Whitening Effect Of Novel Peptide Mixture By Regulating Melanosome Biogenesis, Transfer And Degradation Part 1

ABSTRACT 

Peptides are short chains of amino acids linked by peptide bonds. They are widely used as effective and biocompatible active ingredients in the cosmetic industry. In this study, we developed a novel peptide mixture and identified its anti-pigmentation effect on melanocytes and keratinocytes. Our results revealed that peptide mixture inhibited melanosome biogenesis through the regulation of microphthalmia-associated transcription factor, a key factor of melanogenesis in melanocytes. And we observed that the peptide mixture inhibited melanosome uptake through the reduction of protease-activated receptor 2, a phagocytosis-related receptor in keratinocytes. Furthermore, the peptide mixture activated the autophagy system resulting in the degradation of transferred melanosomes in keratinocytes. The anti-pigmentation effect of a multi-targeting peptide mixture was assessed in a human skin equivalent model (MelanoDerm). Melanin contents in the epidermal layer were significantly decreased by topical treatment of peptide mixture, suggesting that it can be applied as a novel cosmetics material having a whitening function.

According to relevant studies,cistanche is a common herb known as "the miracle herb that prolongs life". Its main component is cistanoside, which has various effects such as antioxidant, anti-inflammatory, and immune function promotion. The mechanism between cistanche and skin whitening lies in the antioxidant effect of cistanche glycosides. Melanin in human skin is produced by the oxidation of tyrosine catalyzed by tyrosinase. The oxidation reaction requires the participation of oxygen, so the oxygen-free radicals in the body become an important factor affecting melanin production. Cistanche contains cistanoside, which is an antioxidant and can reduce the generation of free radicals in the body, thus inhibiting melanin production.

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Key Words 

Autophagy ·Melanosome ·Microphthalmia-associated transcription factor ·PAR-2· Peptide

INTRODUCTION

Melanin is produced by melanocytes located in the basal layer of the epidermis to protect skin cells against harmful external agents, including ultraviolet (UV) rays, fine dust, and various chemical compounds [1]. The factors secreted from keratinocytes mainly by UV irradiation, including alpha-melanocyte-stimulating hormone (MSH), adrenocorticotropic hormone, stem cell factor, endothelin 1, hepatocyte growth factor, basic fibroblast growth factor, and granulocyte-macrophage colony-stimulating factor, stimulate each receptor on the surface of melanocytes and melanin is produced in a lysosome-related organelle called melanosome [2-7]. Among these factors, MSH is a small peptide hormone derived from pro-opiomelanocortin. The binding of -MSH to melanocortin 1 receptor increases the cyclic adenosine monophosphate (cAMP) level that activates protein kinase A (PKA) [8,9]. PKA then phosphorylates cAMP-responsive element binding protein (CREB) and transcription of microphthalmia-associated transcription factor (MITF) is induced by phospho-CREB [10]. MITF acts as a transcription factor for tyrosinase, tyrosinase-related protein 1 (TYRP1), and dopachrome tautomerase (Dct or TYRP2), key enzymes in melanogenesis, and induces their expression [11]. The matured melanosome moves toward the dendrite tip, and the melanosome motor protein complex consisting of Rab27a, Melanophilin, and Myosin Va plays a role in this process [12-14]. Melanophilin, which has binding domains for Rab27a, Myosin Va, and actin, simultaneously binds to Rab27a on the melanosome surface and to Myosin Va interacting with the actin filament. The assembly of these motor proteins is required for melanosome transport to the dendrite tips along the actin filament [15- 19]. Subsequently, melanosomes transferred from the dendrite tips of melanocytes to neighboring keratinocytes and incorporated melanosomes locate in the perinuclear area of keratinocytes [20,21]. Although proper melanin formation is important for normal cell protection, excessive melanin production induces hyperpigmentation, including melasma, freckles, and solar lentigo, which can lead to mental stress and poor quality of life [22,23]. Thus, there are demands for the development of satisfactory whitening agents without side effects such as skin irritation, allergy induction, and carcinogenesis, and many studies targeting various whitening mechanisms have been conducted [24,25].

