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Inhibitory effect of carvacrol on melanin synthesis via suppression of tyrosinase expression
Journal of Functional Foods 45 (2018) 199–205
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Inhibitory effect of carvacrol on melanin synthesis via suppression of tyrosinase expression
T
Nam-Joo Jeona,b, Yon-Suk Kima,c, Eun-Kyung Kimd, Xin Donga,b, Jae-Woong Leea,b, ⁎ Jin-Su Parka,b, Woen-Bin Shina,b, Sang-Ho Moonc,d, Byong-Tae Jeone, Pyo-Jam Parka,b,c, a
Department of Biotechnology, Konkuk University, Chungju 27478, Republic of Korea Department of Applied Life Science, Konkuk University, Chungju 27478, Republic of Korea c Medicinal Biological Resources Research Institute, Konkuk University, Chungju 27478, Republic of Korea d Division of Food Bio Science, Konkuk University, Chungju 27478, Republic of Korea e Hasung Corp., Seoul 05832, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Carvacrol Melanogenesis B16F10 cells IBMX
Carvacrol (2-methyl-5-(1-methylethyl)-phenol) compound derived from oils of Thymus vulgaris is a natural member of monoterpene phenol. It has been shown that carvacrol exhibit anti-microbial, anti-inflammatory, anti-oxidant activities. However, no studies have reported its anti-melanogenesis effect. Therefore, the objective of this study was to investigate the anti-melanogenic potential of carvacrol. Results of this study confirmed that carvacrol could regulate protein expression levels of microphthalmia-associated transcription factor [MITF, a protein closely related to transcription of cAMP response element-binding protein (CREB)] and various enzymes involved in melanin synthesis. Moreover, this study provided evidence that carvacrol could regulate the degradation of MITF protein by extracellularly responsive kinases (ERK) phosphorylation. Carvacrol strongly inhibited the synthesis of CREB protein, tyrosinase-related protein 1 (TRP-1), and tyrosinase known to be important enzymes involved in melanogenesis. These results indicate that carvacrol can inhibit melanin synthase by decreasing enzyme expression levels important for melanin synthesis.
1. Introduction Carvacrol is a monoterpenoid compound derived from oils of Thymus vulgaris, Origanum, Carum copticum. It is a natural member of monoterpene phenol (Hussein, El-Bana, Refaat, & El-Naggar, 2017; Kisk & Roller, 2005; Lampronti, Saab, & Gambari, 2006; Martins, Neves, Silvestre, Silva, & Cavaleiro, 1999). Carvacrol has been used for a wide variety of applications in daily life. It is contained in various products such as cosmetics and wide range of foods. In addition, carvacrol has been shown to exhibit anti-microbial, anti-mutagenic, anti-platelet, analgesic, anti-inflammatory, anti-angiogenic, anti-oxidant, anti-elastase, insecticidal, anti-parasitic, cell-protective and anti-tumor activities (Sokmen et al., 2004; Can Baser, 2008). Carvacrol has also been used as an ingredient in various cosmetics. However, no study has reported the whitening effect of carvacrol. Many inflammatory cytokines as melanogens have been reported. Histamine, a ubiquitous inflammatory mediator, is a representative melanogen induced by inflammation (Yoshida, Takahashi, & Inoue, 2000). Studies have shown that reactive oxygen species (ROS)
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generation can activate melanin production (Kim et al., 2014). Based on this knowledge, the objective of this study was to investigate the antimelanogenesis potential of carvacrol. Melanin is synthesized by epidermal melanocytes in the skin. Melanin works in physiological defence to protect skin from ultraviolet radiation. However, continuous UV irradiation induces increased accumulation of melanin, leading to skin hyperpigmentation (Agar and Young, 2005). Melanogenesis is induced by isobutylmethylxanthine (IBMX) and α-melanocyte-stimulating hormone (α-MSH). Binding of αMSH to its receptor, melanocortin-1 receptor (MC1R), induces enhanced expression of microphthalmia-associated transcription factor (MITF) and, cyclic-AMP (cAMP), leading to increased expression of tyrosinase and other melanogenesis-related enzymes such as tyrosinaserelated protein 1 (TRP-1), dopachrome tautomerase (TRP-2). Hyperpigmented skin has an unaesthetic appearance, showing melasma and freckles (Plensdorf and Martinez, 2009). TRP-1, TRP-2, and tyrosinase are three representative enzymes that are important for melanin regulation. Tyrosinase is a copper-containing glycoprotein that is important in melanin synthesis. It can catalyze three different reactions:
Corresponding author at: Department of Biotechnology, Konkuk University, Chungju 27478, Republic of Korea. E-mail address: [email protected] (P.-J. Park).
https://doi.org/10.1016/j.jff.2018.03.043 Received 23 January 2018; Received in revised form 31 March 2018; Accepted 31 March 2018 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
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2.2. Cell culture
the hydroxylation of tyrosine to 3, 4-dihydroxyphenylalanine (DOPA), oxidation of DOPA to dopaquinone and conversion of dopaquinone to dopachrome and then to dihydro-indolizine (DHI) or indole 5,6-quinone-2-carboxylic acid (DHICA) (Ando, Kondoh, Ichihashi, & Hearing, 2007; Chung et al., 2009; Lee et al., 2010; Shimoda et al., 2010). TRP-1 and TRP-2 also play important roles in the synthesis of melanogenesis. TRP-1 catalyzes oxidation of DHICA while TRP-2 catalyzes conversion of dopachrome to DHICA (Sato, Morita, Ichikawa, Takahashi, & Toriyama, 2008). These enzymes are also regulated by a specific transcription factor, MITF (Hasegawa et al., 2010; Widlund & Fisher, 2003). MITF is regulated by a variety of signaling pathways. It is generally known to be controlled by MAPK. Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases that regulate cell differentiation, proliferation, and cellular activities. The MAPK superfamily contains three well-characterized subfamilies: ERKs, p38, and the c-Jun N-terminal kinases (JNKs). MAPKs are known to play a major role in the regulation of melanogenesis (Jiang et al., 2009; Ye et al., 2010). Furthermore, it has been reported that activation of ERK by c-Kit stimulation phosphorylates MITF at its 73rd serine residue. Phosphorylation of MITF at 73rd of serine is followed by MITF ubiquitination and degradation (Hemesath, Price, Takemoto, Badalian, & Fisher, 1998; Xu et al., 2000). Moreover, p38 MAPK pathway activation can increase melanin synthesis (Hirata et al., 2007; Singh et al., 2005). Activation of ERK signaling could also inhibit melanogenesis by inhibiting tyrosinase activity (Jang et al., 2009). There are numerous melanogenesis inhibiting agents, including arbutin, kojic acid, and linoleic acid. Arbutin is a glycosylated hydroquinone extracted from bearberry plant. It has been used as a cure for hyperpigmentation illnesses in the past. Kojic acid and arbutin are extensively used as cosmetic ingredients due to their anti-tyrosinase activity (Nishimura, Kometani, Okada, Ueno, & Yamamoto, 1995). However, some of these agents can cause skin irritation. It has been described that kojic acid causes skin irritation with side effects such as cytotoxicity, dermatitis, and skin cancer (Busca and Ballotti, 2000). Arbutin has been also banned due to its side effects involving exogenous ochronosis and perdurable depigmentation (Draelos, 2007; O'Donoghue, 2006). Therefore, there were huge interests in finding new potential compounds extracted from natural sources without or with very limited side effects. The present study aimed to investigate such a potential melanogenesis inhibiting compound from a natural source. In this study, we examined the anti-melanogenic effect of carvacrol on IBMX-induced melanogenesis in B16F10 mouse melanoma cells. We also explored the underlying molecular mechanisms involved in this process. Many previous studies on cavacrol have mainly determined its antibacterial and antioxidant properties. However, in present study, we performed a melanogenesis study using carvacrol for the first time. Results of this study provide basic research data for developing cosmetics using multifunctional resources
