Materials
AZ was purchased from Sigma-Aldrich (St. Louis, MO, USA). AZ was dissolved in dimethyl sulfoxide (DMSO) and the maximum concentration of DMSO was 0.1%. Dulbecco's Modified Eagle Medium (DMEM), dulbecco’s phosphate buffered saline (DPBS), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA were purchased from WelGENE (Daegu, South Korea).
Cell lines and cell culture
The mouse melanoma cell line, B16F10, was obtained from Korean Cell Line Bank (KCLB, Seoul, South Korea). The cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin. Cultures were routinely maintained at 37°C in a humidified atmosphere of 5% CO2.
Determination of cytotoxicity
The effect of drugs on the proliferation rate/cytotoxicity of cancer cells was assessed by using a colorimetric MTT assay. Briefly, cells were grown in 96-well flat-bottomed plates in media with 10% FBS allowed attach overnight. Then media was removed and replaced with fresh media with various concentrations of drugs. At the end of the treatment, medium was replaced by MTT (2.5 mg/mL) solution and cells were incubated at 37°C. Following 4 hr of incubation, MTT solution was discarded formazan crystal was solubilized with DMSO. The optical densities were measured at 570 nm. Results were calculated as percentage of unexposed control.
Measurement of melanin contents
B16F10 melanoma cells were seeded at a density of 2.5×105 cells/60 mm culture dish. The cells were treated with AZ combined with Tau for 24 hr. The cells pellets were dissolved in 1 N NaOH at 60°C for 1 hr. The relative melanin content was determined by measuring the absorbance at 475 nm in ELISA reader.
Tyrosinase activity assay
The tyrosinase activity was evaluated by measuring the rated of dopachrome formation of L-DOPA (L-3, 4-dihydroxyphenylalanine). After incubation of AZ with Tau for 24 hr, the cells were washed in ice-cold PBS twice and lysed in phosphate buffer (0.1 M, pH 6.8) containing 1% (w/v) Triton X-100. The cellular extract was clarified by centrifuged at 14000 rpm for 20 min.
Western blot analysis
After treatment with drugs, cells were washed with phophate-buffered saline, harvested, and were lysed in RIPA buffer [50 mM Tris-HCl (pH 8.0) with 150 mM NaCl, 1.0% nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate] containing protease inhibitor and phosphatase inhibitor (Roche, Indianapolis, IN, USA). After centrifugation, the supernatant was separated and stored at -70 C until use. Protein concentration was quantified by using a protein assay kit (Bio-Rad, Hercules, CA). Equal amount of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked and incubated with primary antibody overnight in Tris-buffered saline with 0.2% Tween-20 and 2.5% nonfat dry milk (or 2.5% bovine serum albumin). The primary antibodies used in the study are as follows: ERK 1/2 and phospho-ERK 1/2 antibodies were purchased from Cell signaling (Danvers, MA, USA) and were used at a 1:1000 dilution. Phospho-ERK1/2 antibody detects endogenous levels of ERK 1/2 when phosphorylated Thr/Tyr of ERK1/2. TRP1, TRP2, tyrosinase and MITF antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and used at a 1:500 dilution. Following three washes of 10 minutes with Tris-buffered saline with 0.2% Tween-20, blots were incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz, CA, USA). The blots were washed again three times in Tris-buffered saline with 0.2% Tween-20 and visualized with an ECL advance detection system.
Statistical analysis
All experiments were done at least 3 independent times and values were expressed as means ± S.D. Significant differences between groups were analyzed by using Student t-test. A P-value of < 0.05 was considered statistically significant.