Peptides are short chains of amino acids, the smallest unit of protein, linked to each other by peptide bonds. Recently, extensive research and development regarding peptides as active ingredients have been performed in the cosmetics and pharmaceutical industries because of their high biocompatibility and protein-mimicking ability [26,27].

We screened our peptide library and found four peptides affecting the inhibition of melanin synthesis, migration, and transfer as well as melanosome degradation-promoting activity. Subsequently, this study aimed to investigate the whitening activity of a peptide mixture containing four peptides with the same molar ratio. 

METHODS

Materials

The 2-CTC resin used in the peptide synthesis was purchased from BeadTech (Seoul, Korea). Fmoc-amino acid, hydroxy benzotriazole (HOBt), and N, N, N´, N´-tetramethyl-O-(1H-benzotriazole- 1-yl)uronium hexafluorophosphate (HBTU) were purchased from CS Bio Co. (San Francisco, CA, USA). The reagents used in the synthesis, including dimethylformamide (DMF), N, N-diisopropylethylamine (DIEA), piperidine, trifluoroacetic acid (TFA), thioanisole, phenol, ethane dithiol (EDT), triisopropylsilane (TIS), and diethyl ether, were purchased from Daejung Chemical & Metal Co., Ltd. (Seoul, Korea).

Dulbecco's Modified Eagle Medium (DMEM) powder for melanocytes and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA), and liquid DMEM media and penicillin/streptomycin were purchased from Welgene Inc. (Gyeongsan, Korea). Sodium bicarbonate, sodium hydroxide, arbutin, 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT), 3,4-dihydroxy-L-phenylalanine (LDOPA), trypsin, and rapamycin were purchased from Sigma-Aldrich (St. Louis, MO, USA), and Solvable was purchased from PerkinElmer (Waltham, MA, USA). Antibodies against Rab27A, Melanophilin, MITF, Tyrosinase, and Actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and antibodies against Beclin-1, LC3, p62, p-CREB, and p-ERK1/2 were purchased from Cell Signaling Technology (Danvers, MA, USA). Primer synthesis was performed in Cosmo GENETECH (Seoul, Korea) and further used in the experiment (Table 1).

Peptide synthesis

CTC resin with a capacity of 0.84 mmol/g was subjected to a swelling reaction in a reactor containing DMF solvent. Then, 2 equivalents of Fmoc-C terminal amino acid and 2.5 equivalents of DIEA were added in DMF to the reactor and further subjected to reaction for 2 h. The termination of the reaction was confirmed with a Kaiser test kit (Sigma-Aldrich), and the resin was washed with DMF. The Fmoc was removed by adding 20% piperidine/ DMF twice to the washed resin. The termination of the reaction was confirmed with the Kaiser test kit, and the resin was washed with DMF. Subsequently, the following process was repeated according to the sequence of amino acids in the C terminal to N terminal direction. After 2 equivalents of Fmoc-amino acid, 2 equivalents of HBTU, 2 equivalents of HOBt, and 2.5 equivalents of DIEA were added in DMF, the reaction was performed for 2 h. The termination of the reaction was confirmed with the Kaiser test kit, and the resin was washed with DMF. The Fmoc was removed from an amino acid by adding 20% piperidine/DMF twice to the washed resin. The termination of the reaction was confirmed with the Kaiser test kit, and the resin was washed with DMF. In the case of Pep-3, Ferulic acid conjugation was performed through the following procedure. After 2 equivalents of Ferulic acid, 2 equivalents of HBTU, 2 equivalents of HOBt, and 2.5 equivalents of DIEA were added in DMF, the reaction was performed for 2 h. The termination of the reaction was confirmed with the Kaiser test kit, and the resin was washed with DMF. After the final synthesis was completed, a cleavage solution (TFA:H2O: Ohioans ole:phenol:EDT: TIS = 81.5:5:5:5:2.5:1) was added to separate the peptide from the resin. The peptide was then precipitated using diethyl ether and washed five times. Subsequently, drying was performed to recover the final peptide product.

