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A novel peptide purified from the fermented microalga Pavlova lutheri attenuates oxidative stress and melanogenesis in B16F10 melanoma cells
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A novel peptide purified from the fermented microalga Pavlova lutheri attenuates oxidative stress and melanogenesis in B16F10 melanoma cells Gun-Woo Oh a,1 , Seok-Chun Ko b,1 , Seong-Yeong Heo a , Van-Tinh Nguyen a , GeunHyung Kim c , Chul Ho Jang d,e , Won Sun Park f , Il-Whan Choi g , Zhong-Ji Qian h,∗∗ , Won-Kyo Jung a,∗ a Department of Biomedical Engineering, and Center for Marine-Integrated Biomedical Technology (BK21 Plus) Pukyong National University, Busan 608-737, Republic of Korea b Institute of Marine Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea c Department of Biomechatronic Engineering College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Republic of Korea d Department of Otolaryngology, Chonnam National University Hospital, Gwangju, Republic of Korea e Research Center for Resistant Cells, Chosun University, Gwangju, Republic of Korea f Department of Physiology, Kangwon National University School of Medicine, Chuncheon, Republic of Korea g Department of Microbiology, College of Medicine Inje University, Busan, Republic of Korea h College of Food Science and Technology, Guangdong Ocean University, Zhanjiang 524088, China
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Article history: Received 3 January 2015 Received in revised form 1 May 2015 Accepted 5 May 2015 Available online xxx Keywords: Pavlova lutheri Purified peptide Melanogenesis Reactive oxygen species
a b s t r a c t A novel peptide was purified from marine microalga, Pavlova lutheri fermented by yeast Hansenula polymorpha, and its protective effect against oxidative stress as well as inhibitory effect on melanogenesis was investigated. The purified peptide has a molecular mass of 526 Da and its amino acid sequence was determined as Met-Gly-Arg-Tyr by Q-TOF mass spectroscopy. The purified peptide scavenged DPPH, hydroxyl radicals and hydrogen peroxide (H2 O2 ) at the IC50 values of 0.285, 0.068 and 0.988 mM, respectively. Intracellular reactive oxygen species (ROS) induced H2 O2 was attenuated by addition of the purified peptide. The purified peptide demonstrated inhibitory properties against ␣-MSH-induced melanogenesis via melanin content and tyrosinase (TYR) inhibition in B16F10 melanoma cells. We also found that the purified peptide decreased melanogenesis-related proteins; microphthalmia-associated transcription factor (MITF) and TYR protein expressions. Moreover, the purified peptide activated extracellular signalregulated kinase (ERK) but not that of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). A specific ERK inhibitor significantly blocks the purified peptide-inhibited melanin synthesis and TYR activity. Hence, these results indicated that the purified peptide isolated from P. lutheri has potential whitening effects and prominent protective effects on oxidative stress-induced cell damages, which might be used in pharmaceutical and cosmeceutical industries. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae are the most important and prominent primary producers in marine ecosystem. The fine structure, higher protein content, faster growth even in harsh environment conditions,
∗ Corresponding author. Tel.: +82 51 629 5775; fax: +82 51 629 5775. ∗∗ Corresponding author. Tel.: +86 759 2396270; fax: +86 759 2396270. E-mail addresses: [email protected] (Z.-J. Qian), [email protected] (W.-K. Jung). 1 These authors contributed equally to this study.
makes microalgae an excellent source of bioactive molecules [1,2]. Moreover, microalgae have gain higher attention than ever before due to high availability of proteins. For instance, some microalgae species such as Chlorella sp., Dunaliella sp., Scenedesmus obliquus, Arthrospira sp. and cyanobacteria Spirulina sp. have recently shown higher protein content [3]. A microalga haptophyte, P. lutheri is widely used live feed in aquaculture especially marine invertebrates in particular molluscs, crustaceans, and zooplanktons [4–6]. The specific yeast species kwon as thermotolerant methlotrophic yeast only use methanol as source of carbon and energy. Hansenula polymorpha (Pichia angusta) belongs to
http://dx.doi.org/10.1016/j.procbio.2015.05.007 1359-5113/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Oh G-W, et al. A novel peptide purified from the fermented microalga Pavlova lutheri attenuates oxidative stress and melanogenesis in B16F10 melanoma cells. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.05.007
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a limited number of methanol-utilizing yeast species such as Candida boidinii, P. pastoris, P. methanolica and H. polymorpha [7] and used to fermentation of P. lutheri. The H. polymorpha is generally using to attractive platform for production of recombinant protein, in particular pharmaceuticals, for examples hepatitis B vaccines, interferon alpha-2a, hirudin, insulin, and phytase [8]. In our previous study, fermented P. lutheri by H. polymorpha was exhibited antioxidant activity [7]. However, the bioactivities of fermented P. lutheri by H. polymorpha used in this study have been rarely reported. In addition, the antioxidative peptide of fermented P. lutheri has not yet been reported. Additionally, P. lutheri protein can be converted into value-added products by fermentation by yeast, which may be widely applied to improve the functional properties. Melanin synthesis induced by various inner factors such as ␣-melanocyte-stimulating hormone (␣-MSH) and environmental factors such as ultraviolet radiation (UV). Further, melanin synthesis is mainly controlled by tyrosinase (TYR) produced in specialized dendritic cells known as melanocyte, which located in the basal layer of skin [9]. Melanin is responsible for hair, eyes, skin color and plays a pivotal role in protection the skin against UV-induced DNA damage [10]. However, results of the abnormal melanin synthesis in the skin can leads problematic conditions such as a freckle, liver spots, and melasma known as hyperpigmentary disorders [11]. TYR is copper-containing, the most important enzyme involved in melanin synthesis in mammalian [12]. The microphtalmia-associated transcription factor plays an important role in expression of melanogenesis-relatd protein such as TYR [13]. The enzyme can convert L -tyrosine into two types of melanin such as black-brown eumelanin and red-yellow pheomelanin within membrane-bound cytoplasmic organelles called melanosomes and the transport of melanosomes to neighboring keratinocytes [14]. Reactive oxygen species (ROS) such as hydroxyl radicals (OH• ), superoxide anion (O2 •− ) and hydrogen peroxide (H2 O2 ) are generated as a consequence of cell metabolism within mitochondria [15]. ROS are highly unstable and rapidly react with nearby biomacromolecules such as proteins, lipids and DNA, resulting cells, tissue and entire organs damage in the body. A previous study has shown that ROS could play an important role in regulating melanogenesis, while ROS scavengers and inhibitors reduced hyper-pigmentation or suppressed UV-induced melanogenesis on melanocytes [16,17]. In addition, antioxidants such as vitamin C and E inhibited UV radiation-induced melanocyte proliferation and melanogenesis on mice skin [18]. Hence, antioxidant compounds are excellent agents that can be employed as inhibitors against abnormal production of melanin in melanocytes. The objective of this study was to isolate a novel peptide from fermented P. lutheri and identify the purified peptide with regards to anti-oxidant and anti-melanogenesis effects. Furthermore, we investigated the anti-melanogenesis action by expression of melanogenesis-related proteins and mitogen-activated protein kinases (MAPKs) signaling pathway in ␣-MSH-induced B16F10 mouse melanoma cells.
2. Materials and methods 2.1. Materials P. lutheri (KMCC H-006) used in this study was generously provided by Korea Marine Microalgae Culture Center. The H. polymorpha (KCTC 7538) from Korea Biological Resource Center and culture medium were maintained in standard F/2 (Guillard’s) medium. Dulbecco’s modified eagle’s medium (DMEM), trypsinethylenediaminetetraacetic acid (trypsin-EDTA), fetal bovine serum (FBS) and 5-(and-6)-carboxy-2 ,7 -dichlorofluorescein
diacetate (carboxy-DCF-DA) were obtained from Gibco BRL Life Technologies (Grand Island, NY, USA). Dimethyl sulfoxide (DMSO), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT), ␣-MSH, dihydroxylphenylalanine (L-DOPA) and Hoechst 33342 (HO33342) were purchased from Sigma Co. (St. Louis, MO, USA). The specific antibodies used for western blot analysis were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The other chemicals and reagents used were of analytical grade. 2.2. Preparation and identification of fermented P. lutheri Fermentation of P. lutheri by H. polymorpha was performed according to the method used by Qian et al. [7]. For the preparation of fermented P. lutheri, P. lutheri powder was added to water at a ratio of 1:15 (w/v). Then, the cellulase added to solution at a ratio of 100:1 (w/w) at 37 ◦ C for 8 h (pH 6.0) for degradation of cell wall. After autoclaving at 121 ◦ C for 30 min, the solution was obtained, and the H. polymorpha culture broth was added to the solution at a concentration of 1% (v/v), which well mixed and then incubated at 37 ◦ C for 12 days. The recovered fermented P. lutheri was lyophilized and stored at −20 ◦ C until used. The fermented P. lutheri were loaded onto HiPrep 16/10 CM FF ion-exchange column equilibrated with 20 mM sodium acetate buffer (pH 4.0), and eluted with a linear gradient of NaCl (0–2 M) in the same buffer at a flow rate of 2 mL/min using fast protein liquid chromatography (FPLC). The UV absorbance at 280 nm was monitored in each 4 mL fractions, then pooled and lyophilized, immediately. The lyophilized fractions were further purified on a Primesphere ODS C18 column permeation reversed-phase high performance liquid chromatography (RP-HPLC) with a linear gradient of acetonitrile (0–30% in 30 min) containing 0.1% trifluoroacetic acid (TFA) at a flow rate of 1.0 mL/min. Then the accurate molecular mass and amino acid sequence of purified peptide were ascertained by quadrupole time of flight (Q-TOF) mass spectroscopy (Micromass, Altrincham, UK) coupled to electrospray ionization (ESI) source. 2.3. Radical scavenging activity by electron spin resonance (ESR) spectrometer Radical scavenging activity of the purified peptide was calculated as a scavenging percentage by, S = (H0 − H1 )/H0 × 100%; where, H1 and H0 were ESR signal intensities in the presence and the absence of test sample, respectively. 2.3.1. DPPH radical scavenging activity DPPH radical scavenging activity was measured using the method described by Nanjo et al. [19]. A 30 L sample solution was added to 30 L of DPPH (60 M/L) in ethanol solution. After mixing vigorously for 10 sec, the solution was then transferred into a quartz capillary tube, and the scavenging activity of the purified on DPPH radical was measured using a JES-FA ESR spectrometer (JEOL Ltd., Tokyo, Japan). The spin adduct was measured on an ESR spectrometer exactly 2 min later. Experimental conditions as follows: magnetic field, 336.5 ± 5 mT; power, 5 mW; modulation frequency, 9.41 GHz; amplitude, 1 × 1000; sweep time, 30 sec. DPPH radical scavenging ability was calculated following equation in which H and H0 were relative peak height of radical signals with and without sample, respectively. 2.3.2. Hydroxyl radical scavenging activity Hydroxyl radicals were generated by iron-catalyzed Fenton Haber–Weiss reaction and the generated hydroxyl radicals were rapidly reacted with nitrone spin trap DMPO [20]. The resultant DMPO-OH adducts was detectable with an ESR spectrometer. The sample solution 20 L was mixed with 0.3 M DMPO 20 L, 10 mM
