J Appl Phycol DOI 10.1007/s10811-017-1120-8

Ethanolic extract from Sargassum serratifolium attenuates hyperpigmentation through CREB/ERK signaling pathways in α-MSH-stimulated B16F10 melanoma cells Mohammed Shariful Azam 1 & Eun-Ji Joung 1 & Jinkyung Choi 2 & Hyeung-Rak Kim 1

Received: 1 October 2016 / Revised and accepted: 5 March 2017 # Springer Science+Business Media Dordrecht 2017

Abstract Hyperpigmentation is an increased deposition of melanin in the skin. The effects of ethanolic extract from the brown alga Sargassum serratifolium (ESS) on melanogenic protein expressions in B16F10 mouse melanoma cells were examined to elucidate its hypopigmenting properties. ESS remarkably reduced melanin synthesis in α-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. Western blot analysis revealed that ESS attenuated the expression of melanogenic enzymes, tyrosinase, and tyrosinase-related protein 1, by cyclic adenosine monophosphate (cAMP)-responsive element-binding protein (CREB)-mediated downregulation of microphthalmiaassociated transcription factor (MITF). ESS inhibited accumulation of cellular cAMP that leads to inhibition of CREB phosphorylation. Moreover, ESS activated extracellular signal-regulated kinase (ERK), but not Akt and other mitogen-activated protein kinases, which is responsible for posttranslational downregulation of MITF. Therefore, ESS attenuated α-MSH-stimulated hyperpigmentation in B16F10 cells through modulation of CREB/ ERK signaling pathways. Three antimelanogenic compounds such as sargahydroquinoic acid, sargachromenol, and sargaquinoic acid were identified in ESS depending on inhibition of melanin synthesis. These findings suggest that ESS could be a potential agent in the treatment of hyperpigmentation-related skin disorders.

* Hyeung-Rak Kim [email protected] 1

Department of Food Science and Nutrition, Pukyong National University, 45, Yongso-Ro, Nam-Gu, Busan 48513, Republic of Korea

2

Department of Foodservice Management, Woosong University, Daejeon 34606, Republic of Korea

Keywords B16F10 melanoma cells . CREB . ERK . MITF . Sargassum serratifolium . Phaeophyceae . Tyrosinase

Introduction Melanocytes, at the stratum basale of epidermis, synthesize melanin pigments which are vital factors in determining skin color. Melanin synthesis in melanocytes is related to various stimuli including UV irradiation, α-melanocyte stimulating hormone (α-MSH), or cyclic adenosine monophosphate (cAMP)-enhancing agents (Busca and Ballotti 2000; Yamaguchi and Hearing 2009). These melanin-inducing factors activate the classical cAMP signaling pathway in which the most vital target is the microphthalmia-associated transcription factor (MITF), the regulator of melanogenic enzymes. When α-MSH binds to G-protein-coupled melanocortin-1 receptor, it causes the activation of adenylate cyclase and the elevation of cAMP level activates protein kinase A (PKA). Activated catalytic subunit of PKA is translocated to the nucleus where it phosphorylates and activates cAMP-responsive element-binding protein (CREB). Phosphorylated CREB binds to the cAMP-response element sequence of MITF promoter and upregulates the transcription of MITF and thereby regulate the expression of melanogenic proteins such as tyrosinase, tyrosinase-related protein 1 (TRP1), and TRP2 (Bertolotto et al. 1996; Busca and Ballotti 2000). Dysregulation of this mechanism may be responsible for pigmentation-related disorders including hyperpigmentation (Lehraiki et al. 2014). Several studies also reported that posttranslational downregulation of MITF is caused by the activation of extracellular signal-regulated kinase (ERK) (Song et al. 2015) and phosphatidylinositol 3kinase (PI3K)/Akt is associated with hypopigmentation (Lee et al. 2012). Moreover, other mitogen-activated protein