The purity of the synthesized peptide was identified by HPLC (Thermo Fisher Scientific/U-3000) analysis, and it was detected at UV 214 nm under the flow rate of 1 ml/min under mobile phase 0.1% TFA in water/0.1% TFA in acetonitrile using a C18 (Agilent, Pursuit XRs, 250 × 4.6 mm, 5 m, 100Å) column.

To identify the molecular weight, LC-MS/MS (AB SCIEX, 3200 Q-trap) analysis was performed, and it was detected by MS/MS at the flow rate of 0.25 ml/min under mobile phase 0.1% formic acid in water/0.1% formic acid in acetonitrile gradient using a C18 (Agilent, Pursuit XRs, 100 × 2.0 mm, 5 m, 100Å) column. The MS/MS analysis conditions included ESI Positive mode, Source/ Gas: CUR = 20, CAD = High, IS = 5500, TEM = 350, GS1 = 50, GS2 = 50/Compound DP = 50–80, EP = 10, CE = 10–50, and CES = 1–10 (Table 2).

Cell culture

The B16F10 melanoma cells used in this experiment were purchased from ATCC (Manassas, VA, USA). B16F10 cells were incubated in DMEM media containing 10% heat-inactivated FBS, 1% penicillin/streptomycin, and 1.5 g/L sodium bicarbonate at 37°C and under the condition of 5% CO2.

HaCaT keratinocytes were purchased from CLS (Eppelheim, Germany). HaCaT cells were incubated in DMEM media containing 10% heat-inactivated FBS and 1% penicillin/streptomycin at 37°C and under the condition of 5% CO2.

Cell viability

B16F10 cells were seeded in a 48-well plate at a density of 8 × 103 cells/well and incubated for 16 hours. They were then treated with various concentrations of peptide mixture in 2% FBS-containing media for 3 days. After incubation, 20 l of 4 mg/ml MTT was treated in each well for 4 hours and the media was removed. Subsequently, cells were treated with 500 l of DMSO to dissolve formazan. Absorbance was measured at 570 nm using a spectrophotometer (Molecular Devices, San Jose, CA, USA).

Melanin content assay

B16F10 cells were seeded in a 6-well plate at a density of 5 × 104 cells/well and incubated for 16 h. They were then treated with various concentrations of peptide mixture in 2% FBS-containing media for 3 days. To induce melanin production, 100 ng/ml -MSH was co-treated and 200 M arbutin was used as a positive control. The cells were collected and lysed by treating with 200 l of 1 M NaOH and absorbance for lysate at 490 nm was measured with a spectrophotometer.

Tyrosinase activity test

B16F10 cells were seeded in a 6-well plate at a density of 5 × 104 cells/well and incubated for 16 h. They were then treated with various concentrations of peptide mixture in 2% FBS-containing media for 3 days. To induce melanin production, 100 ng/ml-MSH was co-treated and 200 M arbutin was used as a positive control. Each well was treated with 200 l of lysis buffer (Sigma-Aldrich) to collect the lysate. After quantitation of the protein using a BCA kit (Thermo Fisher Scientific), 90 g of protein from each group was incubated with 20 l of 10 mM L-DOPA in 96- a well plate for 30 min at 37°C. Absorbance for the mixture at 475 nm was measured with a spectrophotometer.