Mouse melanoma cell line B16F10 was obtained from the Korean cell line bank (Seoul, Korea) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 units/mL of penicillin and 100 μg/mL of streptomycin) at 37 °C in a humidified incubator containing 5% CO2. For maintenance, these cells were sub-cultured every two days. 2.3. Determination of cell viability The effect of carvacrol on viability of mouse melanoma cell line B16F10 viability was evaluated using MTT colorimetric assay. Briefly, B16F10 cells were plated into 96-well plates at a density of 5 × 103 cells/well. These cells were treated with various concentrations (25, 50, 100, 200, 400 and 600 μM) of carvacrol and stimulated with or without IBMX (100 μM) at 37 °C for 48 h. After treatments, the medium was replaced by 100 μL of DMEM medium containing MTT (200 µg/mL) in each well followed by incubation at 37 °C for 2 h. After discarding MTT solution, the intracellular formazan product in each well was dissolved in 200 μL DMSO. The absorbance was then measured at 540 nm using a microplate reader (Tecan, Grödig, Austria). Values were calculated in comparison with those of control cells. 2.4. Measurement of melanin content Intracellular melanin contents were determined according to published method (Hosoi, Abe, Suda, & Kuroki, 1985) with slight modifications. Briefly, B16F10 cells were stimulated with IBMX and incubated with carvacrol for 48 h. Cell pellets were harvested and then dissolved in 1 N NaOH containing 10% DMSO at 80 °C for 1 h. Melanin contents were analyzed by measuring absorbance at 475 nm using an ELISA reader (Multiskan GO, Thermo Fisher, Massachsetts, USA). For accurate calculation of melanin contents, each level of melanin was normalized to protein content. 2.5. Tyrosinase activity Tyrosinase activity was determined using published method (Hosoi et al., 1985). Briefly, B16F10 cells were co-treated with IBMX and different concentrations of carvacrol. After 48 h incubation, cells were washed with cold PBS and suspended in a lysis buffer (150 mM NaCl, 10 mM Tris pH 7.5, 5 mM EDTA), and Triton-X 100 1.0% in the presence of protease inhibitors (1 μg/mL leupeptin and 100 μg/mL PMSF) and incubated at 4 °C for 20 min to yield cell lysates. Cell lysates were then centrifuged at 12,000 rpm for 10 min. Protein contents in supernatants were then determined using a protein assay kit (Bio-Rad, Laboratories, Inc., Hercules, CA, USA). Then 90 μL of cell extract was transferred to a 96-well containing 10 μL of L-DOPA (final concentration of 1 mmol/L) prepared in 25 mM phosphate buffer (pH 6.8) and incubated at 37 °C for 20 min. Absorbance was then measured at 475 nm using an ELISA reader (Multiskan GO, Thermo Fisher, MA, USA).
2. Materials and methods 2.1. Chemicals Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Hyclone (Thermo Scientific, Waltham, MA, USA). Phosphorylated-ERK, tyrosinase, TRP-1, and TRP-2 antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). MITF, phosphorylated-CREB and β-actin antibodies were purchased from Cell Signaling Technology Inc. (Denvers, MA, USA). Carvacrol, IBMX, L-DOPA, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
2.6. Western blot analysis B16F10 cells were seeded in 60 mm dishes at a density of 3 × 105 cells/dish. After 24 h of culture, they were co-treated with IBMX (100 μM) and different concentrations (100, 200 and 400 μM) of carvacrol or Kojic acid (1 mM). After incubating at 37 °C for 48 h, cells were subsequently washed with PBS, collected and suspended in a lysis buffer (150 mM NaCl, 10 mM Tris (pH 7.5), 5 mM EDTA, and 1% Triton X-100) containing protease inhibitors (1 μg/mL leupeptin and 100 μg/ mL PMSF). After incubating at 4 °C for 20 min, cell lysates were centrifuged at 12,000 rpm for 10 min. Protein concentration in each cell 200
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lysate (supernatant) were measured using a protein assay kit (Bio-Rad, Laboratories, Inc., Hercules, CA, USA). Proteins (20 μg of B16F10 lysates) were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Buckinghamshire, UK). These membranes were then blocked by Tris-buffered saline-Tween 20 solutions (TBS-T) containing 5% non-fat dry milk, incubated with primary antibodies at 4 °C for 24 h, washed with TBST, and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Bio-Rad, Hercules, CA, USA) at room temperature for 1 h. Protein bands were visualized using an ECL detection kit and a luminescent image analyzer (LAS3000, Fujifilm, Tokyo, Japan). 2.7. Reverse transcription and real-time quantitative PCR Total RNAs were isolated from B16F10 cells using TRIzol Reagent (QIAGEN, CA, USA). Isolated RNAs were reverse-transcribed into cDNAs using cDNA reverse transcription kit (Applied Biosystems, USA). These synthesized cDNAs were subjected to real-time quantitative PCR on a Light Cycler® (Roche, Switzerland) using SYBR qPCR pre-mix (Dyne Bio, SN, KOR). PCR reactions were performed with gene-specific primers for MITF, tyrosinase and endogenous reference 36B4 as follows (forward and reverse, respectively): Tyrosinase(F), 5′-CCT CCT GGC AGA TCA TTT GT-3′, Tyrosinase(R), 5′-GGC AAA TCC TTC CAG TGT GT-3′, 36B4(F), 5′-TGG GCT CCA AGC AGA TGC-3′, 36B4(R), 5′-GGC TTC GCT GGC TCC CAC-3′, MITF(F), 5′-ATG CTG GAA ATG CTA GAA TAC AGT-3′, and MITF(R), 5′-ATC ATC CAT CTG CAT GCA C-3′. Data were analyzed using the 2-ΔΔCT method. Data are expressed as fold change of gene expression. 2.8. Statistical analysis Data are expressed as mean ± standard deviation for triplicate determinations. Analysis of variance (ANOVA) together with Tukey’s test and Dunnett’s test (GraphPad Prism 5) were conducted to identify significant differences between samples. Statistical significance was considered when p values were < 0.05 (p < 0.05). 3. Results Fig. 1. Effects of carvacrol on the cell viability of B16F10 mouse melanoma cells. (A) Cell viability was analyzed by MTT assay. B16F10 cells were treated with different carvacrol concentrations (25, 50, 100, 200, 400, and 600 μM) for 48 h. (B) Cells were treated with carvacrol and IBMX (100 μM) simultaneously. (C) Chemical structure of carvacrol. Each bar represents the mean ± S.E.M of three independent experiments.
3.1. Cytotoxicity of carvacrol to B16F10 mouse melanoma cells To investigate whether carvacrol had cytotoxic effect on B16F10 cells, B16F10 mouse melanoma cells were treated with various doses of carvacrol in the absence or presence of IBMX. Cytotoxic effect of carvacrol on B16F10 cells was measured by MTT assay. MTT assay results revealed that carvacrol had no significant effect on cell viability at concentrations below 400 μM in the presence or absence of IBMX (Fig. 1A and B). Therefore, concentrations of 100, 200, and 400 μM were selected as carvacrol concentration for further experiments. Fig. 1C shows chemical structure of carvacrol.