Please cite this article in press as: Oh G-W, et al. A novel peptide purified from the fermented microalga Pavlova lutheri attenuates oxidative stress and melanogenesis in B16F10 melanoma cells. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.05.007
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FeSO4 20 L and 10 mM H2 O2 20 L in a phosphate buffer solution (pH 7.4), and then transferred into a quartz capillary tube. After 2.5 min, the ESR spectrum was recorded using an ESR spectrometer. The experimental conditions employed were as follows: magnetic field, 336.5 ± 5 mT; power, 1 mW; modulation frequency, 9.41 GHz; amplitude, 1 × 200; sweep time, 4 min. Hydroxyl radical scavenging ability was calculated following equation in which H and H0 were relative peak height of radical signals with and without sample, respectively. 2.4. ABTS radical scavenging assay H2 O2 scavenging activity was determined by a colorimetric assay according to the method of Müller [21]. Phosphate buffer (100 L, 0.1 M, pH 5.0) and sample solution were mixed in a 96microwell plate. H2 O2 (20 L) was added to the mixture, which was then incubated for 5 min at 37 ◦ C. After incubation, 30 L of 1.25 mM ABTS and 30 L of peroxidase (1 unit/mL) were added to the mixture and it was then incubated for 10 min at 37 ◦ C. The absorbance was read with an ELISA plate reader (BioTek Instruments, Winooski, VT) at 405 nm. 2.5. Cell culture and cytotoxic assessment using MTT assay B16F10 melanoma cells were cultured in DMEM containing 10% (v/v) FBS, 1% penicillin/streptomycin, and 5% CO2 humidified atmosphere at 37 ◦ C. The cytotoxic effect of the purified peptide on cells was determined by the MTT assay. The cells were seeded in 96-well plates. After 24 h, cells were treated with various concentrations of the purified peptide for 48 at 37 ◦ C. The MTT stock solution (50 L) was then added to each well to obtain a total reaction volume of 250 L. After 4 h of incubation, the plates were centrifuged (800 × g, 5 min), and the supernatants were aspirated. The formazan crystals in each well were dissolved in 150 L of DMSO, and the absorbance was measured with an ELISA plate reader at 540 nm. 2.6. Genomic DNA extraction Genomic high molecular weight DNA was extracted from B16F10 melanoma cells using standard phenol/proteinase K procedure with slight modifications [22]. Briefly, cells culturing in 10 cm dishes were washed twice with PBS and scraped into 1 mL of PBS containing 5 mM EDTA. After centrifugation cells were dissolved in RNase (0.5 mg/mL), 0.2 M sodium acetate, proteinase K (10 mg/mL), and 10% sodium dodecyl sulfate (SDS). The mixture was incubated for 30 min at 37 ◦ C and 1 h at 55 ◦ C. Following incubation, phenol: chloroform: isoamylalcohol (25:24:1) was added at 1:1 ratio and mixture was centrifuged at 12,000 × g for 5 min at 4 ◦ C. Supernatants were mixed with 100% ice cold ethanol at 1:1.5 ratio and kept for 15 min at −20 ◦ C. After centrifugation at 12,000 × g for 5 min, the pellet was dissolved in TE buffer and purity of DNA was ELISA reader determined at 260/280 nm. Further, the quality of isolated DNA was evaluated with 1% agarose gel electrophoresis in 40 mM Tris–acetate buffer. 2.7. Protective effect of the purified peptide against hydroxyl radical-induced DNA damage Hydroxyl radical mediated DNA oxidation was determined according to Ngo et al. [23]. Briefly, 40 L of DNA reaction mixture were prepared by adding pre-determined concentrations of test sample (or same volume of distilled water as control), 100 M final concentration of EDTA. An aliquot of the reaction mixture containing about 1 g of DNA was electrophoresed on a 1% agarose gel
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for 40 min at 100 V. Gels were then stained with 1 mg/mL ethidium bromide and visualized under UV light. 2.8. Intracellular radical measurement Intracellular production of ROS was detected by oxidation of the cell permeable fluorescence dye 5-(and-6)-carboxy-2 ,7 dichlorofluorescein diacetate (carboxy-DCFH-DA) to fluorescent DCF by H2O2 [24]. B16F10 melanoma cells were seeded in 24-well plate. After 24 h, cells were pre-treated with various concentrations of the purified peptide. After 1 h, H2 O2 was added at a concentration of 1 mM, and then cells incubated for an additional 1 h at 37 ◦ C under a humidified atmosphere. Finally, 40 M carboxy-DCFH-DA was introduced to the cells, and carboxy-DCFH-DA was imaged using fluorescence microscopy. 2.9. Measurement of cellular melanin content The melanin content was measured using a slight modification of a method reported previously [25]. Briefly, B16F10 melanoma cells plated on 6-well plates were pre-incubated and subsequently treated with ␣-MSH coupled with aliquots of the purified peptide at 37 ◦ C. After 48 h, the cells were washed twice with cold PBS and lysed in PBS buffer containing 1% (v/v) Triton X-100. The lysates were clarified by centrifugation at 13,000 × g for 10 min. The supernatants removed and dissolved in 1 M NaOH containing 10% DMSO at 90 ◦ C for 1 h. The relative melanin content was determined by measuring the absorbance at 405 nm in an ELISA reader. 2.10. Measurement of cellular tyrosinase activity Cellular TYR activity was estimated by measuring the formation rate of DOPA chrome from DOPA [26]. B16F10 melanoma cells plated on 6-well plates were pre-incubated and subsequently treated with ␣-MSH coupled with aliquots of the purified peptide at 37 ◦ C. After 48 h, the cells were washed twice with cold PBS and lysed in PBS buffer containing 1% (v/v) Triton X-100. The lysates were clarified by centrifugation at 13,000 × g for 10 min. After protein quantification and normalization, cell lysates (each sample contained the same amount of protein) were incubated in duplicate with 10 mM L-DOPA at 37 ◦ C for 1 h. After incubation, DOPA chrome was monitored by measuring the absorbance at 475 nm. 2.11. Western blot analysis The cells were lysed in lysis buffer (20 mM Tris, 5 mM EDTA, 10 mM Na4 P2 O7 , 100 mM NaF, 2 mM Na3 VO4 , 1% NP-40, 10 mg/mL aprotinin, 10 mg/mL leupeptin and 1 mM PMSF) for 60 min and then centrifuged at 12,000 rpm and 4 ◦ C for 15 min. The protein concentrations were determined using the BCATM protein assay kit. The lysate containing 40 g of protein was subjected to electrophoresis on a SDS-polyacrylamide gel, and the gel was transferred onto a nitrocellulose membrane. The membrane was blocked with 5% non fat dry milk in TBS-T (25 mM Tris–HCl, 137 mM NaCl, 2.65 mM KCl, and 0.05% Tween 20, pH 7.4) for 2 h. The primary antibodies were used at a 1:1000 dilution. The membranes were incubated with the primary antibodies at 4 ◦ C overnight, washed with TBS-T and then incubated with the secondary antibodies at 1:3000 dilutions. The signals were developed using an ECL western blotting detection kit and quantified using the western imaging system (Fujifilm Life Science, Tokyo, Japan). 2.12. Statistical analysis All of data are presented as the mean ± standard deviation (SD) of three determinations. Statistical comparisons of the mean
Please cite this article in press as: Oh G-W, et al. A novel peptide purified from the fermented microalga Pavlova lutheri attenuates oxidative stress and melanogenesis in B16F10 melanoma cells. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.05.007
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Fig. 1. Identification of molecular mass and amino acid sequence of purified peptide. MS/MS experiments were performed on a Q-TOF tandem mass spectrometer equipped with a nano-ESI source. Sequencing of the prurified peptide was acquired over the m/z range 0–1000 and sequenced by using the PepSeq de nove sequencing algorithm.
values were performed by analysis of variance (ANOVA), followed by Duncan’s multiple range test using SPSS software. The statistical significance of the differences was defined at the p < 0.05 level.
3. Results 3.1. Identification of the purified peptide A novel peptide was purified from the fermented P. lutheri using chromatographic methods, combining FPLC on a HiPrep 16/10 DEAE FF anion-exchange column and repeated RP-HPLC on a Primesphere column (data not shown). The molecular mass of the fermented P. lutheri purified a novel peptide was determined to be 526 Da by Q-TOF mass spectroscopy coupled to an ESI source. Its full amino acid sequence was composed of Met-Gly-Arg-Tyr (Fig. 1).
3.4. DNA protective effect of the purified peptide from hydroxyl-Radical Genomic DNA was isolated from B16F10 melanoma cells to study protective effects of purified peptide against DNA oxidative damage. In this experiment the combined effects of 100 M FeSO4 and 50 M H2 O2 on the integrity of genomic DNA was studied by DNA electrophoresis in the presence or absence of the purified peptide. After reaction, almost all DNA was degraded in the control group treated only with Fe (II)–H2 O2 . However, the treatment with the purified peptide exhibited a dose-dependent suppression of DNA degradation against • OH radical mediated damage at concentrations ranging from 25 to 200 M (Fig. 3A). The results of this study clearly explain that the purified peptide can prevent oxidative
3.2. ROS scavenging activity of the purified peptide To explore cell-free system scavenging activities of the purified peptide against DPPH and hydroxyl radicals, we measured radical signals by electron spin-trapping technique. Also, the purified peptide was evaluated for its H2 O2 scavenging activity by ABTS assay. The IC50 value was used an index for the concentration of the purified peptide at 50% reducing of radical. IC50 values of the purified peptide against DPPH, hydroxyl radical and H2 O2 were 0.285 ± 0.003, 0.068 ± 0.001 and 0.988 ± 0.011 mM, respectively. 3.3. Effect of the purified peptide on viability in B16F10 melanoma cells In this study, B16F10 melanoma cells were treated with different concentrations of the purified peptide to determine non-cytotoxic effects for further experiments. The cell viability data confirmed that the purified peptide was non-cytotoxic in B16F10 melanoma cells (Fig. 2). Therefore, it was determined that the purified peptide could be used in further experiments.