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kinases (MAPKs) including c-Jun N-terminal kinase (JNK) (Bu et al. 2008) and p38 MAPK (Wu et al. 2011) regulate melanogenesis via MITF phosphorylation. Hyperpigmentation or abnormal concentrations of melanin in different parts of the body are considered as skin problem or diseases like melasma, leukoplakia, freckles, moles, and lentigo (Han et al. 2015; Kim et al. 2015). Besides, some drug-induced causes, cosmetics, hormonal factors, and some inflammation cause hyperpigmentation (Lehraiki et al. 2014). Various commercial antimelanogenic compounds are usually applied to treat these skin pigmentary disorders. Currently used antimelanogenic agents such as kojic acid and arbutin have little effect, and sometimes, they exhibit severe side effects (Takizawa et al. 2004; Solano et al. 2006). Therefore, research interest is growing to discover nontoxic compounds from natural sources having remarkable skin whitening effects. In recent years, marine natural resources have become promising sources for the development of drugs, functional foods, and cosmeceuticals because of their diversified composition and safety profiles. Brown algae are known to be a rich source of health beneficial compounds, including pigments, phlorotannins, and meroterpinoids (Alghazwi et al. 2016; Wei et al. 2016). As meroterpinoids, sargahydroquinoic acid (SHQA) and sargaquinoic acid (SQA) isolated from Sargassum yezoense and sargahydroquinoic acid inhibited age-related inflammation by suppressing AP-1 and NF-κB pathway (Jeon et al. 2014). Sargachromenol (SCM) from Sargassum horneri suppressed the expression of matrix metalloproteinases in UVA-irradiated dermal fibroblasts (Kim et al. 2012). Recently, we found that Sargassum serratifolium contains large amount of meroterpenoids. Thus, we hypothesized that ethanolic extract from S. serratifolium (ESS) would be a promising skin whitening agent that downregulated the expression of melanogenic proteins like MITF and tyrosinase. In this study, we investigated the effect of ESS on the melanin synthesis and its underlying mechanism using α-MSH-stimulated B16F10 mouse melanoma cells.

(Ser133), Akt1, ERK1/2, JNK, p-JNK, PARP-1 and β-actin, and HRP-conjugated secondary antibodies were from Santa Cruz Biotechnology Inc. (USA). The primary antibodies for p-Akt (Ser473), p-ERK1/2, p38 MAPK, and p-p38 MAPK were from Cell Signaling Technology Inc. (USA). ERKspecific inhibitor PD98059 was from Abcam (USA). Dimethyl sulfoxide (DMSO), α-MSH, and arbutin were from Sigma-Aldrich Co. (USA). Preparation of ESS and isolation of chemical compounds Sargassum serratifolium was harvested from coastal areas of Busan, Republic of Korea, in May 2015. The specimen identification was confirmed by an algal taxonomist (C.G. Choi), at the Department of Ecological Engineering, Pukyong National University, Busan, Republic of Korea. Voucher specimen was deposited in Dr. Choi’s laboratory (CCG51814). Collected sample was air-dried and ground. One and half kilogram of dried sample was extracted twice with 70% ethanol (6 L each time) at 70 °C for 3 h. The combined extract was filtered with ultrafiltration unit (MWCO, 50 kDa) and concentrated until lipophilic fraction was separated with salt water. Lipophilic fraction was concentrated by a rotary vacuum evaporator (Eyela N3010) at 50 °C until water content is less than 5.5% and used for this study. From 1.5 kg of dried algae, 120 g of the ethanolic extract of S. serratifolium (ESS) was obtained. SHQA, SCM, and SQA were isolated according to the method described previously (Joung et al. 2017). Cell culture and treatment with ESS

Materials and methods

B16F10 mouse melanoma cells were cultured in high-glucose DMEM, supplemented with 10% FBS, in a humidified atmosphere containing 5% CO2 in air at 37 °C. For stimulation of melanin synthesis, cells were treated with 1.0 μmol L−1 αMSH. B16F10 cells were pretreated with different concentrations of ESS for 1 h before stimulation with α-MSH. All cellbased assays were conducted at least three times.