3.3. Inhibitory effect of carvacrol on tyrosinase enzyme activity in B16F10 mouse melanoma cells Tyrosinase is encoded by albino locus of mouse. It is a key enzyme responsible for the formation of melanin pigment in animals (Hearing and Tsukamoto, 1991; Pawelek, 1991). Therefore, we treated B16F10 mouse melanoma cells with various doses of carvacrol or kojic acid (1 mM) in the absence and presence of IBMX and then measured tyrosinase activity. Results indicated that carvacrol inhibited tyrosinase enzymatic activity in IBMX-induced melanocytes in a concentrationdependent manner. Moreover, carvacrol inhibited tyrosinase enzyme activity significantly more than kojic acid (Fig. 3).
3.2. Inhibitory effect of carvacrol on melanin synthesis in B16F10 mouse melanoma cells IBMX is known to increase melanin production and regulate melanogenesis pathway (Busca and Ballotti, 2000). Therefore, we investigated melanin inhibitory effect of carvacrol in IBMX-induced B16F10 mouse melanoma cells. We treated B16F10 mouse melanoma cells with various doses of carvacrol or kojic acid (1 mM) in the absence or presence of IBMX. Carvacrol inhibited melanin production in melanocytes significantly more than kojic acid in a concentration-dependent manner. In addition, the color of the cell pellet was changed in a concentration-dependent manner due to melanin production (Fig. 2A and B).
3.4. Inhibitory effect of carvacrol on the expression of melanin-related enzymes in B16F10 cells Previous studies have shown that tyrosinase, TRP1 and TRP2 are involved in the melanogenesis pathway to mediate important responses 201
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Fig. 3. Inhibitory effect of carvacrol on tyrosinase activity in melanocytes. B16F10 mouse melanoma cells were co-treated with IBMX (100 μM) and carvacrol (100, 200, and 400 μM) or kojic acid (1 mM). After 48 h incubation, absorbance was measured at 475 nm. Data are represented as mean ± S.E.M (n = 3). ###p < 0.001 compared to control group, ***p < 0.001 compared to IBMX group.
Fig. 4. Effects of carvacrol on the expression levels of tyrosinase, TRP-1 and TRP-2 in IBMX-induced B16F10 cells. B16F10 mouse melanoma cells were treated with different doses of carvacrol (100, 200, and 400 μM) or kojic acid (1 mM) in the absence or presence of IBMX for 48 h. After harvesting cells, cell lysates were prepared and subjected to Western blot analysis with anti-tyrosinase, anti-TRP-1, and anti-TRP-2 antibody.
Fig. 2. Inhibitory effect of carvacrol on melanin production in melanocytes. B16F10 mouse melanoma cells were co-treated with IBMX (100 μM) and carvacrol (100, 200, and 400 μM) or kojic acid (1 mM). After 48 h incubation, absorbance was measured at 475 nm. (A) Melanin contents expressed as fold values. (B) Photographs of cell culture dishes showing color changes after different sample treatments. Data are represented as mean ± S.E.M (n = 3). ### p < 0.001 compared to control group, ***p < 0.001 compared to IBMX group.
was down-regulated by carvacrol in a dose-dependent manner (Fig. 5B). The activity of CREB was inhibited more by carvacrol at 400 µM than by kojic acid. These results indicate that carvacrol can effectively inhibit phosphorylation of CREB protein.
to melanogenesis (Jin, Oh, Hyun, Kwon, & Kim, 2014). Therefore, effects of carvacrol on tyrosinase, TRP1 and TRP2 protein expression levels were analyzed by Western blotting. Our results showed that carvacrol decreased TRP1 and tyrosinase protein expression levels in IBMX-stimulated B16F10 cells in a concentration-dependent manner. However, carvacrol had no effect on the expression of TRP-2 protein level (Fig. 4).
3.6. Inhibitory effect of carvacrol on MITF protein expression To understand whether transcriptional regulation of tyrosinase and TRP-1 mRNA expressions was involved in the effect of carvacrol, the effect of carvacrol on the expression of MITF, a major transcription factor for tyrosinase and TRP-1, was investigated. After treatment with carvacrol, protein levels of MITF were down-regulated in a dose-dependent manner (Fig. 6B). Carvacrol treatment also decreased IBMXinduced MITF protein expression in a time-dependent manner (Fig. 6A). These results indicate that carvacrol has inhibitory activity on MITF protein expression.
3.5. Inhibitory effects of carvacrol on CREB protein phosphorylation It has been reported that activation of CREB increases MITF gene expression and transcription of MITF target gene including tyrosinase (Hearing, 1999; Screaton et al., 2004). To identify the effect of carvacrol on activity of CREB protein in the pathway regulating MITF expression, effect of carvacrol on CREB phosphorylation level was investigated. IBMX-induced CREB activity was reduced by carvacrol in a time-dependent manner (Fig. 5A). In addition, protein level of p-CREB 202
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Fig. 5. Inhibitory effect of carvacrol on CREB protein phosphorylation in IBMX induced B16F10 cells. (A) The maximum expression level of p-CREB protein was measured after time course treatment with IBMX. Protein modulation was measured in a time-dependent manner. (B) p-CREB protein expression was measured after co-treatment with IBMX (100 μM) and carvacrol (100, 200, and 400 μM) or kojic acid (1 mM) for 2 h. Protein expression was determined by Western blotting.
Fig. 6. Inhibitory effect of carvacrol on MITF protein expression associated with melanin production in IBMX-induced B16F10 cells. B16F10 cells were cotreated with 100 μM IBMX and carvacrol (100, 200, and 400 μM) or kojic acid (1 mM) for 2 h. (A) The maximum expression level of MITF protein was measured by treating IBMX for different time durations. In addition, the level of MITF expression was measured at different time points after treatment with carvacrol. (B) MITF protein expression was measured after 2 h of treatment with various concentration of carvacrol. Protein expression was determined by Western blotting.
Fig. 7. Inhibitory effect of carvacrol on tyrosinase and MITF mRNA expression levels in B16F10 cells induced by IBMX. B16F10 mouse melanoma cells were co-treated with carvacrol (100, 200, and 400 μM) or Kojic acid (1 mM) and IBMX (100 μM). (A) After treatment with carvacrol for 24 h, the expression of tyrosinase gene was measured. (B) The mRNA expression level of MITF was measured after treatment with carvacrol or kojic acid and IBMX for 2 h. Expression levels of tyrosinase and MITF mRNA associated with melanin production were measured by real-time PCR. Data are presented as mean ± S.E.M (n = 3). ### p < 0.001 compared to control group, *p < 0.05, **p < 0.01 and *** p < 0.001 compared to IBMX group.
