MATERIALS AND METHODS
Dimethyl sulfoxide (DMSO), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), Earle’s balanced salts solution (EBSS), insulin, hydrocortisone, minoxidil, dithiothreitol (DTT), phenylmethylsulfonylfluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), sodium orthovanadate (Na3VO4) and mouse monoclonal anti-β-actin antibody were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, U.S.A.). Aprotinin and leupeptin were purchased from Calbiochem (LaJolla, CA, U.S.A.). Nonidet-P40 (NP-40) was purchased from Roche Diagnostics (Mannheim, Germany). [1,2,6,7-3H] testosterone was purchased from American Radiolabeled Chemicals, Inc. (St. Louis). Testosterone and DHT were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Williams medium E and L-glutamine were purchased from Gibco BRL (Grand Island, NY, U.S.A.). Penicillin/streptomycin solution and trypsin–EDTA solution were purchased from Invitrogen-Life Technologies (Carlsbad, CA, U.S.A.). Fetal bovine serum (FBS) and Dulbecco’s modification of Eagle’s medium (DMEM) were purchased from Hyclone Inc. (Logan, UT, U.S.A.). Finasteride was purchased from Merck-Sharpe-Dohme (Whitehouse Station, NJ, U.S.A.). Silica gel 60 F254 TLC plate was purchased from Merck (Darmstadt, Germany). ULTIMA GOLD™ Cocktail was purchased from PerkinElmer, Inc. (Waltham, MA, U.S.A.). Five percent Minoxidil (MINOXYL™) was purchased from Hyundai Pharmaceutical Inc. (Chungnam, Korea). Polyvinylidene fluoride (PVDF) membranes and Protein Assay Dye Reagent Concentrate were purchased from Bio-Rad (Hercules, CA, U.S.A.). Mouse-monoclonal anti-VEGF-R2, α-tubulin, rabbit-polyclonal anti-β-catenin and Smad2/3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Rabbit-polyclonal anti-phospho-(ser552)-β-catenin, phospho-(ser675)-β-catenin, phospho(ser9)-GSK3β and GSK3β were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). Rabbit-polyclonal anti-Lamin B1 was purchased from Abcam (Cambridge, U.K.). NE-PER nuclear extraction kit was purchased from Pierce (Rockford, IL, U.S.A.). Enhanced chemiluminescence solution (West-zol™ Plus) was purchased from iNtRon Biotechnology (Seoul, Korea).
The Preparation of S. muticum Extract and Isolation of Apo-9′-fucoxanthinone
S. muticum were collected from Udo in Jeju Island, Korea and identified by Dr. Dong Sam Kim. A voucher specimen (A10-0000107) has been deposited at the herbarium of Jeju Biodiversity Research Institute. S. muticum was dried in the well-ventilated shade and then 2 kg of S. muticum was extracted with 80% aqueous methanol (MeOH) for 2 d at room temperature. The 80% MeOH extract was concentrated using a vacuum evaporator and dried to produce powder 200 g. The powdered extract was suspended in water and partitioned into n-hexane, methylene chloride (CH2Cl2), ethyl acetate (EtOAc), n-butanol (BuOH) and aqueous fractions. The CH2Cl2 fraction (5.4 g) was chromatographed over celite with n-hexane, CH2Cl2, EtOAc and MeOH successively (hexane–CH2Cl2 1 : 0, 10 : 1, 5 : 1, 2 : 1, CH2Cl2, EtOAc, MeOH). The (hexane–CH2Cl2 1 : 0) fraction was chromatographed with normal phase silica gel (4×50 cm) and eluted with hexane–EtOAc–MeOH (2 : 1 : 0.1). The fraction yielded apo-9′-fucoxanthinone (1.8 mg). The purified compound was identified by comparing its 1H- and 13C-NMR data with those reported in the literature.38) Apo-9′-fucoxanthinone (Fig. 1) was dissolved in DMSO (Amresco, Solon, OH, U.S.A.).
|Fig. 1. The Chemical Structure of Apo-9′-fucoxanthinone|
Male Wistar rats (3 weeks of age), female C57BL/6 mice (6 weeks of age) and male Sprague-Dawley (SD) rats (8 weeks of age) were supplied by Orient Bio (Seongnam, Gyeonggi, Korea). All animals were provided with a standard laboratory diet and water ad libitum. All animals were cared for according to protocols (20110014) approved by the Institutional Animal Care and Use Committee (IACUC) of the Jeju National University.