Melanosome isolation

B16F10 cells were seeded in a 6-well plate at a density of 5 × 104 cells/well and incubated for 16 h. Cells were treated with 100 ng/ ml-MSH for 72 h to induce melanin synthesis and harvested by trypsin/EDTA treatment. Cells were then washed with 10 ml of 0.25 M sucrose (in 10 mM HEPES buffer) and resuspended in 10 ml of 0.25 M sucrose solution. Subsequently, after freezing with liquid nitrogen, thawing was performed at 37°C, which was repeated 10 times. After centrifugation at 1,000 g for 10 min, the supernatant was collected and further centrifuged for 45 min at 20,630 g and 4°C. Subsequently, the pellet was subjected to resuspension with DPBS and stored at –80°C before use.

Melanosome uptake test

HaCaT cells were seeded in a 6-well plate at a density of 1 × 105 cells/well and incubated for 16 h. They were then treated with the peptide mixture in serum-free media for 1 h and further treated with isolated melanosomes for 40 h. The cells were lysed by treating with 200 l of 1 M NaOH and absorbance for lysate at 490 nm was measured with a spectrophotometer. Furthermore, melanosome staining was performed using a Fontana-Masson Stain Kit (ScyTek Laboratories, Logan, UT, USA), and images of cells were detected by a light microscope.

Melanosome degradation test

HaCaT cells were seeded in a 6-well plate at a density of 1 × 105 cells/well and incubated for 16 h. They were pretreated with isolated melanosomes in serum-free media for 48 h and additionally treated with the peptide mixture for 72 h. One micromole of rapamycin was used as a positive control. The cells were lysed by treating with 200 l of 1 M NaOH and absorbance for lysate at 490 nm was measured with a spectrophotometer. Furthermore, melanosome staining was performed using a Fontana-Masson Stain Kit, and images of cells were detected by a light microscope.

Gene expression analysis (RT-PCR)

B16F10 cells were seeded in a 6-well plate at a density of 5 × 104 cells/well and incubated for 16 h. They were then treated with various concentrations of peptide mixture in 2% FBS-containing media for 3 days. To induce melanin production, 100 ng/ml-MSH was co-treated and 200 M arbutin was used as a positive control. RNA was isolated from cells using an RNA extraction kit (Qiagen, Germany), and cDNA synthesis was performed using RT-PCR premix (iNtRON Biotechnology, Seongnam, Korea). After the preparation of the reaction mixture with PCR premix (iNtRON Biotechnology) and primers for each gene, PCR was performed using a PCR machine (Eppendorf, Germany). Subsequently, mRNA expression patterns were determined by agarose gel electrophoresis.

HaCaT cells were seeded in a 6-well plate at a density of 3 × 105 cells/well and incubated for 16 h. They were then treated with various concentrations of peptide mixture in the serum-free media for 1 h and additionally treated with 4U trypsin for 16 h. The cells were collected and RT-PCR was performed in the same manner as described above.

Protein expression analysis (Western blotting)

B16F10 cells were seeded in a 6-well plate at a density of 5 × 104 cells/well and incubated for 1 day. They were then treated with various concentrations of peptide mixture in 2% FBS-containing media for 3 days. To induce melanin production, 100 ng/ml -MSH was co-treated and 200 M arbutin was used as a positive control. The cells were lysed with lysis buffer and the protein was quantified using a BCA kit. Twenty micrograms of protein were separated on 8% polyacrylamide gel and electrophoretically transferred to polyvinylidene fluoride membranes. Membranes were blocked in 5% w/v skim milk in PBS with 0.5% Tween 20 (PBS-T). Incubation with primary antibodies diluted in blocking solution was performed overnight at 4°C and was followed by washing with PBS-T. The appropriate secondary antibodies were diluted in blocking solutions and incubated with the membranes for 1 h at room temperature followed by washing with PBS-T. The membranes were visualized via Western detection reagent (Elpis Biotech, Daejeon, Korea) with Gel Doc (Bio-Rad, Hercules, CA, USA).