3.7. Inhibitory effect of carvacrol on tyrosinase and TRP-1 mRNA expression in B16F10 cells
kojic acid (Fig. 7A and B). 3.8. Degradation effect of carvacrol on MITF protein through ERK protein phosphorylation
MITF protein is the master regulator of melanocyte function and melanogenesis. Tyrosinase has been shown to be an important enzyme in the final production of melanin (Garcia-Borron, Sanchez-Laorden, & Jimenez-Cervantes, 2005; Vance and Goding, 2004). Our results in previous sections confirmed that carvacrol could inhibit tyrosinase activity and modulate MITF expression in IBMX-induced melanocytes. Here, we further determined whether carvacrol could affect tyrosinase and MITF at mRNA level. Our results confirmed that expression levels of tyrosinase and MITF mRNAs were inhibited by carvacrol in a concentration-dependent. We also found that carvacrol had higher inhibitory activity on the expression of tyrosinase and MITF mRNA than
Previous studies have shown that MITF transcription can be effectively regulated by MAPK. For instance, it has been reported that phosphorylation of MITF at serine 73 residue by ERK can cause MITF ubiquitination and degradation (Kim et al., 2002; Vachtenheim and Borovansky, 2010; Wu et al., 2000). Therefore, in this experiment, ERK phosphorylation levels were examined after treatments with carvacrol and IBMX at different time durations. Simultaneous treatment of carvacrol and IBMX increased MITF expression compared to IBMX alone (up to 2 h). However, phosphorylation of ERK protein was significantly 203
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was induced at the initial stage after treatment. In conclusion, carvacrol could inhibit MITF expression by ERK phosphorylation-dependent manner. It can suppress melanin production. Thus, carvacrol could be potentially useful as an important substance for skin whitening or developing other beauty products by inhibiting melanin production. Acknowledgement This work was supported by a special grant from Konkuk University in 2018.
Fig. 8. Effects of carvacrol on ERK phosphorylation and MITF protein levels in B16F10 cells induced by IBMX. B16F10 mouse melanoma cells were co-treated with carvacrol (400 μM) and IBMX (100 μM) and the expression of each protein was determined over time. Expression of phosphorylated-ERK and MITF protein in B16F10 cells was assessed by Western blotting.
Conflicts of interest The authors declare no conflict of interest.
higher after co-treatment with carvacrol and IBMX compared to that after treatment with IBMX alone (Fig. 8). Also, a strict correlation of ERK and MITF was observed after 1 h of treatment of carvacrol but the correlation of ERK and MITF was reduced after 2 h.
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4. Discussion Tyrosinase, a copper-containing enzyme, is important for controlling the production of melanin (Hearing and Tsukamoto, 1991). Hence, molecules controlling tyrosinase expression and activity have been considered for treatment of hyperpigmentation (Ando et al., 2007). In addition, it has been reported that hyperpigmentation and melanin production are increased as a result of ROS generation (Tomita, Hariu, Kato, & Seiji, 1984). Therefore, this experiment was carried out using carvacrol known to possess antioxidant effect. Results of this study showed that carvacrol possessed anti-melanogenic effect in a dose-dependent manner without exerting significant cytotoxicity to B16F10 mouse melanoma cells. In the present study, we found that tyrosinase expression and its activity in B16F10 mouse melanoma cells were decreased by carvacrol treatment. It has been reported that IBMX can elevate cellular cAMP level by inhibiting phosphodiesterase, a cAMP-degrading enzyme (Beavo et al., 1970). In addition, protein kinase A (PKA), phosphorylates CREB, which binds to cAMP response element in the promoter region of MITF gene (Levy, Khaled, & Fisher, 2006; Tachibana, 2000). Therefore, we investigated the effect of carvacrol on the CREB protein regulation. When cells were treated with different concentrations of carvacrol, CREB protein activity was inhibited by carvacrol in a concentration and time-dependent manner. Furthermore, the effect of carvacrol on the expression level of MITF protein closely related to transcription of CREP protein was investigated. MITF is a transcription factor that is closely related to the development of melanocytes and osteoclasts. As a major transcription factor in melanogenesis, MITF can regulate the expression of several genes, tyrosinase, TRP-1, and TRP-2, which play important roles in converting tyrosine to melanin. Therefore, in this study, expression levels of enzymes involved in oxidization of tyrosine were measured. Carvacrol reduced tyrosinase and TRP-1 enzyme expression in a concentration-dependent manner. Moreover, our results confirmed that carvacrol could significantly reduce MITF protein expression in a concentration and time-dependent manner. The MAPK/ERK pathway plays an important role in proliferation and differentiation of various types of cells (Cowley, Paterson, Kemp, & Marshall, 1994; Leppa, Saffrich, Ansorge, & Bohmann, 1998). In melanocytes, the ERK pathway can control melanogenesis by regulating MITF expression. Several studies have reported that phosphorylation of ERK attenuates a-MSH and IBMX-induced MITF protein expression at posttranslational level (Lakshmikuttyamma, Scott, Decoteau, & Geyer, 2010). Therefore, the effect of carvacrol on ERK phosphorylation levels was investigated. When IBMX-induced B16F10 cells were treated with carvacrol, the expression of MITF was decreased and ERK protein phosphorylation 204
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keratinocytes and on mela- nogenesis in mouse B16 cells. Biological and Pharmaceutical Bulletin, 33, 862–868. Singh, S. K., Sarkar, C., Mallick, S., Saha, B., Bera, R., & Bhadra, R. (2005). Human placental lipid induces melanogenesis through p38 MAPK in B16F10 mouse melanoma. Pigment Cell & Melanoma Research, 18, 113–121. Sokmen, A., Gulluce, M., Akpulat, A. H., Dagerera, D., Tepe, B., Polissiou, M., ... Sahin, F. (2004). The in vitro antimicrobial and antioxidant activities of the essential oils and methanol extracts of endemic Thymus spathulifolius. Food Control, 15, 627–634. Tachibana, M. (2000). MITF: A stream flowing for pigment cells. Pigment Cell & Melanoma Research, 13, 230–240. Tomita, Y., Hariu, A., Kato, C., & Seiji, M. (1984). Radical production during tyrosinase reaction, dopa-melanin formation, and photoirradiation of dopa-melanin. Journal of Investigative Dermatology, 82, 573–576. Vachtenheim, J., & Borovansky, J. (2010). “Transcription physiology” of pigment formation in melanocytes: Central role of MITF. Experimental Dermatology, 19, 617–627. Vance, K. W., & Goding, C. R. (2004). The transcription network regulating melanocyte development and melanoma. Pigment Cell & Melanoma Research, 17, 318–325. Widlund, H. R., & Fisher, D. E. (2003). Microphthalamia-associated transcription factor: A critical regulator of pigment cell development and survival. Oncogene, 22, 3035–3041. Wu, M., Hemesath, T. J., Takemoto, C. M., Horstamann, M. A., Wells, A. G., Price, E. R., ... Fisher, D. E. (2000). c-Kit triggers dual phosphorylations, which couple activation and degradation of the essential melanocyte factor Mi. Genes & Development, 14, 301–312. Xu, W., Gong, L., Haddad, M. M., Bischof, O., Campisi, J., Yeh, E. T., & Medrano, E. E. (2000). Regulation of microphthalmia-associated transcription factor MITF protein levels by association with the ubiquitinconjugating enzyme hUBC9. Experimental Cell Research, 255, 135–143. Ye, Y., Chu, J. H., Wang, H., Xu, H., Chou, G. X., Leung, A. K., ... Yu, Z. L. (2010). Involvement of p38 MAPK signaling pathway in the anti-melanogenic effect of Sanbai-tang, a Chinese herbal formula, in B16 cells. Journal of Ethnopharmacology, 132, 533–535. Yoshida, M., Takahashi, Y., & Inoue, S. (2000). Histamine induces melanogenesis and morphologic changes by protein kinase A activation via H2 receptors in human normal melanocytes. Journal of Investigative Dermatology, 114, 334–342.