Isolation and Culture of Rat Vibrissa Follicles
Isolation of rat vibrissa follicles was performed as described previously.39) Briefly, rat vibrissa follicles were harvested from male Wistar rats (23 d of age). First of all, the rats were sacrificed under carbon dioxide (CO2). Both mystacial pads were removed from the rats and placed in a 1 : 1 mixture of solution of EBSS and phosphate buffered saline (PBS) containing 100 units/mL of penicillin and 100 µg/mL of streptomycin. Anagen vibrissa follicles were then carefully dissected using sterile dissecting forceps and blade under a stereomicroscope (Olympus, Tokyo, Japan) from the mystacial pads. The isolated follicles were cultured in 24-well plates containing Williams E medium supplemented with 2 mM L-glutamine, 10 µg/mL insulin, 50 nM hydrocortisone, 100 units/mL penicillin and 100 µg/mL streptomycin at 37°C in an atmosphere comprised of 5% CO2 and 95% air. The isolated follicles were treated with S. muticum extract (0.1, 1, 10 µg/mL) or minoxidil (10 µM) as a positivie control.40) The culture medium containing S. muticum extract or minoxidil was changed every 3 d and the length of the vibrissa follicles was measured using a DP controller (Olympus).
Assay for Prostatic 5α-Reductase Activity
Male SD rats (8-week-old) were sacrificed with CO2. The rat prostates were removed from their capsules, washed with PBS before stored at −80°C. Frozen tissues were placed in a culture dish. The tissues were homogenized with a tacoTM Prep Bead Beater (GeneReach Corp., Taichung, Taiwan) in 5 tissue volumes of homogenizing buffer [20 mM potassium phosphate buffer (pH 6.6), 0.32 M sucrose, 25 µg/mL leupeptin, 25 µg/mL aprotinin, 1 mM DTT and 0.2 mM PMSF]. The homogenates were centrifuged at 700×g for 3 min. The pellets were washed twice with homogenizing buffer. The pellets were suspended in homogenizing buffer and stored at −80°C until use. The suspension (2.5 mg protein/mL as determined by the Bradford dye binding method) was used for 5α-reductase assay. 5α-Reductase activities were analyzed as previously described.41) The reaction were initiated with S. muticum extract (0.1, 1, 10, 100 µg/mL), the solvent fraction of S. muticum extract (0.1, 1, 10 µg/mL) or apo-9′-fucoxanthinone (0.01, 0.1, 1, 10 µM) containing reaction buffer [40 mM potassium phosphate buffer (pH 6.6), 1 mM DTT, 2 mM reduced nicotinamide adenine dinucleotide phosphate, and 120 nCi [1,2,6,7-3H] testosterone]. An independent set of the reaction (n=3) was examined and finasteride (2 nM) was used as a positive control. The mixture was incubated at 37°C for 60 min, and then the reaction was stopped by adding 1 mL of EtOAc. The samples were centrifuged at 1000×g for 5 min. The supernatant was carefully aspirated, dried on the heating plate. The residues were dissolved in 50 µL of EtOAc containing 500 µg/mL of testosterone and 500 µg/mL of DHT, and applied to a silica gel 60 F254 TLC plate. The plate was developed in a 1 : 1 mixture of solution of EtOAc–cyclohexane, and the plate was air dried. The TLC spot of testosterone (T) was visualized under UV light (254 nm). The plate was soaked in 10% H2SO4 solution to confirm the spot of DHT before heating the plate. The plate was clipped off to get area containing androgen, the strips were soaked in 5 mL of ULTIMA GOLD™ Cocktail. Radioactivity was measured by a liquid scintillation counter (Packard Bioscience, Meriden, CT, U.S.A.). The activity of 5α-reductase was expressed as a ratio calculated with the equation: [DHT/(T+DHT)]×100.