Materials

Cell viability assay

Dulbecco’s Modified Eagle’s Medium (DMEM) was from Welgene Inc. (South Korea). Fetal bovine serum (FBS), trypsin-EDTA, and penicillin-streptomycin solution (100×) were from GenDEPOT Inc. (USA). CellTiter 96 AQueous One Solution Cell Proliferation assay kit was from Promega (USA). BCA protein assay kit and enhanced chemiluminescence (ECL) detection kit were from Thermo Scientific (USA). cAMP Parameter Assay Kit was from R&D Systems, Inc. (USA). Nitrocellulose membrane was from GE Healthcare Bio-Science (USA). The primary antibodies for tyrosinase, TRP1, TRP2, MITF, CREB-1, p-CREB-1

The effect of ESS on cell viability was determined using MTS assay with a CellTiter 96 AQueous One Solution Cell Proliferation assay kit according to the manufacturer’s instructions. B16F10 cells were seeded in 96-well plates at a density of 1 × 104 cells per well and incubated for 24 h in DMEM containing 10% FBS. Then, cells were treated with different concentrations of ESS for 24 h in DMEM containing 2% FBS. The culture medium was discarded and replaced with 95 μL of FBS-free DMEM and 5 μL of MTS solution. After 1 h of incubation, the absorbance was measured at 490 nm by a microplate reader.

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Measurement of melanin content

Statistical analysis

The melanin content was determined in accordance with the procedure described previously with some modifications (Hosoi et al. 1985). Briefly, the B16F10 cells were seeded at a density of 2 × 104 cells per well in a 24-well culture plate and incubated for 24 h in DMEM supplemented with 10% FBS. Cells were pretreated with various concentrations of ESS for 1 h and then exposed to 1.0 μmol L−1 of α-MSH for 72 h. The cells washed with phosphate-buffered saline (PBS) were dissolved in 1 N NaOH containing 10% DMSO by boiling at 80 °C for 30 min. The cell lysates were centrifuged for 10 min at 14,000 rpm, and absorbance of the supernatant was measured spectrophotometrically at 405 nm. The value of each measurement was expressed as percentage changes from the α-MSH-treated cells. Arbutin (500 μmol L−1) was used as a positive control.

The results are presented as mean ± standard deviation (SD) of three separate experiments. One-way analysis of variance (ANOVA) followed by Duncan’s multiple range tests were performed using statistical analysis software SPSS (USA). The significance of differences was defined as p < 0.05 level.

Intracellular cAMP assay Intracellular cAMP was quantified using cAMP ELISA kit according to the manufacturer’s instruction. Briefly, B16F10 cells were pretreated with ESS for 1 h and then stimulated with α-MSH for 6 h. Cells were lysed using the lysis buffer provided in the kit. Cell lysis supernatants were used to quantify cAMP. The streptavidin-coated 96-well plate was incubated for 1 h with biotinylated cAMP antibody. Excess antibody was removed by repeated washing with wash buffer. cAMP conjugate, standards, and cell lysates were added to appropriate wells and incubated for 2 h on an orbital shaker at room temperature. The washing process was repeated and then incubated with substrate solution for 30 min at room temperature. After adding stop solution to each well, the concentration of cAMP was quantified by measuring the absorbance at 450 nm using a microplate reader.

Western blot analysis After treatment of B16F10 cells with α-MSH (1.0 μmol L−1) in the presence and absence of various concentrations of ESS for various time periods, the cells were washed with PBS and lysed with lysis buffer containing protease and phosphatase inhibitors. Aliquots of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and transferred onto a nitrocellulose membrane. The membrane was blocked with 10% nonfat milk powder (w/v) in TBST (0.1 mol L−1 Tris-HCl, pH 7.5, 1.5 mol L−1 NaCl, 1% Tween 20) for 1 to 2 h and incubated for 18 to 20 h with primary antibody in TBST. The blots were treated with secondary antibody in TBST for 1 h. The immunoblots were detected using an ECL detection kit.