Kisk, G., & Roller, S. (2005). Carvacrol and p-cymene inactivate Escherichia coli O157: H7 in apple juice. BMC Microbiology, 5, 36. Lakshmikuttyamma, A., Scott, S. A., Decoteau, J. F., & Geyer, C. R. (2010). Reexpression of epigenetically silenced AML tumor suppressor genes by SUV39H1 inhibition. Oncogene, 29, 576–588. Lampronti, I., Saab, A. M., & Gambari, R. (2006). Antiproliferative activity of essential oils derived from plants belonging to the Magnoliophyta division. International Journal of Oncology, 29, 989–995. Lee, Y. S., Kim, H. K., Lee, K. J., Jeon, H. W., Cui, S., Lee, Y. M., ... Kim, Y. H. (2010). Inhibitory effect of glyceollin isolated from soybean against melanogenesis in B16 melanoma cells. BMB Report, 43, 461–467. Leppa, S., Saffrich, R., Ansorge, W., & Bohmann, D. (1998). Differential regulation of cJun by ERK and JNK during PC12 cell differentiation. The EMBO Journal, 17, 4404–4413. Levy, C., Khaled, M., & Fisher, D. E. (2006). MITF: Master regulator of melanocyte development and melanoma oncogene. Trends in Molecular Medicine, 12, 406–414. Martins, R. R. L., Neves, M. G., Silvestre, A. J. D., Silva, A., & Cavaleiro, J. A. S. (1999). Oxidation of aromatic monoterpenes with hydrogen peroxide catalysed byMn(III) porphyrin complexes. Journal of Molecular Catalysis A: Chemical, 137, 41–47. Nishimura, T., Kometani, T., Okada, S., Ueno, N., & Yamamoto, T. (1995). Inhibitory effects of hydroquinone-alpha-glucoside on melanin synthesis. Europe PMC, 115(8), 626–632. O'Donoghue, J. L. (2006). Hydroquinone and its analogues in dermatology-a risk-benefit viewpoint. Journal of Cosmetic Dermatology, 5, 196–203. Pawelek, J. M. (1991). After DOPA chrome. Pigment Cell & Melanoma Research, 4, 53–62. Plensdorf, S., & Martinez, J. (2009). Common pigmentation disorders. American Family Physician Journal, 79, 109–116. Sato, K., Morita, M., Ichikawa, C., Takahashi, H., & Toriyama, M. (2008). Depigmenting mechanisms of all-trans retinoic acid and retinol on B16 melanoma cells. Bioscience, Biotechnology, and Biochemistry, 72, 2589–2597. Screaton, R. A., Conkright, M. D., Katoh, Y., Best, J. L., Canettieri, G., Jeffries, S., ... Takemori, H. (2004). The CREB coactivator TORC2 functions as a calcium-and cAMPsensitive coincidence detector. Cell, 119, 61–74. Shimoda, N., Mutou, Y., Shimura, N., Tsukimoto, M., Awaya, A., & Kojima, S. (2010). Effect of heterocyclic pyrimidine compounds on UVB-induced cell damage in human
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Inhibitory effect of carvacrol on melanin synthesis via suppression of tyrosinase expression
T
Nam-Joo Jeona,b, Yon-Suk Kima,c, Eun-Kyung Kimd, Xin Donga,b, Jae-Woong Leea,b, ⁎ Jin-Su Parka,b, Woen-Bin Shina,b, Sang-Ho Moonc,d, Byong-Tae Jeone, Pyo-Jam Parka,b,c, a
Department of Biotechnology, Konkuk University, Chungju 27478, Republic of Korea Department of Applied Life Science, Konkuk University, Chungju 27478, Republic of Korea c Medicinal Biological Resources Research Institute, Konkuk University, Chungju 27478, Republic of Korea d Division of Food Bio Science, Konkuk University, Chungju 27478, Republic of Korea e Hasung Corp., Seoul 05832, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Carvacrol Melanogenesis B16F10 cells IBMX
Carvacrol (2-methyl-5-(1-methylethyl)-phenol) compound derived from oils of Thymus vulgaris is a natural member of monoterpene phenol. It has been shown that carvacrol exhibit anti-microbial, anti-inflammatory, anti-oxidant activities. However, no studies have reported its anti-melanogenesis effect. Therefore, the objective of this study was to investigate the anti-melanogenic potential of carvacrol. Results of this study confirmed that carvacrol could regulate protein expression levels of microphthalmia-associated transcription factor [MITF, a protein closely related to transcription of cAMP response element-binding protein (CREB)] and various enzymes involved in melanin synthesis. Moreover, this study provided evidence that carvacrol could regulate the degradation of MITF protein by extracellularly responsive kinases (ERK) phosphorylation. Carvacrol strongly inhibited the synthesis of CREB protein, tyrosinase-related protein 1 (TRP-1), and tyrosinase known to be important enzymes involved in melanogenesis. These results indicate that carvacrol can inhibit melanin synthase by decreasing enzyme expression levels important for melanin synthesis.
1. Introduction Carvacrol is a monoterpenoid compound derived from oils of Thymus vulgaris, Origanum, Carum copticum. It is a natural member of monoterpene phenol (Hussein, El-Bana, Refaat, & El-Naggar, 2017; Kisk & Roller, 2005; Lampronti, Saab, & Gambari, 2006; Martins, Neves, Silvestre, Silva, & Cavaleiro, 1999). Carvacrol has been used for a wide variety of applications in daily life. It is contained in various products such as cosmetics and wide range of foods. In addition, carvacrol has been shown to exhibit anti-microbial, anti-mutagenic, anti-platelet, analgesic, anti-inflammatory, anti-angiogenic, anti-oxidant, anti-elastase, insecticidal, anti-parasitic, cell-protective and anti-tumor activities (Sokmen et al., 2004; Can Baser, 2008). Carvacrol has also been used as an ingredient in various cosmetics. However, no study has reported the whitening effect of carvacrol. Many inflammatory cytokines as melanogens have been reported. Histamine, a ubiquitous inflammatory mediator, is a representative melanogen induced by inflammation (Yoshida, Takahashi, & Inoue, 2000). Studies have shown that reactive oxygen species (ROS)
⁎
generation can activate melanin production (Kim et al., 2014). Based on this knowledge, the objective of this study was to investigate the antimelanogenesis potential of carvacrol. Melanin is synthesized by epidermal melanocytes in the skin. Melanin works in physiological defence to protect skin from ultraviolet radiation. However, continuous UV irradiation induces increased accumulation of melanin, leading to skin hyperpigmentation (Agar and Young, 2005). Melanogenesis is induced by isobutylmethylxanthine (IBMX) and α-melanocyte-stimulating hormone (α-MSH). Binding of αMSH to its receptor, melanocortin-1 receptor (MC1R), induces enhanced expression of microphthalmia-associated transcription factor (MITF) and, cyclic-AMP (cAMP), leading to increased expression of tyrosinase and other melanogenesis-related enzymes such as tyrosinaserelated protein 1 (TRP-1), dopachrome tautomerase (TRP-2). Hyperpigmented skin has an unaesthetic appearance, showing melasma and freckles (Plensdorf and Martinez, 2009). TRP-1, TRP-2, and tyrosinase are three representative enzymes that are important for melanin regulation. Tyrosinase is a copper-containing glycoprotein that is important in melanin synthesis. It can catalyze three different reactions:
Corresponding author at: Department of Biotechnology, Konkuk University, Chungju 27478, Republic of Korea. E-mail address: [email protected] (P.-J. Park).