For analysis of p-CREB and p-ERK, B16F10 cells were seeded in a 6-well plate at a density of 3 × 105 cells/well and incubated for 1 day. They were then treated with various concentrations of peptide mixture in 2% FBS-containing media. The incubation times were 30 min for the analysis of p-CREB and 10 min for the analysis of p-ERK. To induce melanin production, 100 ng/ml -MSH was co-treated, and 200 M arbutin was used as a positive control. Western blotting was performed using isolated proteins in the same manner as described above.

HaCaT cells were seeded in a 6-well plate at a density of 3 × 105 cells/well and incubated for 16 h. They were then treated with various concentrations of peptide mixture in serum-free media for 3 days. Two hundred nanograms of rapamycin were treated for 3 hours as a positive control. Western blotting was performed using isolated proteins in the same manner as described above.

Skin equivalent analysis

To test the whitening effect of peptide mixture using skin equivalent, a liposome sample was prepared as follows. The peptide mixture was dissolved in distilled water at a concentration of 2,000 g/ml and adjusted to pH 7. After filtration using a 0.22-m pore size filter, 1% hydrogenated phosphatidylcholine buffer was mixed at a ratio of 10%. The nano-sized liposome sample was prepared by passing through the 75-m cells five times at 1,000 bar using the Microfluidizer.

MelanoDerm MEL-300-B (MatTek Corporation, Ashland, MA, USA) was topically treated with 25 l or 50 l of liposome containing 2,000 g/ml peptide mixture three times a week for 2 weeks. The same volume of vehicle was applied to control tissue. The treated skin equivalents were washed with DPBS and light microscopy was performed to check the morphology of melanocytes. Subsequently, to analyze the production of melanin, skin equivalents were frozen at –80°C for 30 min and thawed at room temperature. Tissue samples were incubated with 1% sodium bicarbonate for 30 min and dried. After treatment with 300 l of Solvable, the samples were left overnight at 95°C and then cooled at room temperature. The absorbance of the tissue extract at 490 nm was measured with a spectrophotometer.

To observe the melanin distribution within the tissues through histology, all samples were fixed in 4% phosphate-buffered formalin for 24 h. Fixed samples were paraffin-embedded and cut into a 5-m section using a microtome (Leica Biosystems, Wetzlar, Germany). Paraffin sections were stained using Fontana-Masson Stain Kit according to the manufacturer's instructions and light microscopy was performed.

Statistics

The experiments conducted in this study were repeated three times or more. The statistical significance of the data was tested through Student's t-test. The resulting values were expressed as mean ± standard deviation and were considered statistically significant when the p-value was less than 0.05.

RESULTS

Peptide mixture inhibits-MSH-induced melanin synthesis and tyrosinase activity in B16F10 cells

To determine the effect of the peptide mixture on cell viability, B16F10 melanoma cells, and HaCaT keratinocytes were treated with the peptide mixture at concentrations of 10, 50, 100, and 200 M, and an MTT assay was performed. The results showed that the peptide mixture was not cytotoxic in the overall treatment concentrations, and subsequent experiments were performed in the above concentration range (Fig. 1A).

To identify the inhibitory effect of the peptide mixture on melanin synthesis, B16F10 cells were treated at concentrations of 10, 50, 100, and 200 M to compare melanin production rates. The results showed dose-dependent inhibition of melanin production by the peptide mixture. Inhibition rates were 3% at the lowest concentration of 10 M and 26% at the highest concentration of 200 M (Fig. 1B).

Tyrosinase is one of the key enzymes in melanin synthesis. The tyrosinase activity assay was performed to verify that the melanogenesis inhibitory effect of the peptide mixture was caused by the regulation of tyrosinase. The results showed significant inhibition of tyrosinase activity by the peptide mixture in B16F10 cells. Inhibition rates were 8% at the lowest concentration of 10 M and 33% at the highest concentration of 200 M (Fig. 1C).

Ask for more: david.deng@wecistanche.com  WhatApp:86 13632399501