Results ESS attenuated melanin synthesis in α-MSH-stimulated B16F10 melanoma cells The effect of ESS on cell viability was determined using MTS assay. B16F10 cells were treated at a concentration of 0– 10 μg mL−1of ESS for 24 h. Viability of B16F10 was not affected by ESS up to 10 μg mL −1 of concentration (Fig. 1a). Thus, we used 0–5 μg mL−1 of ESS in further experiments. To investigate the effect of ESS on melanin synthesis in B16F10 cells, cells were pretreated with various concentrations of ESS (0–5 μg mL−1) for 1 h. After stimulation with α-MSH for 72 h, the intracellular production of melanin was dose-dependently inhibited (EC50 2.72 ± 0.17 μg mL−1) by ESS (Fig. 1b). Inhibition of α-MSH-stimulated hyperpigmentation by ESS was also evident by extracellular melanin content (Fig. 1c) and cell pellets after 72 h of ESS treatment (Fig. 1d). ESS suppressed the α-MSH-stimulated expression of melanogenic enzymes To assess the expression levels of melanogenic enzymes (tyrosinase, TRP1 and TRP2), whole cell lysates were subjected to Western blot analysis after treatment with ESS. As shown in Fig. 2a, ESS treatment dose-dependently attenuated the expression of both tyrosinase and TRP1, while that of TRP2 was not affected by ESS. These results suggest that ESS can attenuate α-MSH-stimulated hyperpigmentation in B16F10 cells. ESS downregulated MITF expression through inhibition of CREB activation As ESS attenuated α-MSH-induced expression of melanogenic proteins, we tested whether ESS affects their transcription factor, MITF, and its upstream signal molecule, CREB. Stimulation of B16F10 cells with α-MSH resulted in increased expression of MITF that was significantly downregulated in a dose-dependent manner by ESS treatment (p < 0.05, Fig. 2b). We also found that after stimulation with α-MSH, B16F10 cells markedly increased the phosphorylation of CREB. However, ESS

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Fig. 1 Effects of ESS on cell viability and melanin content in B16F10 mouse melanoma cells. Cells were treated with 1–10 μg mL−1 of ESS for 24 h, and viabilities were determined using MTS assay kit (a). Cells were pretreated with ESS (1–5 μg mL−1) for 1 h and then exposed to α-MSH for 72 h in the presence of ESS. Relative melanin contents were measured as described in BMaterials and methods^ (b). Photograph of culture

medium representing extracellular melanin content after 72 h of ESS treatments (c). Photograph of cell pellet after 72 h of ESS treatments (d). Data show mean ± standard deviation (SD) of three independent experiments. Different letters indicate treatment groups with significant (P < 0.05) difference (b)

significantly suppressed PKA-dependent CREB phosphorylation in α-MSH-stimulated B16F10 cells in a dose-dependent manner that resulted in the downregulation of MITF expression (p < 0.05, Fig. 2b). Moreover, anti-p-CREB-1 (Ser133) antibody can also detect phosphorylated form of activating transcription factor-1 (pATF-1) (Fig. 2b) as it belongs to the same CREB/ATF family of transcription factor, and its structure and function are closely related to p-CREB (Hummler et al. 1994).

ESS attenuated MITF through ERK signaling pathway

ESS suppressed intracellular cAMP cAMP plays an important role in PKA-dependent CREB activation in melanogenesis. Therefore, we examined the effect of ESS on intracellular cAMP level. As shown in Fig. 3, upon 6 h treatment, α-MSH significantly enhanced cAMP level in B16F10 cells (p < 0.05). ESS suppressed intracellular cAMP level in α-MSH-stimulated B16F10 cells in a dose-dependent manner. This result implies that the suppression of MITF by ESS was caused by reduced cAMP in α-MSH-stimulated B16F10 cells.

To understand the molecular mechanisms on the regulation of MITF by ESS, we determined the phosphorylation level of MAPKs and Akt in α-MSH-stimulated B16F10 cells upon treatment with different concentrations of ESS. We found that 2.5 and 5.0 μg mL−1 of ESS significantly phosphorylated ERK (p < 0.05, Fig. 4a) that might be responsible for decreased melanin synthesis via posttranslational degradation of MITF. However, ESS has no effect on the phosphorylation of Akt, JNK, and p38 MAPK. Treatment with ERK inhibitor, PD98059 (15 μmol L−1), remarkably restored the expression of tyrosinase (Fig. 4b). These data further confirmed the involvement of ERK in the hypopigmenting mechanism of ESS in α-MSH-stimulated B16F10 cells. Identification of hypopigmenting compounds in EES The elution profile of ESS is presenting separated four peaks at different retention times (Fig. 5). With repeated chromatography, we isolated SHQA, SCM, and SQA from S. serratifolium.