https://doi.org/10.1016/j.jff.2018.03.043 Received 23 January 2018; Received in revised form 31 March 2018; Accepted 31 March 2018 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
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2.2. Cell culture
the hydroxylation of tyrosine to 3, 4-dihydroxyphenylalanine (DOPA), oxidation of DOPA to dopaquinone and conversion of dopaquinone to dopachrome and then to dihydro-indolizine (DHI) or indole 5,6-quinone-2-carboxylic acid (DHICA) (Ando, Kondoh, Ichihashi, & Hearing, 2007; Chung et al., 2009; Lee et al., 2010; Shimoda et al., 2010). TRP-1 and TRP-2 also play important roles in the synthesis of melanogenesis. TRP-1 catalyzes oxidation of DHICA while TRP-2 catalyzes conversion of dopachrome to DHICA (Sato, Morita, Ichikawa, Takahashi, & Toriyama, 2008). These enzymes are also regulated by a specific transcription factor, MITF (Hasegawa et al., 2010; Widlund & Fisher, 2003). MITF is regulated by a variety of signaling pathways. It is generally known to be controlled by MAPK. Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases that regulate cell differentiation, proliferation, and cellular activities. The MAPK superfamily contains three well-characterized subfamilies: ERKs, p38, and the c-Jun N-terminal kinases (JNKs). MAPKs are known to play a major role in the regulation of melanogenesis (Jiang et al., 2009; Ye et al., 2010). Furthermore, it has been reported that activation of ERK by c-Kit stimulation phosphorylates MITF at its 73rd serine residue. Phosphorylation of MITF at 73rd of serine is followed by MITF ubiquitination and degradation (Hemesath, Price, Takemoto, Badalian, & Fisher, 1998; Xu et al., 2000). Moreover, p38 MAPK pathway activation can increase melanin synthesis (Hirata et al., 2007; Singh et al., 2005). Activation of ERK signaling could also inhibit melanogenesis by inhibiting tyrosinase activity (Jang et al., 2009). There are numerous melanogenesis inhibiting agents, including arbutin, kojic acid, and linoleic acid. Arbutin is a glycosylated hydroquinone extracted from bearberry plant. It has been used as a cure for hyperpigmentation illnesses in the past. Kojic acid and arbutin are extensively used as cosmetic ingredients due to their anti-tyrosinase activity (Nishimura, Kometani, Okada, Ueno, & Yamamoto, 1995). However, some of these agents can cause skin irritation. It has been described that kojic acid causes skin irritation with side effects such as cytotoxicity, dermatitis, and skin cancer (Busca and Ballotti, 2000). Arbutin has been also banned due to its side effects involving exogenous ochronosis and perdurable depigmentation (Draelos, 2007; O'Donoghue, 2006). Therefore, there were huge interests in finding new potential compounds extracted from natural sources without or with very limited side effects. The present study aimed to investigate such a potential melanogenesis inhibiting compound from a natural source. In this study, we examined the anti-melanogenic effect of carvacrol on IBMX-induced melanogenesis in B16F10 mouse melanoma cells. We also explored the underlying molecular mechanisms involved in this process. Many previous studies on cavacrol have mainly determined its antibacterial and antioxidant properties. However, in present study, we performed a melanogenesis study using carvacrol for the first time. Results of this study provide basic research data for developing cosmetics using multifunctional resources
Mouse melanoma cell line B16F10 was obtained from the Korean cell line bank (Seoul, Korea) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 units/mL of penicillin and 100 μg/mL of streptomycin) at 37 °C in a humidified incubator containing 5% CO2. For maintenance, these cells were sub-cultured every two days. 2.3. Determination of cell viability The effect of carvacrol on viability of mouse melanoma cell line B16F10 viability was evaluated using MTT colorimetric assay. Briefly, B16F10 cells were plated into 96-well plates at a density of 5 × 103 cells/well. These cells were treated with various concentrations (25, 50, 100, 200, 400 and 600 μM) of carvacrol and stimulated with or without IBMX (100 μM) at 37 °C for 48 h. After treatments, the medium was replaced by 100 μL of DMEM medium containing MTT (200 µg/mL) in each well followed by incubation at 37 °C for 2 h. After discarding MTT solution, the intracellular formazan product in each well was dissolved in 200 μL DMSO. The absorbance was then measured at 540 nm using a microplate reader (Tecan, Grödig, Austria). Values were calculated in comparison with those of control cells. 2.4. Measurement of melanin content Intracellular melanin contents were determined according to published method (Hosoi, Abe, Suda, & Kuroki, 1985) with slight modifications. Briefly, B16F10 cells were stimulated with IBMX and incubated with carvacrol for 48 h. Cell pellets were harvested and then dissolved in 1 N NaOH containing 10% DMSO at 80 °C for 1 h. Melanin contents were analyzed by measuring absorbance at 475 nm using an ELISA reader (Multiskan GO, Thermo Fisher, Massachsetts, USA). For accurate calculation of melanin contents, each level of melanin was normalized to protein content. 2.5. Tyrosinase activity Tyrosinase activity was determined using published method (Hosoi et al., 1985). Briefly, B16F10 cells were co-treated with IBMX and different concentrations of carvacrol. After 48 h incubation, cells were washed with cold PBS and suspended in a lysis buffer (150 mM NaCl, 10 mM Tris pH 7.5, 5 mM EDTA), and Triton-X 100 1.0% in the presence of protease inhibitors (1 μg/mL leupeptin and 100 μg/mL PMSF) and incubated at 4 °C for 20 min to yield cell lysates. Cell lysates were then centrifuged at 12,000 rpm for 10 min. Protein contents in supernatants were then determined using a protein assay kit (Bio-Rad, Laboratories, Inc., Hercules, CA, USA). Then 90 μL of cell extract was transferred to a 96-well containing 10 μL of L-DOPA (final concentration of 1 mmol/L) prepared in 25 mM phosphate buffer (pH 6.8) and incubated at 37 °C for 20 min. Absorbance was then measured at 475 nm using an ELISA reader (Multiskan GO, Thermo Fisher, MA, USA).