Fig. 2. The cytotoxic effect of the purified peptide on viability in B16F10 melanoma cells. Cells were treated with the purified peptide at the indicated concentrations (50, 100 and 200 M). After 24 h to treat the purified peptide cell viability was assessed by MTT assay. Values are expressed as mean ± S.D. in triplicate experiments.
Please cite this article in press as: Oh G-W, et al. A novel peptide purified from the fermented microalga Pavlova lutheri attenuates oxidative stress and melanogenesis in B16F10 melanoma cells. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.05.007
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or H2 O2 . However, the purified peptide pretreatment reduced the fluorescence intensity values in a dose-dependent manner in the cells induced by H2 O2 treatment. These results indicate that the purified peptide significantly reduced the evaluated ROS levels induced by H2 O2 , and suggest that the purified peptide possesses intracellular ROS scavenging activity.
3.6. Inhibitory effect of the purified peptide on melanin content and tyrosinase activity in ˛-MSH-induced B16F10 cells To verify the ability of the purified peptide on ␣-MSH-mediated melanogenesis, determined the quantity of intracellular melanin and TYR activity in the presence of ␣-MSH. As shown in Fig. 4, the purified peptide substantially decreased the ␣-MSH-induced cellular melanin contents in a dose-dependent manner compared to the ␣-MSH alone treated group. Likewise, in the TYR activity, the purified peptide also reduced the ␣-MSH-increased intracellular TYR activity in a dose-dependent manner (Fig. 4B). These results suggest that the purified peptide down-regulated TYR activity and that this inhibitory effect may lead to decrease cellular melanin synthesis in B16F10 cells.
3.7. Effect of the purified peptide on tyrosinase and MITF expression in ˛-MSH-stimulated B16F10 cells To elucidate the mechanisms underlying the anti-TYR and antimelanogenic activities of the purified peptide, we examined the effects of the purified peptide on the expression levels of the melanogenic enzymes such as TYR by Western blot. As shown in Fig. 5, the purified peptide at 50–200 M dose-dependently reduced the expression of TYR at 48 h. Since the TYR is transcriptionally regulated by MITF, we next examined the influence of the purified peptide on MITF expression. Results showed that the purified peptide also dose-dependently inhibited MITF expression in B16F10 cells (Fig. 5).
Fig. 3. (A) DNA oxidative damage protection by the purified peptide. Genomic DNA from B16F10 cells was pre-treated with peptide and exposed to • OH using Fenton chemistry. Line 1, Blank (DNA 5 L); line 2, FeSO4 and H2 O2 (DNA damage control); lines 3–6, FeSO4 and H2 O2 in the presence of peptide with different concentrations (25–200 M), respectively. (B) The intracellular radical generated was detected by DCFH-DA staining. Carboxy-2 ,7 -diclorofluorescein diacetate (Carboxy-DCFDA) was used to evaluate intracellular oxidant formation. Digital images of DCF fluorescence were obtained with a fluorescence microscope system. Bar graph exhibited fluorescence intensity and line graph showed the intracellular radical scavenging activity. Values are expressed as mean ± S.D. in triplicate experiments. Statistical evaluation was performed to compare the experimental groups and H2 O2 -treated cells. *p < 0.05 and **p < 0.01.
damage to DNA when DNA is exposed to hydroxyl radical generated by Fe (II)/H2 O2 . Fe2+ catalyses the conversion of H2 O2 , which is a major route to the synthesis of hydroxyl radical in biological systems. 3.5. Inhibitory effect of the purified peptide on intracellular ROS generated by H2 O2 Intracellular ROS scavenging activity could be detected via measurements of the level of DCF, the results of which are illustrated in Fig. 3B. The fluorescence intensity value in B16F10 melanoma cells increased significantly after treatment with H2 O2 compared to that in the untreated cells, which did not contain either the peptide
3.8. Effect of the purified peptide on phosphorylation of MAPKs pathway in ˛-MSH-stimulated B16F10 cells We examined the influence of the purified peptide treatment on the activation of JNK, ERK and p38 MAPKs in an attempt to further understand the molecular mechanisms involved in the hypopigmentation property of the purified peptide by Western blot. As shown in Fig. 6, ERK phosphorylation was significantly enhanced after the purified peptide treatment. On the other hand, p38 and JNK phosphorylation was remain unchanged by the purified peptide (Fig. 6). Thus, results imply that the purified peptideinduced inhibition of melanogenesis occurs through regulation of ERK phosphorylation. To further confirm and refined the role of ERK signaling on the purified peptide-induced anti-melanogenic effect, we employed a specific inhibitor of ERK, PD98059, which blocks ERK signaling. As shown Fig. 7A and B, PD98059 treatment markedly restored melanin synthesis and TYR activity suppressed by the purified peptide. Additionally, to confirm our results, we examined the effect of PD98059 on the expression of MITF in the presence of ␣-MSH or the purified peptide Fig. 7C shows that the purified peptide-induced suppression of ␣-MSH-induced MITF expression was inhibited by PD98059. We next examined whether PD98059 inhibits the ERK pathway in ␣-MSH-stimulated B16F10 cells, and found that PD98059 does in fact block ERK activation in the purified peptide treated B16F10 cells (Fig. 7D). Thus, results imply that the purified peptide-induced inhibition of melanogenesis occurs through phosphorylation ERK.
Please cite this article in press as: Oh G-W, et al. A novel peptide purified from the fermented microalga Pavlova lutheri attenuates oxidative stress and melanogenesis in B16F10 melanoma cells. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.05.007
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Fig. 4. Effect of the purified peptide on cellular (A) melanin content and tyrosinase activity in B16F10 mouse melanoma cells. Cells were exposed 50 nM ␣-MSH in the presence of the purified peptide (50–200 M). The percentage values of the treated cells are expressed relatively compared to that in the untreated cells. Values are expressed as mean ± S.D. in triplicate experiments. Statistical evaluation was performed to compare the experimental groups and ␣-MSH-treated cells. *p < 0.05 and **p < 0.01.
Fig. 5. Effect of the purified peptide on the expresstion of melanogenesis-related proteins in B16F10 melanoma cells. Cells were exposed 50 nM ␣-MSH in the presence of the purified peptide (50–200 M). The expression levels of the MITF and tyrosinase proteins were examined by western blot. GAPDH was used as an internal control. Values are expressed as mean ± S.D. in triplicate experiments. Statistical evaluation was performed to compare the experimental groups and ␣-MSH-treated cells. *p < 0.05 and **p < 0.01.
Fig. 6. Effect of the purified peptide on phosphorylation of MAPKs in B16F10 melanoma cells. Cells were exposed 50 nM ␣-MSH in the presence of the purified peptide (50–200 M). The expression levels of the p-p38, p38, p-JNK, JNK, p-ERK and ERK proteins were examined by Western blot. Values are expressed as mean ± S.D. in triplicate experiments. Statistical evaluation was performed to compare the experimental groups and ␣-MSH-treated cells. *p < 0.05 and **p < 0.01.
Please cite this article in press as: Oh G-W, et al. A novel peptide purified from the fermented microalga Pavlova lutheri attenuates oxidative stress and melanogenesis in B16F10 melanoma cells. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.05.007
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Fig. 7. Effect of PD98059 on the purified peptide activities in ␣-MSH-stimulated B16F10 melanoma cells. Cells were exposed 50 nM ␣-MSH in the presence and absence of the purified peptide (200 M) and PD98059 (10 M). Effect of PD98059 on (A) melanin content and (B) tyrosinase activity. Melanin content and tyrosinase activity in control cells were regarded as 100%. Values are expressed as mean ± S.D. in triplicate experiments. *p < 0.05. The expression levels of (C) MITF and (D) phosphor-ERK and ERK proteins were examined by Western blot. Data shown are the representative of three independent Western blotting experiments.
