Hydrolyzed Cow Colostrum Extract (BCFM) Manufacturing
Cow colostrum was obtained from Cheong-Won Farm (Cheongju City, Chungcheongbuk-do, Republic of Korea). Hydrolyzed cow colostrum extract, named BCFM (Bio Conversion First Milk) was manufactured in the following way: Alcalase® (EMD Milipore Corp, MA, USA) was added to colostrum whey to obtain hydrolyzed colostrum, and enzyme inactivation was achieved by heating. The hydrolyzed colostrum whey was filtered through a 0.22µm filter (JetBiofil, Guangzhou, China). The filtered colostrum whey was lyophilized to obtain a BCFM powder.
Cell Culture
B16F1 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium/Ham’s medium (Hyclone, UT, USA) supplemented with 10% fetal bovine serum (Gibco, NY, USA) and 1% antibiotics (Gibco, NY, USA). Cells were cultured in a humidified incubator at 37°C in a 5% CO2 atmosphere. B16F1 cells were seeded at ~ 60% confluence in complete growth media. Twenty-four hours later, the culture media were replaced with new media containing the indicated concentrations of BCFM (1, 5, and 10 µg/mL). After 60 minutes, the cells were treated with 100 nM of alpha-MSH (Sigma, MO, USA) and co-cultured for 24, 48, and 72 hours. Following this, the cells were collected for further experiments.
Cell Viability
B16F1 cells were plated at a density of 1.0×103 cells/mL in 96-well plates and incubated at 37°C for 24 hours. Then, the cells were treated at each concentration of BCFM (1, 5, and 10 µg/mL). After incubation for 72 hours, 10 µL of MTT (0.5 mg/mL in PBS) solution was added to each well. The incubation was continued for 4 hours more; the formazan crystals were dissolved in DMSO, and the optical density was measured at 520 nm using a SYNERGY HTX (BioTek, VT, USA).
Melanin Content Assay and Melanin Observations
B16F1 cells were plated at a density of 5.0×103 cells/mL in 24-well plates and incubated at 37°C for 24 hours. Twenty-four hours later, the cells were treated with each concentration of BCFM (1, 5, and 10 µg/mL). After 60 minutes, the cells were treated with 100 nM of alpha-MSH (Sigma, MO, USA) and co-cultured for 72 hours. Then, the cell pellets were collected in 1.5 mL tube with a centrifugation at 1000×g for 5 minutes. After discarding the supernatants, cell pellets were dried for 1 hour in a dry oven. For cell lysis, 300 µL of 1 N NaOH were added to each tube and incubated at 50°C for 1 hour. Total melanin contents were measured at 420 nm using a SYNERGY HTX. After treatment with BCFM (1, 5, and 10 µg/mL) and 100 nM of alpha-MSH for 72 hours, melanin production was visually observed using a microscope (Olympus, Tokyo, Japan).
cAMP Measurement
Cyclic AMP (cAMP) levels were measured using an ELISA kit (#581001; Cayman Chemical, MI, USA) in accordance with the manufacturer’s recommendations. B16F1 cells were seeded at a density of 5.0×103 cells/mL in 24-well plates and incubated at 37°C for 24 hour. Twenty-four hour later, the cells were treated with each concentration of BCFM (1, 5, and 10 µg/mL). After 60 minutes, the cells were treated with 100 nM of alpha-MSH and co-cultured for 72 hours. After 72 hours, 50 µL of cell culture media was used for the ELISA assay. cAMP levels were measured at 420 nm using a SYNERGY HTX.
Western blotting
Samples were prepared from the cultured B16F1 cells with a protein extraction buffer (Cell Lytic™ M; Sigma, MO, USA) according to the instruction manual. The protein concentration of the samples was measured with a Bradford assay (#5000006; Bio-Rad, CA, USA) using a spectrophotometer with an optimal density at 595 nm. The protein samples were separated on precast gradient polyacrylamide gels (#4561033; Bio-Rad, CA, USA) and transferred to nitrocellulose membranes (#4561033; Bio-Rad, CA, USA) using the Mini-PROTEAN Tetra Cell, 4-Gel System (Bio-Rad, CA, USA) according to the manufacturer’s recommendations. The membranes were blocked in 5% skimmed milk. The blocked membranes were probed with a primary antibody and a horseradish peroxidase-conjugated secondary antibody. Following a repeat of the washing step, the membranes were kept in enhanced chemiluminescence detection reagents (#170–5060; Bio-Rad, CA, USA) for 1 minute. Signal intensity was measured with an image analyzer (ChemiDoc™ XRS+; Bio-Rad, CA, USA). The primary antibodies used in the present study were purchased from Abcam BD biosciences (anti-MC1R, ab180776; anti-MITF, ab140606; Cambridge, Cambridgeshire, UK), Cell Signaling Technology (anti-CREB, #9197; anti-phospho-CREB, #9198; anti-PKA, #5842; anti-phospho-PKA, #5661; Danvers, MA, USA), and Santa Cruz Biotechnology (anti-GAPDH, sc-32233; anti-TRP-1, sc-166857; anti-Tyrosinase, sc-20035; Dallas, TX, USA).
RT-qPCR
Total RNA was extracted from B16F1 cells treated with 100 nM of alpha-MSH in the presence or absence of BCFM using a TRI reagent (Molecular Research Center, Cincinnati, USA); total RNA was then reverse transcribed into cDNA using the ReverTra Ace® qPCR RT Master Mix with gDNA Remover (TOYOBO, Co., Ltd, Osaka, Japan) in accordance with the manufacturer’s recommendations. The TaqMan analysis was performed using predesigned and optimized Assays on Demand (Applied Biosystems, MA, USA). The following assays were used: MITF (ID: Mm00434954_m1), Tyrosinase (ID: Mm00495817_m1), TRP-1 (ID: Mm00453201_m1), MC1R (ID: Mm00434851_s1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ID: Hs99999905_m1). The reaction parameters were 95 ℃ for 15 s, followed by 40 cycles of 30 sec at 95 ℃ for melting, and 30 sec at 60 ℃ for annealing/extension. All measurements were performed in duplicate or triplicate, and the results were analyzed using a CFX96™ Real-time system (BIO-RAD, CA, USA). The results were first normalized to the endogenous GAPDH expression level.
Immunocytochemistry
B16F1 cells were grown on chamber slides and fixed with 3.7% paraformaldehyde (Biosolution, Seoul, Republic of Korea) in PBS for 30 minutes. Cells were permeated with 0.5% Triton for 15 minutes and incubated with primary antibodies against MITF (ab140606; Abcam BD biosciences, Cambridge, UK) and tyrosinase (sc-20035; Santa Cruz Biotechnology, TX, USA) for 24 hours at 4 ℃. After washing, they were incubated with the secondary antibodies (green: ab7086, red: ab150160; Abcam BD biosciences, Cambridge, UK) for 30 minutes at room temperature. Cells were counterstained with DAPI mounting solution (ab104139; Abcam BD biosciences, Cambridge, UK). Immunolabeling was examined using an Eclipse Ts2-FL microscope (Nikon, Tokyo, Japan). The data were analyzed with NIS-Elements BR 5.3 software (Nikon, Tokyo, Japan).
Statistical Analysis
One-way ANOVA was performed using Tukey’s post hoc test for comparison of different groups. The results are expressed as means ± S.E. and p < 0.05 was considered statistically significant.