2. Materials and methods 2.1. Chemicals Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Hyclone (Thermo Scientific, Waltham, MA, USA). Phosphorylated-ERK, tyrosinase, TRP-1, and TRP-2 antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). MITF, phosphorylated-CREB and β-actin antibodies were purchased from Cell Signaling Technology Inc. (Denvers, MA, USA). Carvacrol, IBMX, L-DOPA, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
2.6. Western blot analysis B16F10 cells were seeded in 60 mm dishes at a density of 3 × 105 cells/dish. After 24 h of culture, they were co-treated with IBMX (100 μM) and different concentrations (100, 200 and 400 μM) of carvacrol or Kojic acid (1 mM). After incubating at 37 °C for 48 h, cells were subsequently washed with PBS, collected and suspended in a lysis buffer (150 mM NaCl, 10 mM Tris (pH 7.5), 5 mM EDTA, and 1% Triton X-100) containing protease inhibitors (1 μg/mL leupeptin and 100 μg/ mL PMSF). After incubating at 4 °C for 20 min, cell lysates were centrifuged at 12,000 rpm for 10 min. Protein concentration in each cell 200
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lysate (supernatant) were measured using a protein assay kit (Bio-Rad, Laboratories, Inc., Hercules, CA, USA). Proteins (20 μg of B16F10 lysates) were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Buckinghamshire, UK). These membranes were then blocked by Tris-buffered saline-Tween 20 solutions (TBS-T) containing 5% non-fat dry milk, incubated with primary antibodies at 4 °C for 24 h, washed with TBST, and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Bio-Rad, Hercules, CA, USA) at room temperature for 1 h. Protein bands were visualized using an ECL detection kit and a luminescent image analyzer (LAS3000, Fujifilm, Tokyo, Japan). 2.7. Reverse transcription and real-time quantitative PCR Total RNAs were isolated from B16F10 cells using TRIzol Reagent (QIAGEN, CA, USA). Isolated RNAs were reverse-transcribed into cDNAs using cDNA reverse transcription kit (Applied Biosystems, USA). These synthesized cDNAs were subjected to real-time quantitative PCR on a Light Cycler® (Roche, Switzerland) using SYBR qPCR pre-mix (Dyne Bio, SN, KOR). PCR reactions were performed with gene-specific primers for MITF, tyrosinase and endogenous reference 36B4 as follows (forward and reverse, respectively): Tyrosinase(F), 5′-CCT CCT GGC AGA TCA TTT GT-3′, Tyrosinase(R), 5′-GGC AAA TCC TTC CAG TGT GT-3′, 36B4(F), 5′-TGG GCT CCA AGC AGA TGC-3′, 36B4(R), 5′-GGC TTC GCT GGC TCC CAC-3′, MITF(F), 5′-ATG CTG GAA ATG CTA GAA TAC AGT-3′, and MITF(R), 5′-ATC ATC CAT CTG CAT GCA C-3′. Data were analyzed using the 2-ΔΔCT method. Data are expressed as fold change of gene expression. 2.8. Statistical analysis Data are expressed as mean ± standard deviation for triplicate determinations. Analysis of variance (ANOVA) together with Tukey’s test and Dunnett’s test (GraphPad Prism 5) were conducted to identify significant differences between samples. Statistical significance was considered when p values were < 0.05 (p < 0.05). 3. Results Fig. 1. Effects of carvacrol on the cell viability of B16F10 mouse melanoma cells. (A) Cell viability was analyzed by MTT assay. B16F10 cells were treated with different carvacrol concentrations (25, 50, 100, 200, 400, and 600 μM) for 48 h. (B) Cells were treated with carvacrol and IBMX (100 μM) simultaneously. (C) Chemical structure of carvacrol. Each bar represents the mean ± S.E.M of three independent experiments.
3.1. Cytotoxicity of carvacrol to B16F10 mouse melanoma cells To investigate whether carvacrol had cytotoxic effect on B16F10 cells, B16F10 mouse melanoma cells were treated with various doses of carvacrol in the absence or presence of IBMX. Cytotoxic effect of carvacrol on B16F10 cells was measured by MTT assay. MTT assay results revealed that carvacrol had no significant effect on cell viability at concentrations below 400 μM in the presence or absence of IBMX (Fig. 1A and B). Therefore, concentrations of 100, 200, and 400 μM were selected as carvacrol concentration for further experiments. Fig. 1C shows chemical structure of carvacrol.
3.3. Inhibitory effect of carvacrol on tyrosinase enzyme activity in B16F10 mouse melanoma cells Tyrosinase is encoded by albino locus of mouse. It is a key enzyme responsible for the formation of melanin pigment in animals (Hearing and Tsukamoto, 1991; Pawelek, 1991). Therefore, we treated B16F10 mouse melanoma cells with various doses of carvacrol or kojic acid (1 mM) in the absence and presence of IBMX and then measured tyrosinase activity. Results indicated that carvacrol inhibited tyrosinase enzymatic activity in IBMX-induced melanocytes in a concentrationdependent manner. Moreover, carvacrol inhibited tyrosinase enzyme activity significantly more than kojic acid (Fig. 3).
3.2. Inhibitory effect of carvacrol on melanin synthesis in B16F10 mouse melanoma cells IBMX is known to increase melanin production and regulate melanogenesis pathway (Busca and Ballotti, 2000). Therefore, we investigated melanin inhibitory effect of carvacrol in IBMX-induced B16F10 mouse melanoma cells. We treated B16F10 mouse melanoma cells with various doses of carvacrol or kojic acid (1 mM) in the absence or presence of IBMX. Carvacrol inhibited melanin production in melanocytes significantly more than kojic acid in a concentration-dependent manner. In addition, the color of the cell pellet was changed in a concentration-dependent manner due to melanin production (Fig. 2A and B).
3.4. Inhibitory effect of carvacrol on the expression of melanin-related enzymes in B16F10 cells Previous studies have shown that tyrosinase, TRP1 and TRP2 are involved in the melanogenesis pathway to mediate important responses 201
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Fig. 3. Inhibitory effect of carvacrol on tyrosinase activity in melanocytes. B16F10 mouse melanoma cells were co-treated with IBMX (100 μM) and carvacrol (100, 200, and 400 μM) or kojic acid (1 mM). After 48 h incubation, absorbance was measured at 475 nm. Data are represented as mean ± S.E.M (n = 3). ###p < 0.001 compared to control group, ***p < 0.001 compared to IBMX group.
Fig. 4. Effects of carvacrol on the expression levels of tyrosinase, TRP-1 and TRP-2 in IBMX-induced B16F10 cells. B16F10 mouse melanoma cells were treated with different doses of carvacrol (100, 200, and 400 μM) or kojic acid (1 mM) in the absence or presence of IBMX for 48 h. After harvesting cells, cell lysates were prepared and subjected to Western blot analysis with anti-tyrosinase, anti-TRP-1, and anti-TRP-2 antibody.
Fig. 2. Inhibitory effect of carvacrol on melanin production in melanocytes. B16F10 mouse melanoma cells were co-treated with IBMX (100 μM) and carvacrol (100, 200, and 400 μM) or kojic acid (1 mM). After 48 h incubation, absorbance was measured at 475 nm. (A) Melanin contents expressed as fold values. (B) Photographs of cell culture dishes showing color changes after different sample treatments. Data are represented as mean ± S.E.M (n = 3). ### p < 0.001 compared to control group, ***p < 0.001 compared to IBMX group.
was down-regulated by carvacrol in a dose-dependent manner (Fig. 5B). The activity of CREB was inhibited more by carvacrol at 400 µM than by kojic acid. These results indicate that carvacrol can effectively inhibit phosphorylation of CREB protein.
to melanogenesis (Jin, Oh, Hyun, Kwon, & Kim, 2014). Therefore, effects of carvacrol on tyrosinase, TRP1 and TRP2 protein expression levels were analyzed by Western blotting. Our results showed that carvacrol decreased TRP1 and tyrosinase protein expression levels in IBMX-stimulated B16F10 cells in a concentration-dependent manner. However, carvacrol had no effect on the expression of TRP-2 protein level (Fig. 4).
3.6. Inhibitory effect of carvacrol on MITF protein expression To understand whether transcriptional regulation of tyrosinase and TRP-1 mRNA expressions was involved in the effect of carvacrol, the effect of carvacrol on the expression of MITF, a major transcription factor for tyrosinase and TRP-1, was investigated. After treatment with carvacrol, protein levels of MITF were down-regulated in a dose-dependent manner (Fig. 6B). Carvacrol treatment also decreased IBMXinduced MITF protein expression in a time-dependent manner (Fig. 6A). These results indicate that carvacrol has inhibitory activity on MITF protein expression.