4. Discussion Recently, due to the relatively limited natural products of land, many researchers have focused on marine natural products with various biological activities. Microalgae have been popular food stuff worldwide, and there are rich in protein, minerals, carbohydrates, and bioactive substance [2]. Therefore, it is thought that microalgae could be developed as useful functional materials. Additionally, according to previous study, the amino-acids patterns of the microalgal proteins show high nutritive value and availability of essential amino acids [27]. Among the microalgae, P. lutheri is the most important primary producers in aquatic environments and they have high concentration protein. Fermented P. rutheri by yeast C. rugopelliculosa or H. polymorpha have become good candidates for sources of natural antioxidants, as revealed by a number of recent studies [7,27]. According to previous study, ROS is play an important roles in the regulating the melanogenesis and melanocytes proliferation [28,29]. Scavengers and inhibitors of ROS formation can reduce melanogenesis in melanocyte. Although some reports suggest that fermented P. lutheri exhibit antioxidant effect on free radicals, it is no reports in the protective effect of the purified peptide from fermented P. lutheri on the cell damage induced by oxidative stress and its inhibitory effect on mealnogenesis, and the underlying mechanisms. Thus, in this study, the potential whitening effect of the purified peptide isolated from fermented P. lutheri was investigated. Interest has emerged to identify and characterize bioactive peptides from microalgae proteins [2,30]. These bioactive peptides can be derived by fermentation of microalgae proteins. Additionally, fermented substances are also a source of bioactive peptides, which are short peptides released from proteins that have been shown to exert biological activities [31,32]. Biological properties of the peptides were highly influenced by molecular weight and structure [33]. In general, antioxidative peptides containing aromatic acid residues (tyrosine, tryptophan and phenylalanine) at the
C-terminus have strong radical scavenging activities [34]. The aromatic amino acid residues can make active oxygen stable through direct electron transfer [35]. As shown the results of this study, the presence of tyrosine in the sequence could have attributed to the antioxidative effect of the purified peptide from fermented P. lutheri. ROS including free radicals such as hydroxyl radicals, and superoxide radicals and non-free radical species such as H2 O2 perform important functions in cell signaling. Cell damages are normally able to defend themselves against oxidative stress by the use of endogenous antioxidant enzymes, including superoxide dismutases, catalases and glutathione peroxidases [36]. However, an imbalance between ROS and antioxidant defense mechanisms can result in oxidative modification of the intracellular molecules [37]. Hydroxyl radicals generated by the Fenton reaction are known to cause oxidative breaks in DNA strands to yield its open circular or relaxed forms [38]. Also, H2 O2 has been extensively used as an inducer of oxidative stress in vitro models [36]. Therefore, in this study, we report the protective effects of the purified peptide against ROS-induced cell damage for antioxidant. The results presented here indicated that the purified peptide reduced DNA damage by hydroxyl radicals, and suppressed intracellular ROS produced by H2 O2 . These results suggest that the purified peptide from fermented P. lutheri has the ability to protect cells from oxidative stress-related cellular injuries. Melanogenesis is a physiological process resulting in the synthesis of melanin pigment, which plays a crucial protective role against skin photo-carcinogenesis [39]. Among the process of melanogenesis, TYR participates in oxidative steps of melanin synthesis that convert L -tyrosine into L -DOPA and other intermediates through hydroxylation of L -tyrosine on first step in melanocytes [40,41]. Also, melanin content correlates directly with the activity of TYR [42]. Therefore, we investigated whether the purified peptide could inhibit melanin synthesis in the presence of ␣-MSH. We found that the purified peptide inhibited
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melanin synthesis, activity of TYR and protein levels of TYR in a dose-dependent manner. These results suggest that the purified peptide may down-regulate TYR activity and inhibit cellular melanin synthesis in B16F10 melanoma cells. In addition, ␣-MSH potently induces MITF expression, which is regulated at the levels of TYR [42]. Moreover, MITF is crucial role in the control of melanocyte differentiation, proliferation, survival and melanin synthesis [43–45]. Therefore, MITF expression is appropriate target for cosmeceutical treatment of melanogenesis. Thus, we examined the inhibitory effect of the purified peptide on the expression of MITF in ␣-MSH-stimulated B16F10 melanoma cells. The purified peptide attenuated ␣-MSH-induced expression of MITF. These results suggest that the purified peptide does not directly inhibit TYR activity, and that the purified peptide inhibitory effect coordinate MITF down-regulation in B16F10 melanoma cells. To evaluate the mechanism of the inhibitory effect of the purified peptide on melanogenesis, we examined the activation factor MAPKs. One of the extensively investigated transduction mechanisms involved in the melanogenesis process is the MAPKs pathway [42,46]. Previous studies have revealed that phosphorylation of p38 MAPK can induce through activate MITF expression, and also phosphorylation of JNK and ERK can reduce melanogenesis through MITF phosphorylation and its subsequent degradation on B16F10 cells [47–51]. We therefore examined the influence of the purified peptide treatment on the activation of JNK, ERK and p38 MAPK attempting to further understand the molecular mechanisms involved in the pigmentation property of the purified peptide by western blot assay. The results showed that phosphorylation of ERK were significantly enhanced after the purified peptide treatment. On the other hand, JNK and p38 MAPK phosphorylation were no changed by the purified peptide. To determine which ERK plays an important role in regulating the purified peptide-induced melanogenesis, we treated B16F10 cells with the ERK-specific inhibitor PD98059. We found that PD98059 markedly increase the purified peptidesuppressed melanin synthesis and TYR activity. We also confirmed that the purified peptide-induced suppression of MITF expression was inhibited by PD98059 in ␣-MSH-stimulated B16F10 cells. This finding suggested that the purified peptide-induced antimelanogenesis in B16F10 melanoma cells through ERK mediated pathways. In conclusion, we evaluated the anti-oxidant and antimelanogenesis of a purified peptide from the fermented microalga, P. lutheri by yeast H. polymorpha. The purified peptide exerts a profound protective effect against oxidative stress. In addition, the purified peptide suppresses melanin synthesis and TYR activity through ERK pathway-mediated suppression of MITF and TYR in ␣-MSH-stimulated B16F10 melanoma cells. These results indicated that the purified peptide from P. lutheri can be used as an effective natural source to make cosmeceutical and pharmaceutical products.
[2]
[3] [4] [5] [6] [7]
[8] [9]
[10] [11] [12]
[13] [14]
[15]
[16]
[17]
[18]
[19]
[20] [21]
[22]
[23]
[24]
Acknowledgments [25]
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2013R1A1A1A05013577), and also supported by grant from Marine Technology Application Program (PJT200673) and Marine Biotechnology Program (20150220) Funded by Ministry of Oceans and Fisheries, Republic of Korea.
[26]
[27]
[28]
References [29] [1] Nguyen MHT, Qian ZJ, Nguyen VT, Choi IW, Heo SJ, Oh CH, Kang DH, Kim GH, Jung WK. Tetrameric peptide purified from hydrolysates of biodiesel byproducts of Nannochloropsis oculata induces osteoblastic differentiation through
MAPK and Smad pathway on MG-63 and D1 cells. Process Biochemistry 2013;48:1387–94. Ko SC, Kang N, Kim EA, Kang MC, Lee SH, Kang SM, Lee JB, Jeon BT, Kim SK, Park SJ. A novel angiotensin I-converting enzyme (ACE) inhibitory peptide from a marine Chlorella ellipsoidea and its antihypertensive effect in spontaneously hypertensive rats. Process Biochemistry 2012;47:2005–11. Becker E. Micro-algae as a source of protein. Biotechnology Advances 2007;25:207–10. Borowitzka MA. Microalgae for aquaculture: opportunities and constraints. Journal of Applied Phycology 1997;9:393–401. Wikfors GH, Ohno M. Impact of algal research in aquaculture. Journal of Phycology 2001;37:968–74. Volkman JK, Dunstan GA, Jeffrey S, Kearney PS. Fatty acids from microalgae of the genus Pavlova. Phytochemistry 1991;30:1855–9. Qian ZJ, Jung WK, Kang KH, Ryu B, Kim SK, Je JY, Heo SJ, Oh C, Kang DH, Park WS. In Vitro antioxidant activities of the fermented marine microalga Palova lutheri (Haptophyta) with the yeast Hansenula polymorpha. Journal of Phycology 2012;48:475–82. C¸elik E, C¸alık P. Production of recombinant proteins by yeast cells. Biotechnology Advances 2012;30:1108–18. Park H, Kosmadaki M, Yaar M, Gilchrest B. Cellular mechanisms regulating human melanogenesis. Cellular and Molecular Life Sciences 2009;66:1493–506. Ortonne JP. Photoprotective properties of skin melanin. British Journal of Dermatology 2002;146:7–10. Briganti S, Camera E, Picardo M. Chemical and instrumental approaches to treat hyperpigmentation. Pigment Cell Research 2003;16:101–10. Hearing VJ. Biochemical control of melanogenesis and melanosomal organization. Journal of Investigative Dermatology Symposium Proceedings 1999;4:24–8 [Nature Publishing Group]. Yamaguchi Y, Hearing VJ. Physiological factors that regulate skin pigmentation. Biofactors 2009;35:193–9. Ito S, Wakamatsu K. Quantitative analysis of eumelanin and pheomelanin in humans, mice, and other animals: a comparative review. Pigment Cell Research 2003;16:523–31. Feissner RF, Skalska J, Gaum WE, Sheu SS. Crosstalk signaling between mitochondrial Ca2+ and ROS. Frontiers in Bioscience: A Journal and Virtual Library 2009;14:1197–218. Yoshimura M, Watanabe Y, Kasai K, Yamakoshi J, Koga T. Inhibitory effect of an ellagic acid-rich pomegranate extract on tyrosinase activity and ultraviolet-induced pigmentation. Bioscience, Biotechnology, and Biochemistry 2005;69:2368–73. Yamakoshi J, Otsuka F, Sano A, Tokutake S, Saito M, Kikuchi M, Kubota Y. Lightening effect on ultraviolet-induced pigmentation of guinea pig skin by oral administration of a proanthocyanidin-rich extract from grape seeds. Pigment Cell Research 2003;16:629–38. Quevedo Jr WC, Holstein TJ, Dyckman JACOB, McDonald CJ, Isaacson EL. Inhibition of UVR-induced tanning and immunosuppression by topical applications of vitamins C and E to the skin of hairless (hr/hr) mice. Pigment Cell Research/Sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society 2000;13:89–98. Nanjo F, Goto K, Seto R, Suzuki M, Sakai M, Hara Y. Scavenging effects of tea catechins and their derivatives on 1,1-diphenyl-2-picrylhydrazyl radical. Free Radical Biology and Medicine 1996;21:895–902. Rosen GM, Rauckman EJ. Spin trapping of superoxide and hydroxyl radicals. Methods in Enzymology 1984;105:198–209. Müller HE. Detection of hydrogen peroxide produced by microorganisms on an ABTS peroxidase medium. Zentralblatt für Bakteriologie, Mikrobiologie und Hygiene Series A: Medical Microbiology, Infectious Diseases, Virology, Parasitology 1985;259:151–4. Sambrook J, Russell DW, Russell DW. Molecular cloning: a laboratory manual (3-volumeset). Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2001. Ngo DN, Kim MM, Qian ZJ, Jung WK, Lee SH, Kim SK. Free radical-scavenging activities of low molecular weight chitin oligosaccharides lead to antioxidant effect in live cells. Journal of Food Biochemistry 2010;34:161–77. Mendis E, Kim MM, Rajapakse N, Kim SK. An in vitro cellular analysis of the radical scavenging efficacy of chitooligosaccharides. Life Sciences 2007;80:2118–27. Tsuboi T, Kondoh H, Hiratsuka J, Mishima Y. Enhanced melanogenesis induced by tyrosinase gene-transfer increases boron-uptake and killing effect of boron neutron capture therapy for amelanotic melanoma. Pigment Cell Research 1998;11:275–82. Shen T, Heo SI, Wang MH. Involvement of the p38 MAPK and ERK signaling pathway in the anti-melanogenic effect of methyl 3, 5-dicaffeoyl quinate in B16F10 mouse melanoma cells. Chemico-Biological Interactions 2012;199: 106–11. Ryu B, Kang KH, Ngo DH, Qian ZJ, Kim SK. Statistical optimization of microalgaePavlova lutheri cultivation conditions and its fermentation conditions by yeast, Candida rugopelliculosa. Bioresource Technology 2012;107:307–13. Kim YJ. Antimelanogenic and antioxidant properties of gallic acid. Biological and Pharmaceutical Bulletin 2007;30:1052–5. Chou TH, Ding HY, Hung WJ, Liang CH. Antioxidative characteristics and inhibition of ␣-melanocyte-stimulating hormone-stimulated melanogenesis of vanillin and vanillic acid from Origanum vulgare. Experimental Dermatology 2010;19:742–50.
