Accepted Manuscript Sulforaphane promotes murine hair growth by accelerating the degradation of dihydrotestosterone Mari Sasaki, Shohei Shinozaki, Kentaro Shimokado PII:

S0006-291X(16)30288-1

DOI:

10.1016/j.bbrc.2016.02.099

Reference:

YBBRC 35406

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 19 February 2016 Accepted Date: 23 February 2016

Please cite this article as: M. Sasaki, S. Shinozaki, K. Shimokado, Sulforaphane promotes murine hair growth by accelerating the degradation of dihydrotestosterone, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.02.099. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Sulforaphane promotes murine hair growth by accelerating the degradation

Mari Sasaki a, Shohei Shinozaki a,b*, Kentaro Shimokado a

Department of Geriatrics and Vascular Medicine, Tokyo Medical and Dental University

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a

Graduate School of Medicine, Tokyo, Japan

Department of Arteriosclerosis and Vascular Biology, Tokyo Medical and Dental University

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b

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of dihydrotestosterone

* Corresponding author

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Graduate School of Medicine, Tokyo, Japan

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Address: Department of Arteriosclerosis and Vascular Biology, Tokyo Medical and Dental University Graduate School of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan;

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Phone: +81-3-5803-5969; Fax: +81-3-5803-0267; E-mail: [email protected]

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ABSTRACT Dihydrotestosterone (DHT) causes the regression of human hair follicles in the parietal scalp,

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leading to androgenic alopecia (AGA). Sulforaphane (SFN) increases the expression of DHT degrading enzymes, such as 3α-hydroxysteroid dehydrogenases (3α-HSDs), and, therefore, SFN treatment may improve AGA. To determine the effects of SFN on hair growth, we administered

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SFN (10 mg/kg BW, IP) or vehicle (DMSO) to ob/ob mice for six weeks and examined hair regeneration and the plasma levels of testosterone and DHT. We also tested the effects of SFN on

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the expression of two forms of 3α-HSD, aldo-keto reductase 1c21 and dehydrogenase/reductase (SDR family) member 9, both in vitro and in vivo. SNF significantly enhanced hair regeneration in ob/ob mice. The mice treated with SFN showed lower plasma levels of testosterone and DHT than those treated with vehicle. SFN increased the mRNA and protein levels of the two forms of

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3α-HSD in the liver of the mice and in cultured murine hepatocyte Hepa1c1c7 cells. These results suggest that SFN treatment increases the amount of 3α-HSDs in the liver, accelerates the

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degradation of blood DHT, and subsequently blocks the suppression of hair growth by DHT.

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HIGHLIGHTS Sulforaphane is a novel candidate for the treatment of androgenic alopecia.



Sulforaphane improves androgenic alopecia by lowering the plasma dihydrotestosterone

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concentration. 

The induction of hepatic 3α-HSD by sulforaphane leads to the degradation of

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dihydrotestosterone.

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KEYWORDS

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Androgenetic alopecia, dihydrotestosterone, 3α-HSD, sulforaphane, metabolic syndrome

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INTRODUCTION Dihydrotestosterone (DHT) plays an important role in androgenetic alopecia (AGA), which is

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the most common type of hair loss [1-3]. DHT acts through androgen receptors in hair follicles [4], whereby it shortens the anagen (growth) phase and elongates the telogen (resting) phase of hair growth, resulting in increased immature hair [5] and decreased new hair [6].

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Currently, 5α-reductase inhibitors that suppress the conversion of testosterone into DHT are widely used to treat AGA [7, 8], but the effect of DHT degrading enzymes on hair growth is less

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clear and has not been clinically tested. DHT is mainly degraded in the liver by hydroxysteroid dehydrogenases (HSDs), such as 3α-HSD, 3β-HSD, and 17β-HSD [9-11]. There are two protein superfamilies within the 3α-HSDs: aldo-keto reductases (AKRs) [12] and the short-chain dehydrogenase/reductase (SDR) family [13]. AKRs can bind to DHT [14] and degrade DHT by

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acting as a 3-ketosteroid reductase in the presence of dihydronicotinamide adenine dinucleotide phosphate (NADPH) [15]. SDRs convert DHT to 3β,17β-androstanediol (3β-diol) under reduced (NADPH-rich) conditions [16]. AKRs have been extensively studied, whereas SDRs are poorly

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characterized.