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Fig. 2 Effects of ESS on expression of melanogenic proteins and activation of their transcription factor in B16F10 cells. Cells were pretreated with ESS for 1 h and stimulated with α-MSH for 48 h in the presence of ESS. Whole cell lysates were subjected to Western blot analysis with corresponding antibodies. The densitograph is representing relative band density for tyrosinase, TRP1 and TRP2 normalized by β-actin (a). Cells were pretreated with ESS for 1 h and stimulated with

α-MSH for 6 h in the presence of ESS. Nuclear extract of cells was subjected to Western blot analysis with corresponding antibodies. The densitograph represents relative band density for MITF normalized by poly(ADP-ribose) polymerase-1 (PARP-1) and p-CREB normalized by CREB (b). Data show mean ± SD of three independent experiments. Different letters indicate treatment groups with significant (P < 0.05) difference

Identification of three compounds was confirmed by the retention time of our authentic standards (Gwon et al. 2015; Joung et al. 2017). The content of SHQA, SCM, and SQA was estimated to be 18.8 ± 2.1, 3.12 ± 0.36, and 0.95 ± 0.10 g, respectively, in 100 g of ESS. EC50 values of SHQA, SCM, and SQA for the inhibition of melanin synthesis were estimated to be 2.21 ± 0.47, 3.51 ± 0.21, and 1.97 ± 0.15 μmol L−1, respectively (Table 1).

Discussion

Fig. 3 The effect of ESS on intracellular cAMP levels in B16F10 cells. Cells were pretreated with ESS for 1 h and stimulated with α-MSH for 6 h in the presence and absence of ESS. Cellular cAMP levels were quantified using a cAMP ELISA kit according to the manufacturer’s instruction. Data show mean ± SD of three independent experiments. Different letters indicate treatment groups with significant (P < 0.05) difference

Recently, researchers have focused on marine natural resources in the search for safe and effective bioactive compounds having antimelanogenic activity. Among the marine compounds, dioxinodehydroeckol (Lee et al. 2012) and fucoidan (Song et al. 2015) isolated from brown algae Ecklonia stolonifera and Fucus vesiculosus, respectively, have been reported for antimelanogenic activity. Other marine resources like a peptide from fermented microalga Pavlova lutheri (Oh et al. 2015) and geoditin A isolated from marine sponge Geodia japonica (Cheung et al. 2012) have also been reported for antimelanogenic activity. In this study, we investigated the ethanolic extract from S. serratifolium to find out new bioactive natural product from marine sources for the treatment of visible skin hyperpigmentation. We identified three compounds from ESS such as SHQA, SCM, and SQA (Fig. 5). Isolated three compounds exhibited hypopigmenting properties in α-MSH-stimulated B16F10 cells that could be responsible for antimelanogenic activity of ESS (Table 1). Overproduction of melanin leads to skin pigmentation disorders that affect the quality of life. Therefore, screening effective, stable, safe, and affordable antimelanogenic agent is of great importance for the natural cosmotic agent for skin whitening. In our study, we demonstrated 5.0 μg mL−1of ESS efficiently attenuated melanin synthesis in α-MSH-

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Fig. 4 Effects of ESS on activation of Akt and MAPKs in B16F10 cells. Cells were pretreated with ESS for 1 h and stimulated with α-MSH for 4 h in the presence and absence of ESS. Whole cell lysates were subjected to Western blot analysis with corresponding antibodies. The densitograph is representing relative band density for p-ERK normalized by ERK, p-Akt normalized by Akt, p-p38 MAPK normalized by p38 MAPK, and p-JNK normalized by JNK (a). Cells were pretreated with ESS for 1 h in the

presence and absence of PD98059 and stimulated with α-MSH for 48 h. Whole cell lysates were subjected to Western blot analysis with anti-tyrosinase antibody. The densitograph represents relative band density for tyrosinase normalized by β-actin (b). Data show mean ± SD of three independent experiments. Different letters indicate treatment groups with significant (P < 0.05) difference

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J Appl Phycol Table 1 Hypopigmenting activity of ESS and isolated active components on α-MSHstimulated B16F10 mouse melanoma cells

Compounds

EC50

ESS (μg mL−1)