Please cite this article in press as: Oh G-W, et al. A novel peptide purified from the fermented microalga Pavlova lutheri attenuates oxidative stress and melanogenesis in B16F10 melanoma cells. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.05.007
G Model PRBI-10421; No. of Pages 9
ARTICLE IN PRESS G.-W. Oh et al. / Process Biochemistry xxx (2015) xxx–xxx
[30] Ko SC, Kim D, Jeon YJ. Protective effect of a novel antioxidative peptide purified from a marine Chlorella ellipsoidea protein against free radical-induced oxidative stress. Food and Chemical Toxicology 2012;50:2294–302. [31] Rokka T, Syvaoja EL, Tuominen J, Korhonen H. Release of bioactive peptides by enzymatic proteolysis of Lactobacillus GG fermented UHT milk. Milchwissenschaft 1997;52:675–8. [32] Ashar MN, Chand R. Fermented milk containing ACE-inhibitory peptides reduces blood pressure in middle aged hypertensive subjects. Milchwissenschaft 2004;59:363–6. [33] Sheih I, Wu TK, Fang TJ. Antioxidant properties of a new antioxidative peptide from algae protein waste hydrolysate in different oxidation systems. Bioresource Technology 2009;100:3419–25. [34] Samaranayaka AG, Lichan EC. Food-derived peptidic antioxidants: a review of their production, assessment, and potential applications. Journal of Functional Foods 2011;3:229–54. [35] Qian ZJ, Jung WK, Kim SK. Free radical scavenging activity of a novel antioxidativepeptide purified from hydrolysate of bullfrog skin, Rana catesbeiana Shaw. Bioresource Technology 2008;99:1690–8. [36] Heo SJ, Jeon YJ. Radical scavenging capacity and cytoprotective effect of enzymatic digests of Ishige okamurae. Journal of Applied Phycology 2008;20:1087–95. [37] Aruoma OI, Kaur H, Halliwell B. Oxygen free radicals and human diseases. The Journal of the Royal Society for the Promotion of Health 1991;111:172–7. [38] Ngo DH, Qian ZJ, Ryu B, Park JW, Kim SK. In vitro antioxidant activity of a peptide isolated from Nile tilapia (Oreochromis niloticus) scale gelatin in free radicalmediated oxidative systems. Journal of Functional Foods 2010;2:107–17. [39] Heo SJ, Ko SC, Kang SM, Cha SH, Lee SH, Kang DH, Jung WK, Affan A, Oh C, Jeon YJ. Inhibitory effect of diphlorethohydroxycarmalol on melanogenesis and its protective effect against UV-B radiation-induced cell damage. Food and Chemical Toxicology 2010;48:1355–61. [40] Heo SJ, Ko SC, Cha SH, Kang DH, Park HS, Choi YU, Kim D, Jung WK, Jeon YJ. Effect of phlorotannins isolated from Ecklonia cava on melanogenesis and their protective effect against photo-oxidative stress induced by UV-B radiation. Toxicology In Vitro 2009;23:1123–30.
9
[41] Kang SM, Heo SJ, Kim KN, Lee SH, Yang HM, Kim AD, Jeon YJ. Molecular docking studies of a phlorotannin, dieckol isolated from Ecklonia cava with tyrosinase inhibitory activity. Bioorganic & Medicinal Chemistry 2012;20:311–6. [42] Kim KN, Yang HM, Kang SM, Kim D, Ahn G, Jeon YJ. Octaphlorethol A isolated from Ishige foliacea inhibits ␣-MSH-stimulated induced melanogenesis via ERK pathway in B16F10 melanoma cells. Food and Chemical Toxicology 2013;59:521–6. [43] Kim DS, Park SH, Park KC. Transforming growth factor-1 decreases melanin synthesis via delayed extracellular signal-regulated kinase activation. The International Journal of Biochemistry & Cell Biology 2004;36:1482–91. [44] Levy C, Khaled M, Fisher DE. MITF: master regulator of melanocyte development and melanoma oncogene. Trends in Molecular Medicine 2006;12:406–14. [45] Jeong HS, Lee SH, Yun HY, Baek KJ, Kwon NS, Park KC, Kim DS. Involvement of mTOR signaling in sphingosylphosphorylcholine-induced hypopigmentation effects. Journal of Biomedical Science 2011;8:55. [46] Jiang Z, Xu J, Long M, Tu Z, Yang G, He G. 2,3,5,4 -tetrahydroxystilbene-2-O--dglucoside (THSG) induces melanogenesis in B16 cells by MAP kinase activation and tyrosinase upregulation. Life Sciences 2009;85:345–50. [47] Singh SK, Sarkar C, Mallick S, Saha B, Bera R, Bhadra R. Human placental lipid induces melanogenesis through p38 MAPK in B16F10 mouse melanoma. Pigment Cell Research 2005;18:113–21. [48] Li X, Guo L, Sun Y, Zhou J, Gu Y, Li Y. Baicalein inhibits melanogenesis through activation of the ERK signaling pathway. International Journal of Molecular Medicine 2010;25:923–7. [49] Ahn JH, Jin SH, Kang HY. LPS induces melanogenesis through p38 MAPK activation in human melanocytes. Archives of Dermatological Research 2008;300:325–9. [50] Liu WS, Kuan YD, Chiu KH, Wang WK, Chang FH, Liu CH, Lee CH. The extract of Rhodobacter sphaeroides inhibits melanogenesis through the MEK/ERK signaling pathway. Marine Drugs 2013;11:1899–908. [51] Kim EH, Jeong HS, Yun HY, Baek KJ, Kwon NS, Park KC, Kim DS. Geranylgeranylacetone inhibits melanin synthesis via ERK activation in Mel-Ab cells. Life Sciences 2013;93:226–32.
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A novel peptide purified from the fermented microalga Pavlova lutheri attenuates oxidative stress and melanogenesis in B16F10 melanoma cells Gun-Woo Oh a,1 , Seok-Chun Ko b,1 , Seong-Yeong Heo a , Van-Tinh Nguyen a , GeunHyung Kim c , Chul Ho Jang d,e , Won Sun Park f , Il-Whan Choi g , Zhong-Ji Qian h,∗∗ , Won-Kyo Jung a,∗ a Department of Biomedical Engineering, and Center for Marine-Integrated Biomedical Technology (BK21 Plus) Pukyong National University, Busan 608-737, Republic of Korea b Institute of Marine Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea c Department of Biomechatronic Engineering College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Republic of Korea d Department of Otolaryngology, Chonnam National University Hospital, Gwangju, Republic of Korea e Research Center for Resistant Cells, Chosun University, Gwangju, Republic of Korea f Department of Physiology, Kangwon National University School of Medicine, Chuncheon, Republic of Korea g Department of Microbiology, College of Medicine Inje University, Busan, Republic of Korea h College of Food Science and Technology, Guangdong Ocean University, Zhanjiang 524088, China
a r t i c l e
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Article history: Received 3 January 2015 Received in revised form 1 May 2015 Accepted 5 May 2015 Available online xxx Keywords: Pavlova lutheri Purified peptide Melanogenesis Reactive oxygen species
a b s t r a c t A novel peptide was purified from marine microalga, Pavlova lutheri fermented by yeast Hansenula polymorpha, and its protective effect against oxidative stress as well as inhibitory effect on melanogenesis was investigated. The purified peptide has a molecular mass of 526 Da and its amino acid sequence was determined as Met-Gly-Arg-Tyr by Q-TOF mass spectroscopy. The purified peptide scavenged DPPH, hydroxyl radicals and hydrogen peroxide (H2 O2 ) at the IC50 values of 0.285, 0.068 and 0.988 mM, respectively. Intracellular reactive oxygen species (ROS) induced H2 O2 was attenuated by addition of the purified peptide. The purified peptide demonstrated inhibitory properties against ␣-MSH-induced melanogenesis via melanin content and tyrosinase (TYR) inhibition in B16F10 melanoma cells. We also found that the purified peptide decreased melanogenesis-related proteins; microphthalmia-associated transcription factor (MITF) and TYR protein expressions. Moreover, the purified peptide activated extracellular signalregulated kinase (ERK) but not that of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). A specific ERK inhibitor significantly blocks the purified peptide-inhibited melanin synthesis and TYR activity. Hence, these results indicated that the purified peptide isolated from P. lutheri has potential whitening effects and prominent protective effects on oxidative stress-induced cell damages, which might be used in pharmaceutical and cosmeceutical industries. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae are the most important and prominent primary producers in marine ecosystem. The fine structure, higher protein content, faster growth even in harsh environment conditions,
∗ Corresponding author. Tel.: +82 51 629 5775; fax: +82 51 629 5775. ∗∗ Corresponding author. Tel.: +86 759 2396270; fax: +86 759 2396270. E-mail addresses: [email protected] (Z.-J. Qian), [email protected] (W.-K. Jung). 1 These authors contributed equally to this study.
makes microalgae an excellent source of bioactive molecules [1,2]. Moreover, microalgae have gain higher attention than ever before due to high availability of proteins. For instance, some microalgae species such as Chlorella sp., Dunaliella sp., Scenedesmus obliquus, Arthrospira sp. and cyanobacteria Spirulina sp. have recently shown higher protein content [3]. A microalga haptophyte, P. lutheri is widely used live feed in aquaculture especially marine invertebrates in particular molluscs, crustaceans, and zooplanktons [4–6]. The specific yeast species kwon as thermotolerant methlotrophic yeast only use methanol as source of carbon and energy. Hansenula polymorpha (Pichia angusta) belongs to
http://dx.doi.org/10.1016/j.procbio.2015.05.007 1359-5113/© 2015 Elsevier Ltd. All rights reserved.
