oxygen species using DCFH2-DA dye. Cells were treated with different concentrations of etomidate for 48 h and it was evident that etomidate treatment had a considerable impact on generation of reactive oxygen species in N2a cells as presented in Fig. 4. Therefore, these findings clearly indicated that etomidate induced generation of reactive oxygen species that led to apoptosis of N2a cells.

The current study investigated whether etomidate treatment of N2a cells had any influence on mitochondrial membrane potential using Rodamine-123 dye. It was evident that etomidate treatment of N2a cells lead to loss of their mitochondrial membrane potential in a dose-dependent manner which led to quenching of the fluorescence when compared with the untreated cells which retained fluorescence. These findings confirmed that etomidate induced apoptosis in N2a cells by reducing the mitochondrial membrane potential of these cells (Fig. 5).

In the present study, the effect of etomidate on the caspase activation and cleaving of PARP was investigated using western blotting. The N2a cells were treated with different concentrations of etomidate for 48 h. An increase in the expression of cleaved PARP was observed in a dose dependent manner. There was also a decrease in the expression level of the initiator caspase (caspase-9), along with a decrease in the expression of procaspase-3 in a dose-dependent manner. These findings suggested that etomidate induces apoptosis in a dose-dependent manner (Fig. 6).