Sulforaphane (SFN), an isothiocyanate isolated from broccoli, is a potential modulator of

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DHT degradation. SFN is known to have anti-oxidative effects through activation of the nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) pathway [17, 18]. SFN induces DHT degradation enzymes, such as AKR1C1 [19] and AKR1C2 [20] in human cultured cells. SFN also promotes the production of NADPH by inducing NADPH-producing genes, such as glucose-6-phosphate dehydrogenase X-linked (G6pdx) [21]. Therefore, SFN may increase the activity of HSDs by increasing NADPH. 4

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These previous reports prompted us to hypothesize that SFN treatment might promote hair growth by lowering the blood DHT level via induction of 3α-HSDs in the liver. To the best of our

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knowledge, there has been no study that has reported that SFN induces DHT degrading enzymes, decreases plasma levels of DHT, or accelerates hair growth in vivo. To test our hypothesis, we employed leptin-deficient (ob/ob) mice. These mice have an increased plasma concentration of

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testosterone [22, 23], and show impaired hair regeneration due to elongation of the telogen

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(resting) phase [24-26] and inhibition of the anagen transition in the second hair cycle [26].

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MATERIALS AND METHODS Animals.

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Male ob/ob mice at 7 weeks of age, purchased from the Sankyo Laboratory (Tokyo, Japan), were used for this study. The study was approved by the Institutional Animal Care Committee of Tokyo Medical and Dental University. The animal care facility is accredited by the Association

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for Assessment and Accreditation of Laboratory Animal Care. The mice were maintained at 25 °C and illuminated by 12:12-h light-dark cycles. The mice were provided with free access to

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standard rodent chow and water ad libitum. After removal of hair from the dorsal skin using depilatory cream (day 0), the mice were regularly monitored and recorded by photography (EOS kiss X5; Canon, Tokyo, Japan) for 6 weeks. The mice were treated with SNF (10 mg/kg BW, IP, LKT Laboratories, St. Paul, MN) or dimethyl sulfoxide (DMSO) on alternating days for six

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weeks. Regenerated hair could be seen as dark hair on pinkish-white shaved skin [4]. The hair regeneration area was quantified using the NIH ImageJ 1.50b software program (NTIS,

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Springfield, VA).

Measurement of testosterone and dihydrotestosterone concentrations.

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Plasma testosterone and DHT concentrations were measured using mouse testosterone (Cayman, Ann Arbor, MI) and DHT (Abnova, Taipei City, Taiwan) ELISA kits.

Isolation of total RNA and quantitative RT-PCR. Total RNA was isolated with an RNeasy Mini kit (Qiagen, Valencia, CA). First-strand cDNA was synthesized from 1 µg of total RNA using a High Capacity cDNA Reverse Transcription kit 6

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(Applied Biosystems, Carlsbad, CA). Real-time RT-PCR analyses were performed as previously described [27] using 10 ng cDNA and TaqMan probes (Applied Biosystems) for aldo-keto

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reductase 1c21 (Akr1c21), short-chain dehydrogenase/reductase (SDR) family member 9 (Dhrs9) and 18S ribosomal RNA, and were conducted with the TaKaRa PCR Thermal Cycler Dice (Takara Bio, Osaka, Japan). The gene expression of Akr1c21 and Dhrs9 was normalized to that

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of 18S ribosomal RNA.

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Tissue and cell homogenization.

Mouse livers were collected at 18 h after SFN injection under anesthesia with pentobarbital sodium (50 mg/kg BW, IP). Tissue and cell samples were homogenized in ice-cold homogenization buffer (50 mM HEPES, pH 8.0, 150 mM NaCl, 2 mM EDTA, 2.5% lithium

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dodecyl sulfate, 2% CHAPS, 10% glycerol, 10 mM sodium fluoride, 2 mM sodium vanadate, 1 mM PMSF, 10 mM sodium pyrophosphate, 1 mM DTT, protease inhibitor cocktail (Sigma)), as

Immunoblotting.

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described previously [27].

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Immunoblotting was performed as described previously [27]. Anti-AKR1C2, anti-GAPDH (Cell Signaling, Beverly, MA), and anti-DHRS9 (GeneTex, Irvine, CA) antibodies were used as primary antibodies. Anti-rabbit IgG antibody conjugated to horseradish peroxidase (GE Healthcare Life Science, Pittsburgh, PA) was used as the secondary antibody. Amersham ECL Select Western Blotting Detection Reagent (GE Healthcare Life Science) was used to visualize signals. Bands were visualized using a LAS1000 analyzer (Fuji Film, Tokyo, Japan) and bands of 7

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interest were quantified using the Multi Gauge software program (Fuji Film).

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Statistical analysis. The data were compared with one-way ANOVA followed by Scheffe’s multiple comparison test or Student’s t-test. A value of P

Sulforaphane promotes murine hair growth by accelerating the degradation of dihydrotestosterone.

Dihydrotestosterone (DHT) causes the regression of human hair follicles in the parietal scalp, leading to androgenic alopecia (AGA). Sulforaphane (SFN...
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