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a limited number of methanol-utilizing yeast species such as Candida boidinii, P. pastoris, P. methanolica and H. polymorpha [7] and used to fermentation of P. lutheri. The H. polymorpha is generally using to attractive platform for production of recombinant protein, in particular pharmaceuticals, for examples hepatitis B vaccines, interferon alpha-2a, hirudin, insulin, and phytase [8]. In our previous study, fermented P. lutheri by H. polymorpha was exhibited antioxidant activity [7]. However, the bioactivities of fermented P. lutheri by H. polymorpha used in this study have been rarely reported. In addition, the antioxidative peptide of fermented P. lutheri has not yet been reported. Additionally, P. lutheri protein can be converted into value-added products by fermentation by yeast, which may be widely applied to improve the functional properties. Melanin synthesis induced by various inner factors such as ␣-melanocyte-stimulating hormone (␣-MSH) and environmental factors such as ultraviolet radiation (UV). Further, melanin synthesis is mainly controlled by tyrosinase (TYR) produced in specialized dendritic cells known as melanocyte, which located in the basal layer of skin [9]. Melanin is responsible for hair, eyes, skin color and plays a pivotal role in protection the skin against UV-induced DNA damage [10]. However, results of the abnormal melanin synthesis in the skin can leads problematic conditions such as a freckle, liver spots, and melasma known as hyperpigmentary disorders [11]. TYR is copper-containing, the most important enzyme involved in melanin synthesis in mammalian [12]. The microphtalmia-associated transcription factor plays an important role in expression of melanogenesis-relatd protein such as TYR [13]. The enzyme can convert L -tyrosine into two types of melanin such as black-brown eumelanin and red-yellow pheomelanin within membrane-bound cytoplasmic organelles called melanosomes and the transport of melanosomes to neighboring keratinocytes [14]. Reactive oxygen species (ROS) such as hydroxyl radicals (OH• ), superoxide anion (O2 •− ) and hydrogen peroxide (H2 O2 ) are generated as a consequence of cell metabolism within mitochondria [15]. ROS are highly unstable and rapidly react with nearby biomacromolecules such as proteins, lipids and DNA, resulting cells, tissue and entire organs damage in the body. A previous study has shown that ROS could play an important role in regulating melanogenesis, while ROS scavengers and inhibitors reduced hyper-pigmentation or suppressed UV-induced melanogenesis on melanocytes [16,17]. In addition, antioxidants such as vitamin C and E inhibited UV radiation-induced melanocyte proliferation and melanogenesis on mice skin [18]. Hence, antioxidant compounds are excellent agents that can be employed as inhibitors against abnormal production of melanin in melanocytes. The objective of this study was to isolate a novel peptide from fermented P. lutheri and identify the purified peptide with regards to anti-oxidant and anti-melanogenesis effects. Furthermore, we investigated the anti-melanogenesis action by expression of melanogenesis-related proteins and mitogen-activated protein kinases (MAPKs) signaling pathway in ␣-MSH-induced B16F10 mouse melanoma cells.
2. Materials and methods 2.1. Materials P. lutheri (KMCC H-006) used in this study was generously provided by Korea Marine Microalgae Culture Center. The H. polymorpha (KCTC 7538) from Korea Biological Resource Center and culture medium were maintained in standard F/2 (Guillard’s) medium. Dulbecco’s modified eagle’s medium (DMEM), trypsinethylenediaminetetraacetic acid (trypsin-EDTA), fetal bovine serum (FBS) and 5-(and-6)-carboxy-2 ,7 -dichlorofluorescein
diacetate (carboxy-DCF-DA) were obtained from Gibco BRL Life Technologies (Grand Island, NY, USA). Dimethyl sulfoxide (DMSO), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT), ␣-MSH, dihydroxylphenylalanine (L-DOPA) and Hoechst 33342 (HO33342) were purchased from Sigma Co. (St. Louis, MO, USA). The specific antibodies used for western blot analysis were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The other chemicals and reagents used were of analytical grade. 2.2. Preparation and identification of fermented P. lutheri Fermentation of P. lutheri by H. polymorpha was performed according to the method used by Qian et al. [7]. For the preparation of fermented P. lutheri, P. lutheri powder was added to water at a ratio of 1:15 (w/v). Then, the cellulase added to solution at a ratio of 100:1 (w/w) at 37 ◦ C for 8 h (pH 6.0) for degradation of cell wall. After autoclaving at 121 ◦ C for 30 min, the solution was obtained, and the H. polymorpha culture broth was added to the solution at a concentration of 1% (v/v), which well mixed and then incubated at 37 ◦ C for 12 days. The recovered fermented P. lutheri was lyophilized and stored at −20 ◦ C until used. The fermented P. lutheri were loaded onto HiPrep 16/10 CM FF ion-exchange column equilibrated with 20 mM sodium acetate buffer (pH 4.0), and eluted with a linear gradient of NaCl (0–2 M) in the same buffer at a flow rate of 2 mL/min using fast protein liquid chromatography (FPLC). The UV absorbance at 280 nm was monitored in each 4 mL fractions, then pooled and lyophilized, immediately. The lyophilized fractions were further purified on a Primesphere ODS C18 column permeation reversed-phase high performance liquid chromatography (RP-HPLC) with a linear gradient of acetonitrile (0–30% in 30 min) containing 0.1% trifluoroacetic acid (TFA) at a flow rate of 1.0 mL/min. Then the accurate molecular mass and amino acid sequence of purified peptide were ascertained by quadrupole time of flight (Q-TOF) mass spectroscopy (Micromass, Altrincham, UK) coupled to electrospray ionization (ESI) source. 2.3. Radical scavenging activity by electron spin resonance (ESR) spectrometer Radical scavenging activity of the purified peptide was calculated as a scavenging percentage by, S = (H0 − H1 )/H0 × 100%; where, H1 and H0 were ESR signal intensities in the presence and the absence of test sample, respectively. 2.3.1. DPPH radical scavenging activity DPPH radical scavenging activity was measured using the method described by Nanjo et al. [19]. A 30 L sample solution was added to 30 L of DPPH (60 M/L) in ethanol solution. After mixing vigorously for 10 sec, the solution was then transferred into a quartz capillary tube, and the scavenging activity of the purified on DPPH radical was measured using a JES-FA ESR spectrometer (JEOL Ltd., Tokyo, Japan). The spin adduct was measured on an ESR spectrometer exactly 2 min later. Experimental conditions as follows: magnetic field, 336.5 ± 5 mT; power, 5 mW; modulation frequency, 9.41 GHz; amplitude, 1 × 1000; sweep time, 30 sec. DPPH radical scavenging ability was calculated following equation in which H and H0 were relative peak height of radical signals with and without sample, respectively. 2.3.2. Hydroxyl radical scavenging activity Hydroxyl radicals were generated by iron-catalyzed Fenton Haber–Weiss reaction and the generated hydroxyl radicals were rapidly reacted with nitrone spin trap DMPO [20]. The resultant DMPO-OH adducts was detectable with an ESR spectrometer. The sample solution 20 L was mixed with 0.3 M DMPO 20 L, 10 mM
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FeSO4 20 L and 10 mM H2 O2 20 L in a phosphate buffer solution (pH 7.4), and then transferred into a quartz capillary tube. After 2.5 min, the ESR spectrum was recorded using an ESR spectrometer. The experimental conditions employed were as follows: magnetic field, 336.5 ± 5 mT; power, 1 mW; modulation frequency, 9.41 GHz; amplitude, 1 × 200; sweep time, 4 min. Hydroxyl radical scavenging ability was calculated following equation in which H and H0 were relative peak height of radical signals with and without sample, respectively. 2.4. ABTS radical scavenging assay H2 O2 scavenging activity was determined by a colorimetric assay according to the method of Müller [21]. Phosphate buffer (100 L, 0.1 M, pH 5.0) and sample solution were mixed in a 96microwell plate. H2 O2 (20 L) was added to the mixture, which was then incubated for 5 min at 37 ◦ C. After incubation, 30 L of 1.25 mM ABTS and 30 L of peroxidase (1 unit/mL) were added to the mixture and it was then incubated for 10 min at 37 ◦ C. The absorbance was read with an ELISA plate reader (BioTek Instruments, Winooski, VT) at 405 nm. 2.5. Cell culture and cytotoxic assessment using MTT assay B16F10 melanoma cells were cultured in DMEM containing 10% (v/v) FBS, 1% penicillin/streptomycin, and 5% CO2 humidified atmosphere at 37 ◦ C. The cytotoxic effect of the purified peptide on cells was determined by the MTT assay. The cells were seeded in 96-well plates. After 24 h, cells were treated with various concentrations of the purified peptide for 48 at 37 ◦ C. The MTT stock solution (50 L) was then added to each well to obtain a total reaction volume of 250 L. After 4 h of incubation, the plates were centrifuged (800 × g, 5 min), and the supernatants were aspirated. The formazan crystals in each well were dissolved in 150 L of DMSO, and the absorbance was measured with an ELISA plate reader at 540 nm. 2.6. Genomic DNA extraction Genomic high molecular weight DNA was extracted from B16F10 melanoma cells using standard phenol/proteinase K procedure with slight modifications [22]. Briefly, cells culturing in 10 cm dishes were washed twice with PBS and scraped into 1 mL of PBS containing 5 mM EDTA. After centrifugation cells were dissolved in RNase (0.5 mg/mL), 0.2 M sodium acetate, proteinase K (10 mg/mL), and 10% sodium dodecyl sulfate (SDS). The mixture was incubated for 30 min at 37 ◦ C and 1 h at 55 ◦ C. Following incubation, phenol: chloroform: isoamylalcohol (25:24:1) was added at 1:1 ratio and mixture was centrifuged at 12,000 × g for 5 min at 4 ◦ C. Supernatants were mixed with 100% ice cold ethanol at 1:1.5 ratio and kept for 15 min at −20 ◦ C. After centrifugation at 12,000 × g for 5 min, the pellet was dissolved in TE buffer and purity of DNA was ELISA reader determined at 260/280 nm. Further, the quality of isolated DNA was evaluated with 1% agarose gel electrophoresis in 40 mM Tris–acetate buffer. 2.7. Protective effect of the purified peptide against hydroxyl radical-induced DNA damage Hydroxyl radical mediated DNA oxidation was determined according to Ngo et al. [23]. Briefly, 40 L of DNA reaction mixture were prepared by adding pre-determined concentrations of test sample (or same volume of distilled water as control), 100 M final concentration of EDTA. An aliquot of the reaction mixture containing about 1 g of DNA was electrophoresed on a 1% agarose gel
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for 40 min at 100 V. Gels were then stained with 1 mg/mL ethidium bromide and visualized under UV light. 2.8. Intracellular radical measurement Intracellular production of ROS was detected by oxidation of the cell permeable fluorescence dye 5-(and-6)-carboxy-2 ,7 dichlorofluorescein diacetate (carboxy-DCFH-DA) to fluorescent DCF by H2O2 [24]. B16F10 melanoma cells were seeded in 24-well plate. After 24 h, cells were pre-treated with various concentrations of the purified peptide. After 1 h, H2 O2 was added at a concentration of 1 mM, and then cells incubated for an additional 1 h at 37 ◦ C under a humidified atmosphere. Finally, 40 M carboxy-DCFH-DA was introduced to the cells, and carboxy-DCFH-DA was imaged using fluorescence microscopy. 2.9. Measurement of cellular melanin content The melanin content was measured using a slight modification of a method reported previously [25]. Briefly, B16F10 melanoma cells plated on 6-well plates were pre-incubated and subsequently treated with ␣-MSH coupled with aliquots of the purified peptide at 37 ◦ C. After 48 h, the cells were washed twice with cold PBS and lysed in PBS buffer containing 1% (v/v) Triton X-100. The lysates were clarified by centrifugation at 13,000 × g for 10 min. The supernatants removed and dissolved in 1 M NaOH containing 10% DMSO at 90 ◦ C for 1 h. The relative melanin content was determined by measuring the absorbance at 405 nm in an ELISA reader. 2.10. Measurement of cellular tyrosinase activity Cellular TYR activity was estimated by measuring the formation rate of DOPA chrome from DOPA [26]. B16F10 melanoma cells plated on 6-well plates were pre-incubated and subsequently treated with ␣-MSH coupled with aliquots of the purified peptide at 37 ◦ C. After 48 h, the cells were washed twice with cold PBS and lysed in PBS buffer containing 1% (v/v) Triton X-100. The lysates were clarified by centrifugation at 13,000 × g for 10 min. After protein quantification and normalization, cell lysates (each sample contained the same amount of protein) were incubated in duplicate with 10 mM L-DOPA at 37 ◦ C for 1 h. After incubation, DOPA chrome was monitored by measuring the absorbance at 475 nm. 2.11. Western blot analysis The cells were lysed in lysis buffer (20 mM Tris, 5 mM EDTA, 10 mM Na4 P2 O7 , 100 mM NaF, 2 mM Na3 VO4 , 1% NP-40, 10 mg/mL aprotinin, 10 mg/mL leupeptin and 1 mM PMSF) for 60 min and then centrifuged at 12,000 rpm and 4 ◦ C for 15 min. The protein concentrations were determined using the BCATM protein assay kit. The lysate containing 40 g of protein was subjected to electrophoresis on a SDS-polyacrylamide gel, and the gel was transferred onto a nitrocellulose membrane. The membrane was blocked with 5% non fat dry milk in TBS-T (25 mM Tris–HCl, 137 mM NaCl, 2.65 mM KCl, and 0.05% Tween 20, pH 7.4) for 2 h. The primary antibodies were used at a 1:1000 dilution. The membranes were incubated with the primary antibodies at 4 ◦ C overnight, washed with TBS-T and then incubated with the secondary antibodies at 1:3000 dilutions. The signals were developed using an ECL western blotting detection kit and quantified using the western imaging system (Fujifilm Life Science, Tokyo, Japan). 2.12. Statistical analysis All of data are presented as the mean ± standard deviation (SD) of three determinations. Statistical comparisons of the mean
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Fig. 1. Identification of molecular mass and amino acid sequence of purified peptide. MS/MS experiments were performed on a Q-TOF tandem mass spectrometer equipped with a nano-ESI source. Sequencing of the prurified peptide was acquired over the m/z range 0–1000 and sequenced by using the PepSeq de nove sequencing algorithm.
