Dulbecco’s modified Eagle’s medium, fetal bovine serum, and penicillin/streptomycin (100 IU/50 μg/ml) were obtained from Invitrogen (Grand Island, NY). Mellitin, H2O2, 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide, 4′, 6-diamidino-2-phenylindole (DAPI), 2′,7′-dichlorofluorescein diacetate, rabbit anti-Bax, rabbit anti-Bcl-2, and rabbit anti-caspase-3 were purchased from Abcam (Cambridge, MA). Anti-rabbit horseradish peroxidase-linked IgG antibodies were purchased from GE Healthcare Life Science (Buckinghamshire, England, UK). All other chemicals were of analytical grade.
Cell culture and treatment
Human neuroblastoma SH-SY5Y cells, obtained from the Korea Cell Line Bank (Seoul, Korea), were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum, and 1% penicillin/streptomycin at 37°C under 5% CO2 in air. To determine the effect of melittin on H2O2-exposed SH-SY5Y cells, SH-SY5Y cells were treated with various doses of melittin for 1 h before H2O2 exposure for 6 h. H2O2 was prepared immediately before use as a 20 mM stock. Melittin was dissolved in phosphate-buffered saline (PBS) and the stock solutions were added directly to the culture media. In a single experiment, each treatment was performed in triplicate.
Cell viability assay
Cell viability was determined by MTT assay. SH-SY5Y cells were seeded in 96-well plates at density of 8 × 104 cells/well and incubated for 48 h prior to experimental treatments. The cells were pre-incubated with or without melittin following incubation with H2O2 for 24 h. The cultured medium was removed and 50 μl MTT solution (1 mg/ml in PBS) was placed in each well. After incubation at 37°C for 4 h, the solution was carefully removed, and 150 μl dimethyl sulfoxide was added. Absorbance was measured at 570 nm using a microplate reader (Bio-Tek Instruments, Inc., Winooski, VT).
Lactate dehydrogenase release assay
Lactate dehydrogenase (LDH) is released into the cell culture supernatant when cells undergo by apoptosis or necrosis. LDH levels were measured using a Cytotoxicity Cell Death kit (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. Briefly, the cells (8 × 104 cells/well) were seeded in 96-well plates and then incubated with 100 μM H2O2 for 24 h with or without melittin pretreatment for 1 h. For analysis, 100 μl supernatant was transferred to a new 96 well plate, and 100 μl of reaction mixture was added to each well and incubated at 37°C for 30 min. Absorbance was measured at 490 nm using microplate reader (Bio-Tek Instruments, Inc.). LDH release was determined in cells treated with 2% Triton X-100 (high control); the assay medium served as the low control and was subtracted from all absorbance measurements.
Nuclear staining with DAPI
Nuclear morphology was assessed by staining with DAPI. Cells (1 × 105 cells/well) were seeded on coverslips in 6-well plates for 48 h and then treated with 100 μM of H2O2 for 24 h with or without melittin pretreatment for 1 h. The cells were washed twice with PBS and fixed with 1% paraformaldehyde for 15 min. The fixed cells were washed twice with PBS and stained with 4′, 6-DAPI (1 μg/ml) for 10 min at 37°C in the dark. Cells were washed twice with PBS and were observed using a fluorescent microscope.
Activation of caspase-3 was determined according to the protocols recommended for the caspase-3 assay kit (R&D Systems, Minneapolis, MN). In brief, the cells were lysed and centrifuged to obtain the supernatant. The supernatant was added to the reaction mixture containing dithiothreitol and caspase-3 substrate, and incubated for 2 h at 37°C. Absorbance was measured at a wavelength of 405 nm using a microplate reader (Bio-Tek Instruments, Inc.).
Western blot analysis
Cell were lysed with ice-cold lysis buffer containing protease inhibitors and centrifuged at 14,000 rpm for 10 min. The protein content of each supernatant was determined using a Bradford assay with bovine serum albumin as the protein standard. Samples (10 μg) were separated by polyacrylamide gel (10%) electrophoresis, and then transferred to a polyvinylidene difluoride membrane (0.45 μm, Immobilon-P Transfer membrane, Millipore, Billerica, MA). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h. After blocking, membranes were incubated with Bcl-2, Bax, and caspase-3 antibody (Abcam) in TBST overnight at 4°C. After washing in TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare Life Science) at a 1:5000 dilution for 1 h at room temperature. After washing with TBST, proteins were visualized using a Super Signal West Pico Kit (Pierce, Rockford, IL) detection system. Densitometric analysis was performed using Quantity One (Bio-Rad, Hercules, CA) to scan the signals.
RNA extraction and reverse transcription-polymerase chain reaction
Total RNA was isolated using the Total RNA Purification kit (Nanohelix, Daejeon, Korea) according to the manufacturer’s instructions. Reverse transcription of total RNA (1 μg) was performed for 1 h at 45°C using RT Premix kit (Oligo dT primer; iNtRON Biotechnology, Sungnam, Korea). The reaction was terminated by heating at 95°C at 5 min. cDNA was amplified by polymerase chain reaction (PCR) Premix kit (i-Taq) (iNtRON Biotechnology). Sequences of primers for Bcl-2 cDAN are: forward primer 5′-CGACTTCGCCGAGATGTCCAGCCAG-3′ and reverse primer 5′-ACTTGTGGCCCAGATAGGCACCCAG-3′, for Bax cDNA are: forward primer 5′-ACCAAGAAGCTGAGCGAGTGTC-3′ and reverse primer 5′-TGTCCAGCCCATGATGGTTC-3′, and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA are: forward primer 5′-AATGACCCCTTCATTGAC-3′ and reverse primer 5′-TCCACGACGTACTCAGCGC-3′. PCR for Bcl-2 and GAPDH)  was performed with 35 cycles as follows: denaturation at 94°C for 30 s, annealing at 61°C for 1 min, and extension at 72°C for 1 min. PCR for Bax  was performed with 40 cycles and the reaction conditions were: denaturation at 94°C for 45 s, annealing at 61°C for 1 min, and extension at 72°C for 1 min using PCR Thermal Cycler Dice (Takara, Shiga, Japan). The PCR products were analyzed by 2% agarose gel electrophoresis with ethidium bromide. The signal intensity of each band was quantified and normalized against GAPDH. Densitometric analysis was carried out using Quantity One (Bio-Rad) to scan the signals.
All data are expressed as the mean ± standard error of the mean (SEM). Statistical differences among groups were calculated by analysis of variance (ANOVA) followed by Duncan’s multiple range test (SPSS version 18.0, Chicago, IL). Differences with a p value less than 0.05 were considered significant.