Assay for the Proliferation of DPCs
Rat vibrissa immortalized DPCs42) were donated by the Skin Research Institute, Amore Pacific Corporation R & D Center, Korea. The DPCs were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin (100 unit/mL, 100 µg/mL, respectively) at 37°C in a humidified atmosphere under 5% CO2. The proliferation of DPCs was evaluated by measuring the metabolic activity using a MTT assay.43) Briefly, DPCs (1.0×104 cells/mL) were seeded into 96-well plates, cultured for 24 h under 1% serum conditions, and treated with vehicle (DMSO diluted 1 : 500 in DMEM containing 1% FBS), or with the S. muticum extract (0.1, 1, 10 µg/mL), the solvent fraction of S. muticum extract (0.1, 1, 10, 40 µg/mL) or apo-9′-fucoxanthinone (0.001, 0.01, 0.1, 1 µM) for 4 d. After incubation, 0.1 mg (50 µL of a 2 mg/mL solution) of MTT was added to each well, and the cells were incubated at 37°C for 4 h. The plates were centrifuged at 350×g for 5 min and the media was carefully aspirated. DMSO 200 µL was added to each well to dissolve the formazan crystals and the absorbance of the plate at 540 nm was read immediately on a microplate reader (Versamax; Molecular Devices, Sunnyvale, CA, U.S.A.). All experiments were performed in triplicate and the mean absorbance values were calculated. The results were expressed as the percentage of vehicle treated groups. Minoxidil was used as a positive control.
Hair Growth Activity in Vivo
Anagen was induced on the back skin of C57BL/6 mice that were in the telogen phase of the cycle by depilation, as described previously.44) The anagen phase was induced in the back skin of the 7-week-old female C57BL/6 mice by shaving, which led to synchronized development of anagen hair follicles. From the following day (day 1), 0.2 mL of S. muticum EtOAc fraction in 50% ethanol was topically applied every day for 41 d. MINOXYL™ was used as a positive control. The back skin of the mice was then observed and photographed at 1, 14, 20, 33 and 41 d after shaving. For quantitative assessment, dotmatrix planimetry was performed.45)
Western Blot Analysis
The DPCs (1.0×105 cells/mL in 100 mm dishes) were pre-incubated for 24 h under 1% serum conditions, and the cells were treated with apo-9′-fucoxanthinone (1 µM) and minoxidil (10 µM) as a positive control for 72 h. The cells were washed twice with ice-cold PBS. The cells were lysed in lysis buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO3, 10 mM NaF, 1 mM DTT, 1 mM PMSF, 25 µg/mL aprotinin, 25 µg/mL leupeptin and 1% NP-40] to obtain whole cell protein and kept on ice for 30 min. The cell lysates were centrifuged at 21000×g at 4°C for 15 min. Supernatants were stored at −20°C until analysis. On the other hand, to obtain nuclear and cytoplasmic fractions, cells were lysed with NE-PER nuclear and cytoplasmic extraction reagents according to the manufacturer’s protocol. Fractions were stored at −70°C until analysis. Protein concentration was determined by the Bradford method.46) Equal amounts of protein were separated on 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels. The proteins were then transferred onto PVDF membranes. The membrane was blocked for 1 h with 5% nonfat dried milk in Tween-20-TBS (T-TBS) (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween-20), each membrane was incubated with specific primary antibodies against phospho(ser552)-β-catenin (1 : 1000), phospho(ser675)-β-catenin (1 : 1000), β-catenin (1 : 1000), phospho(ser9)-GSK3β (1 : 1000), GSK3β (1 : 1000), Smad2/3 (1 : 1000), α-tubulin (1 : 500), Lamin B1 (1 : 3000), VEGF-R2 (1 : 500) and β-actin (1 : 5000) at 4°C overnight. The membrane was incubated with a secondary horseradish peroxidase antibody (1 : 5000) at room temperature for 1 h. The membrane was exposed on X-ray film, and protein bands were detected using West-zol™ Plus. Band intensities were quantified with NIH Image software (http://rsb.info.nih.gov/ij/).
All results were expressed as the mean±standard deviation (S.D.) of at least three independent experiments. Student’s t-test was used to determine the statistical significance (p-value <0.05) of differences between values for the various experimental and control groups. SigmaStat (Systat Software Inc., San Jose, CA, U.S.A.) was used for statistical analysis.