values were performed by analysis of variance (ANOVA), followed by Duncan’s multiple range test using SPSS software. The statistical significance of the differences was defined at the p < 0.05 level.
3. Results 3.1. Identification of the purified peptide A novel peptide was purified from the fermented P. lutheri using chromatographic methods, combining FPLC on a HiPrep 16/10 DEAE FF anion-exchange column and repeated RP-HPLC on a Primesphere column (data not shown). The molecular mass of the fermented P. lutheri purified a novel peptide was determined to be 526 Da by Q-TOF mass spectroscopy coupled to an ESI source. Its full amino acid sequence was composed of Met-Gly-Arg-Tyr (Fig. 1).
3.4. DNA protective effect of the purified peptide from hydroxyl-Radical Genomic DNA was isolated from B16F10 melanoma cells to study protective effects of purified peptide against DNA oxidative damage. In this experiment the combined effects of 100 M FeSO4 and 50 M H2 O2 on the integrity of genomic DNA was studied by DNA electrophoresis in the presence or absence of the purified peptide. After reaction, almost all DNA was degraded in the control group treated only with Fe (II)–H2 O2 . However, the treatment with the purified peptide exhibited a dose-dependent suppression of DNA degradation against • OH radical mediated damage at concentrations ranging from 25 to 200 M (Fig. 3A). The results of this study clearly explain that the purified peptide can prevent oxidative
3.2. ROS scavenging activity of the purified peptide To explore cell-free system scavenging activities of the purified peptide against DPPH and hydroxyl radicals, we measured radical signals by electron spin-trapping technique. Also, the purified peptide was evaluated for its H2 O2 scavenging activity by ABTS assay. The IC50 value was used an index for the concentration of the purified peptide at 50% reducing of radical. IC50 values of the purified peptide against DPPH, hydroxyl radical and H2 O2 were 0.285 ± 0.003, 0.068 ± 0.001 and 0.988 ± 0.011 mM, respectively. 3.3. Effect of the purified peptide on viability in B16F10 melanoma cells In this study, B16F10 melanoma cells were treated with different concentrations of the purified peptide to determine non-cytotoxic effects for further experiments. The cell viability data confirmed that the purified peptide was non-cytotoxic in B16F10 melanoma cells (Fig. 2). Therefore, it was determined that the purified peptide could be used in further experiments.
Fig. 2. The cytotoxic effect of the purified peptide on viability in B16F10 melanoma cells. Cells were treated with the purified peptide at the indicated concentrations (50, 100 and 200 M). After 24 h to treat the purified peptide cell viability was assessed by MTT assay. Values are expressed as mean ± S.D. in triplicate experiments.
Please cite this article in press as: Oh G-W, et al. A novel peptide purified from the fermented microalga Pavlova lutheri attenuates oxidative stress and melanogenesis in B16F10 melanoma cells. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.05.007
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or H2 O2 . However, the purified peptide pretreatment reduced the fluorescence intensity values in a dose-dependent manner in the cells induced by H2 O2 treatment. These results indicate that the purified peptide significantly reduced the evaluated ROS levels induced by H2 O2 , and suggest that the purified peptide possesses intracellular ROS scavenging activity.
3.6. Inhibitory effect of the purified peptide on melanin content and tyrosinase activity in ˛-MSH-induced B16F10 cells To verify the ability of the purified peptide on ␣-MSH-mediated melanogenesis, determined the quantity of intracellular melanin and TYR activity in the presence of ␣-MSH. As shown in Fig. 4, the purified peptide substantially decreased the ␣-MSH-induced cellular melanin contents in a dose-dependent manner compared to the ␣-MSH alone treated group. Likewise, in the TYR activity, the purified peptide also reduced the ␣-MSH-increased intracellular TYR activity in a dose-dependent manner (Fig. 4B). These results suggest that the purified peptide down-regulated TYR activity and that this inhibitory effect may lead to decrease cellular melanin synthesis in B16F10 cells.
3.7. Effect of the purified peptide on tyrosinase and MITF expression in ˛-MSH-stimulated B16F10 cells To elucidate the mechanisms underlying the anti-TYR and antimelanogenic activities of the purified peptide, we examined the effects of the purified peptide on the expression levels of the melanogenic enzymes such as TYR by Western blot. As shown in Fig. 5, the purified peptide at 50–200 M dose-dependently reduced the expression of TYR at 48 h. Since the TYR is transcriptionally regulated by MITF, we next examined the influence of the purified peptide on MITF expression. Results showed that the purified peptide also dose-dependently inhibited MITF expression in B16F10 cells (Fig. 5).
Fig. 3. (A) DNA oxidative damage protection by the purified peptide. Genomic DNA from B16F10 cells was pre-treated with peptide and exposed to • OH using Fenton chemistry. Line 1, Blank (DNA 5 L); line 2, FeSO4 and H2 O2 (DNA damage control); lines 3–6, FeSO4 and H2 O2 in the presence of peptide with different concentrations (25–200 M), respectively. (B) The intracellular radical generated was detected by DCFH-DA staining. Carboxy-2 ,7 -diclorofluorescein diacetate (Carboxy-DCFDA) was used to evaluate intracellular oxidant formation. Digital images of DCF fluorescence were obtained with a fluorescence microscope system. Bar graph exhibited fluorescence intensity and line graph showed the intracellular radical scavenging activity. Values are expressed as mean ± S.D. in triplicate experiments. Statistical evaluation was performed to compare the experimental groups and H2 O2 -treated cells. *p < 0.05 and **p < 0.01.
damage to DNA when DNA is exposed to hydroxyl radical generated by Fe (II)/H2 O2 . Fe2+ catalyses the conversion of H2 O2 , which is a major route to the synthesis of hydroxyl radical in biological systems. 3.5. Inhibitory effect of the purified peptide on intracellular ROS generated by H2 O2 Intracellular ROS scavenging activity could be detected via measurements of the level of DCF, the results of which are illustrated in Fig. 3B. The fluorescence intensity value in B16F10 melanoma cells increased significantly after treatment with H2 O2 compared to that in the untreated cells, which did not contain either the peptide
3.8. Effect of the purified peptide on phosphorylation of MAPKs pathway in ˛-MSH-stimulated B16F10 cells We examined the influence of the purified peptide treatment on the activation of JNK, ERK and p38 MAPKs in an attempt to further understand the molecular mechanisms involved in the hypopigmentation property of the purified peptide by Western blot. As shown in Fig. 6, ERK phosphorylation was significantly enhanced after the purified peptide treatment. On the other hand, p38 and JNK phosphorylation was remain unchanged by the purified peptide (Fig. 6). Thus, results imply that the purified peptideinduced inhibition of melanogenesis occurs through regulation of ERK phosphorylation. To further confirm and refined the role of ERK signaling on the purified peptide-induced anti-melanogenic effect, we employed a specific inhibitor of ERK, PD98059, which blocks ERK signaling. As shown Fig. 7A and B, PD98059 treatment markedly restored melanin synthesis and TYR activity suppressed by the purified peptide. Additionally, to confirm our results, we examined the effect of PD98059 on the expression of MITF in the presence of ␣-MSH or the purified peptide Fig. 7C shows that the purified peptide-induced suppression of ␣-MSH-induced MITF expression was inhibited by PD98059. We next examined whether PD98059 inhibits the ERK pathway in ␣-MSH-stimulated B16F10 cells, and found that PD98059 does in fact block ERK activation in the purified peptide treated B16F10 cells (Fig. 7D). Thus, results imply that the purified peptide-induced inhibition of melanogenesis occurs through phosphorylation ERK.