3.5. Inhibitory effects of carvacrol on CREB protein phosphorylation It has been reported that activation of CREB increases MITF gene expression and transcription of MITF target gene including tyrosinase (Hearing, 1999; Screaton et al., 2004). To identify the effect of carvacrol on activity of CREB protein in the pathway regulating MITF expression, effect of carvacrol on CREB phosphorylation level was investigated. IBMX-induced CREB activity was reduced by carvacrol in a time-dependent manner (Fig. 5A). In addition, protein level of p-CREB 202
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Fig. 5. Inhibitory effect of carvacrol on CREB protein phosphorylation in IBMX induced B16F10 cells. (A) The maximum expression level of p-CREB protein was measured after time course treatment with IBMX. Protein modulation was measured in a time-dependent manner. (B) p-CREB protein expression was measured after co-treatment with IBMX (100 μM) and carvacrol (100, 200, and 400 μM) or kojic acid (1 mM) for 2 h. Protein expression was determined by Western blotting.
Fig. 6. Inhibitory effect of carvacrol on MITF protein expression associated with melanin production in IBMX-induced B16F10 cells. B16F10 cells were cotreated with 100 μM IBMX and carvacrol (100, 200, and 400 μM) or kojic acid (1 mM) for 2 h. (A) The maximum expression level of MITF protein was measured by treating IBMX for different time durations. In addition, the level of MITF expression was measured at different time points after treatment with carvacrol. (B) MITF protein expression was measured after 2 h of treatment with various concentration of carvacrol. Protein expression was determined by Western blotting.
Fig. 7. Inhibitory effect of carvacrol on tyrosinase and MITF mRNA expression levels in B16F10 cells induced by IBMX. B16F10 mouse melanoma cells were co-treated with carvacrol (100, 200, and 400 μM) or Kojic acid (1 mM) and IBMX (100 μM). (A) After treatment with carvacrol for 24 h, the expression of tyrosinase gene was measured. (B) The mRNA expression level of MITF was measured after treatment with carvacrol or kojic acid and IBMX for 2 h. Expression levels of tyrosinase and MITF mRNA associated with melanin production were measured by real-time PCR. Data are presented as mean ± S.E.M (n = 3). ### p < 0.001 compared to control group, *p < 0.05, **p < 0.01 and *** p < 0.001 compared to IBMX group.
3.7. Inhibitory effect of carvacrol on tyrosinase and TRP-1 mRNA expression in B16F10 cells
kojic acid (Fig. 7A and B). 3.8. Degradation effect of carvacrol on MITF protein through ERK protein phosphorylation
MITF protein is the master regulator of melanocyte function and melanogenesis. Tyrosinase has been shown to be an important enzyme in the final production of melanin (Garcia-Borron, Sanchez-Laorden, & Jimenez-Cervantes, 2005; Vance and Goding, 2004). Our results in previous sections confirmed that carvacrol could inhibit tyrosinase activity and modulate MITF expression in IBMX-induced melanocytes. Here, we further determined whether carvacrol could affect tyrosinase and MITF at mRNA level. Our results confirmed that expression levels of tyrosinase and MITF mRNAs were inhibited by carvacrol in a concentration-dependent. We also found that carvacrol had higher inhibitory activity on the expression of tyrosinase and MITF mRNA than
Previous studies have shown that MITF transcription can be effectively regulated by MAPK. For instance, it has been reported that phosphorylation of MITF at serine 73 residue by ERK can cause MITF ubiquitination and degradation (Kim et al., 2002; Vachtenheim and Borovansky, 2010; Wu et al., 2000). Therefore, in this experiment, ERK phosphorylation levels were examined after treatments with carvacrol and IBMX at different time durations. Simultaneous treatment of carvacrol and IBMX increased MITF expression compared to IBMX alone (up to 2 h). However, phosphorylation of ERK protein was significantly 203
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was induced at the initial stage after treatment. In conclusion, carvacrol could inhibit MITF expression by ERK phosphorylation-dependent manner. It can suppress melanin production. Thus, carvacrol could be potentially useful as an important substance for skin whitening or developing other beauty products by inhibiting melanin production. Acknowledgement This work was supported by a special grant from Konkuk University in 2018.
Fig. 8. Effects of carvacrol on ERK phosphorylation and MITF protein levels in B16F10 cells induced by IBMX. B16F10 mouse melanoma cells were co-treated with carvacrol (400 μM) and IBMX (100 μM) and the expression of each protein was determined over time. Expression of phosphorylated-ERK and MITF protein in B16F10 cells was assessed by Western blotting.
Conflicts of interest The authors declare no conflict of interest.
higher after co-treatment with carvacrol and IBMX compared to that after treatment with IBMX alone (Fig. 8). Also, a strict correlation of ERK and MITF was observed after 1 h of treatment of carvacrol but the correlation of ERK and MITF was reduced after 2 h.
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4. Discussion Tyrosinase, a copper-containing enzyme, is important for controlling the production of melanin (Hearing and Tsukamoto, 1991). Hence, molecules controlling tyrosinase expression and activity have been considered for treatment of hyperpigmentation (Ando et al., 2007). In addition, it has been reported that hyperpigmentation and melanin production are increased as a result of ROS generation (Tomita, Hariu, Kato, & Seiji, 1984). Therefore, this experiment was carried out using carvacrol known to possess antioxidant effect. Results of this study showed that carvacrol possessed anti-melanogenic effect in a dose-dependent manner without exerting significant cytotoxicity to B16F10 mouse melanoma cells. In the present study, we found that tyrosinase expression and its activity in B16F10 mouse melanoma cells were decreased by carvacrol treatment. It has been reported that IBMX can elevate cellular cAMP level by inhibiting phosphodiesterase, a cAMP-degrading enzyme (Beavo et al., 1970). In addition, protein kinase A (PKA), phosphorylates CREB, which binds to cAMP response element in the promoter region of MITF gene (Levy, Khaled, & Fisher, 2006; Tachibana, 2000). Therefore, we investigated the effect of carvacrol on the CREB protein regulation. When cells were treated with different concentrations of carvacrol, CREB protein activity was inhibited by carvacrol in a concentration and time-dependent manner. Furthermore, the effect of carvacrol on the expression level of MITF protein closely related to transcription of CREP protein was investigated. MITF is a transcription factor that is closely related to the development of melanocytes and osteoclasts. As a major transcription factor in melanogenesis, MITF can regulate the expression of several genes, tyrosinase, TRP-1, and TRP-2, which play important roles in converting tyrosine to melanin. Therefore, in this study, expression levels of enzymes involved in oxidization of tyrosine were measured. Carvacrol reduced tyrosinase and TRP-1 enzyme expression in a concentration-dependent manner. Moreover, our results confirmed that carvacrol could significantly reduce MITF protein expression in a concentration and time-dependent manner. The MAPK/ERK pathway plays an important role in proliferation and differentiation of various types of cells (Cowley, Paterson, Kemp, & Marshall, 1994; Leppa, Saffrich, Ansorge, & Bohmann, 1998). In melanocytes, the ERK pathway can control melanogenesis by regulating MITF expression. Several studies have reported that phosphorylation of ERK attenuates a-MSH and IBMX-induced MITF protein expression at posttranslational level (Lakshmikuttyamma, Scott, Decoteau, & Geyer, 2010). Therefore, the effect of carvacrol on ERK phosphorylation levels was investigated. When IBMX-induced B16F10 cells were treated with carvacrol, the expression of MITF was decreased and ERK protein phosphorylation 204
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