2.72 ± 0.17 2.21 ± 0.47 3.51 ± 0.21 1.97 ± 0.15

SHQA (μmol L−1) SCM (μmol L−1) SQA (μmol L−1)

EC50 was measured by inhibitory activity of melanin production in α-MSHstimulated B16F10 cells. Values are mean ± SD, n = 3

stimulated B16F10 cells which is more effective than that of 500 μmol L−1 of arbutin (Fig. 1b). Downregulation of tyrosinase enzyme expression is an important approach to inhibition of melanogenesis as these enzymes catalyzes melanin biosynthesis. In this study, marked suppression of MITF expression by ESS dose-dependently downregulated the expression of tyrosinase and TRP1, but not the TRP2 (Fig. 2a). Downregulation of tyrosinase and TRP1 should be in line with suppressed expression of MITF as it is the master transcription factor for these melanogenic enzymes (Cheung et al. 2012). A previous study reported that MITF sufficiently regulates the production of tyrosinase and TRP1 genes but not the TRP2 gene (Yasumoto et al. 1997). This finding is also consistent with the previous study that reported downregulation of tyrosinase and TRP1 expressions via inhibition of MITF

Fig. 6 The proposed scheme of hypopigmenting action mechanism of ESS in α-MSH-stimulated B16F10 cells, where downwards arrow indicates decrease and upwards arrow indicates increase. The scheme represents downregulation of MITF by ESS via inhibition of cAMPdependent CREB activation and through activation of ERK. These mechanisms ultimately downregulated the expression of tyrosinase/ TRP1 genes leading to decreased melanin synthesis

expression, but no change in TRP2 expression (Lin et al. 2012). On the while, MITF alone is not capable of transactivation of TRP2 (Potterf et al. 2001), and the association of MITF with other transcription factors plays a vital role to activate theTRP2 promoter (Ludwig et al. 2004; Vachtenheim and Borovansky 2010). cAMP plays a key role in MITF expression via phosphorylation of CREB (Roh et al. 2013). Ser133 is a major site of CREB phosphorylation which is required to its activation for the expression of downstream genes (Sakamoto et al. 2011). A previous study reported that the inhibition of CREB phosphorylation at Ser133 residue in α-MSH-stimulated B16 cells by bisabolangelone resulted in the downregulation of MITF expression (Roh et al. 2013). In this experiment, we demonstrated the ability of ESS to inhibit cellular cAMP accumulation in α-MSH-stimulated B16F10 cells (Fig. 3) that suppressed CREB phosphorylation at Ser133 residue leading to the suppression of MITF and tyrosinase expression (Fig. 2). Previous studies reported the involvement of protein kinases in the regulation of melanin synthesis via proteolytic degradation of phosphorylated MITF (Tu et al. 2012). Activation of ERK is thought to be a feedback mechanism for the inhibition of melanogenesis, as excessive production of melanin would be toxic for the cell (Khaled et al. 2002). Inhibition of melanogenesis via ERK activation was also reported by some other researchers (Lee et al. 2015; Oh et al. 2015; Song et al. 2015). We found that ERK phosphorylation is associated with downregulation of MITF leading to decreased melanogenesis (Kim et al. 2010). We further confirmed the involvement of ERK in the hypopigmenting mechanism of ESS using ERK-specific inhibitor, PD98059. The PD98059 treatment ameliorated the suppressive effect of ESS on tyrosinase expression, suggesting that in association with cAMP, the ERK signaling also plays an important role in the depigmenting properties of ESS. In conclusion, inhibition of melanin synthesis is an appropriate approach to skin-whitening or to treat hyperpigmentary disorders. In this study, we demonstrated the capability of ESS to inhibit α-MSH-stimulated hyperpigmentation in B16F10 cells through modulation of MITF via CREB/ERK signaling pathways and proposed its action mechanism (Fig. 6). Further investigation is going on to elucidate the action mechanisms of active compounds from ESS which is responsible for its antimelanogenic activity to ensure its potential application in skin hyperpigmentary disorders. Acknowledgements This research was supported by the project "Development of nutraceuticals from Sargassum serratifolium" funded by the Ministry of Oceans and Fisheries, Republic of Korea. Compliance with ethical standards Conflict of interest The authors have declared that there is no conflict of interest.

J Appl Phycol

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