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Fig. 4. Effect of the purified peptide on cellular (A) melanin content and tyrosinase activity in B16F10 mouse melanoma cells. Cells were exposed 50 nM ␣-MSH in the presence of the purified peptide (50–200 M). The percentage values of the treated cells are expressed relatively compared to that in the untreated cells. Values are expressed as mean ± S.D. in triplicate experiments. Statistical evaluation was performed to compare the experimental groups and ␣-MSH-treated cells. *p < 0.05 and **p < 0.01.
Fig. 5. Effect of the purified peptide on the expresstion of melanogenesis-related proteins in B16F10 melanoma cells. Cells were exposed 50 nM ␣-MSH in the presence of the purified peptide (50–200 M). The expression levels of the MITF and tyrosinase proteins were examined by western blot. GAPDH was used as an internal control. Values are expressed as mean ± S.D. in triplicate experiments. Statistical evaluation was performed to compare the experimental groups and ␣-MSH-treated cells. *p < 0.05 and **p < 0.01.
Fig. 6. Effect of the purified peptide on phosphorylation of MAPKs in B16F10 melanoma cells. Cells were exposed 50 nM ␣-MSH in the presence of the purified peptide (50–200 M). The expression levels of the p-p38, p38, p-JNK, JNK, p-ERK and ERK proteins were examined by Western blot. Values are expressed as mean ± S.D. in triplicate experiments. Statistical evaluation was performed to compare the experimental groups and ␣-MSH-treated cells. *p < 0.05 and **p < 0.01.
Please cite this article in press as: Oh G-W, et al. A novel peptide purified from the fermented microalga Pavlova lutheri attenuates oxidative stress and melanogenesis in B16F10 melanoma cells. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.05.007
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Fig. 7. Effect of PD98059 on the purified peptide activities in ␣-MSH-stimulated B16F10 melanoma cells. Cells were exposed 50 nM ␣-MSH in the presence and absence of the purified peptide (200 M) and PD98059 (10 M). Effect of PD98059 on (A) melanin content and (B) tyrosinase activity. Melanin content and tyrosinase activity in control cells were regarded as 100%. Values are expressed as mean ± S.D. in triplicate experiments. *p < 0.05. The expression levels of (C) MITF and (D) phosphor-ERK and ERK proteins were examined by Western blot. Data shown are the representative of three independent Western blotting experiments.
4. Discussion Recently, due to the relatively limited natural products of land, many researchers have focused on marine natural products with various biological activities. Microalgae have been popular food stuff worldwide, and there are rich in protein, minerals, carbohydrates, and bioactive substance [2]. Therefore, it is thought that microalgae could be developed as useful functional materials. Additionally, according to previous study, the amino-acids patterns of the microalgal proteins show high nutritive value and availability of essential amino acids [27]. Among the microalgae, P. lutheri is the most important primary producers in aquatic environments and they have high concentration protein. Fermented P. rutheri by yeast C. rugopelliculosa or H. polymorpha have become good candidates for sources of natural antioxidants, as revealed by a number of recent studies [7,27]. According to previous study, ROS is play an important roles in the regulating the melanogenesis and melanocytes proliferation [28,29]. Scavengers and inhibitors of ROS formation can reduce melanogenesis in melanocyte. Although some reports suggest that fermented P. lutheri exhibit antioxidant effect on free radicals, it is no reports in the protective effect of the purified peptide from fermented P. lutheri on the cell damage induced by oxidative stress and its inhibitory effect on mealnogenesis, and the underlying mechanisms. Thus, in this study, the potential whitening effect of the purified peptide isolated from fermented P. lutheri was investigated. Interest has emerged to identify and characterize bioactive peptides from microalgae proteins [2,30]. These bioactive peptides can be derived by fermentation of microalgae proteins. Additionally, fermented substances are also a source of bioactive peptides, which are short peptides released from proteins that have been shown to exert biological activities [31,32]. Biological properties of the peptides were highly influenced by molecular weight and structure [33]. In general, antioxidative peptides containing aromatic acid residues (tyrosine, tryptophan and phenylalanine) at the
C-terminus have strong radical scavenging activities [34]. The aromatic amino acid residues can make active oxygen stable through direct electron transfer [35]. As shown the results of this study, the presence of tyrosine in the sequence could have attributed to the antioxidative effect of the purified peptide from fermented P. lutheri. ROS including free radicals such as hydroxyl radicals, and superoxide radicals and non-free radical species such as H2 O2 perform important functions in cell signaling. Cell damages are normally able to defend themselves against oxidative stress by the use of endogenous antioxidant enzymes, including superoxide dismutases, catalases and glutathione peroxidases [36]. However, an imbalance between ROS and antioxidant defense mechanisms can result in oxidative modification of the intracellular molecules [37]. Hydroxyl radicals generated by the Fenton reaction are known to cause oxidative breaks in DNA strands to yield its open circular or relaxed forms [38]. Also, H2 O2 has been extensively used as an inducer of oxidative stress in vitro models [36]. Therefore, in this study, we report the protective effects of the purified peptide against ROS-induced cell damage for antioxidant. The results presented here indicated that the purified peptide reduced DNA damage by hydroxyl radicals, and suppressed intracellular ROS produced by H2 O2 . These results suggest that the purified peptide from fermented P. lutheri has the ability to protect cells from oxidative stress-related cellular injuries. Melanogenesis is a physiological process resulting in the synthesis of melanin pigment, which plays a crucial protective role against skin photo-carcinogenesis [39]. Among the process of melanogenesis, TYR participates in oxidative steps of melanin synthesis that convert L -tyrosine into L -DOPA and other intermediates through hydroxylation of L -tyrosine on first step in melanocytes [40,41]. Also, melanin content correlates directly with the activity of TYR [42]. Therefore, we investigated whether the purified peptide could inhibit melanin synthesis in the presence of ␣-MSH. We found that the purified peptide inhibited
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melanin synthesis, activity of TYR and protein levels of TYR in a dose-dependent manner. These results suggest that the purified peptide may down-regulate TYR activity and inhibit cellular melanin synthesis in B16F10 melanoma cells. In addition, ␣-MSH potently induces MITF expression, which is regulated at the levels of TYR [42]. Moreover, MITF is crucial role in the control of melanocyte differentiation, proliferation, survival and melanin synthesis [43–45]. Therefore, MITF expression is appropriate target for cosmeceutical treatment of melanogenesis. Thus, we examined the inhibitory effect of the purified peptide on the expression of MITF in ␣-MSH-stimulated B16F10 melanoma cells. The purified peptide attenuated ␣-MSH-induced expression of MITF. These results suggest that the purified peptide does not directly inhibit TYR activity, and that the purified peptide inhibitory effect coordinate MITF down-regulation in B16F10 melanoma cells. To evaluate the mechanism of the inhibitory effect of the purified peptide on melanogenesis, we examined the activation factor MAPKs. One of the extensively investigated transduction mechanisms involved in the melanogenesis process is the MAPKs pathway [42,46]. Previous studies have revealed that phosphorylation of p38 MAPK can induce through activate MITF expression, and also phosphorylation of JNK and ERK can reduce melanogenesis through MITF phosphorylation and its subsequent degradation on B16F10 cells [47–51]. We therefore examined the influence of the purified peptide treatment on the activation of JNK, ERK and p38 MAPK attempting to further understand the molecular mechanisms involved in the pigmentation property of the purified peptide by western blot assay. The results showed that phosphorylation of ERK were significantly enhanced after the purified peptide treatment. On the other hand, JNK and p38 MAPK phosphorylation were no changed by the purified peptide. To determine which ERK plays an important role in regulating the purified peptide-induced melanogenesis, we treated B16F10 cells with the ERK-specific inhibitor PD98059. We found that PD98059 markedly increase the purified peptidesuppressed melanin synthesis and TYR activity. We also confirmed that the purified peptide-induced suppression of MITF expression was inhibited by PD98059 in ␣-MSH-stimulated B16F10 cells. This finding suggested that the purified peptide-induced antimelanogenesis in B16F10 melanoma cells through ERK mediated pathways. In conclusion, we evaluated the anti-oxidant and antimelanogenesis of a purified peptide from the fermented microalga, P. lutheri by yeast H. polymorpha. The purified peptide exerts a profound protective effect against oxidative stress. In addition, the purified peptide suppresses melanin synthesis and TYR activity through ERK pathway-mediated suppression of MITF and TYR in ␣-MSH-stimulated B16F10 melanoma cells. These results indicated that the purified peptide from P. lutheri can be used as an effective natural source to make cosmeceutical and pharmaceutical products.
[2]
[3] [4] [5] [6] [7]
[8] [9]
[10] [11] [12]
[13] [14]
[15]
[16]
[17]
[18]
[19]
[20] [21]
[22]
[23]
[24]
Acknowledgments [25]
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2013R1A1A1A05013577), and also supported by grant from Marine Technology Application Program (PJT200673) and Marine Biotechnology Program (20150220) Funded by Ministry of Oceans and Fisheries, Republic of Korea.
[26]
[27]
[28]
References [29] [1] Nguyen MHT, Qian ZJ, Nguyen VT, Choi IW, Heo SJ, Oh CH, Kang DH, Kim GH, Jung WK. Tetrameric peptide purified from hydrolysates of biodiesel byproducts of Nannochloropsis oculata induces osteoblastic differentiation through
MAPK and Smad pathway on MG-63 and D1 cells. Process Biochemistry 2013;48:1387–94. Ko SC, Kang N, Kim EA, Kang MC, Lee SH, Kang SM, Lee JB, Jeon BT, Kim SK, Park SJ. A novel angiotensin I-converting enzyme (ACE) inhibitory peptide from a marine Chlorella ellipsoidea and its antihypertensive effect in spontaneously hypertensive rats. Process Biochemistry 2012;47:2005–11. Becker E. Micro-algae as a source of protein. Biotechnology Advances 2007;25:207–10. Borowitzka MA. Microalgae for aquaculture: opportunities and constraints. Journal of Applied Phycology 1997;9:393–401. Wikfors GH, Ohno M. Impact of algal research in aquaculture. Journal of Phycology 2001;37:968–74. Volkman JK, Dunstan GA, Jeffrey S, Kearney PS. Fatty acids from microalgae of the genus Pavlova. Phytochemistry 1991;30:1855–9. Qian ZJ, Jung WK, Kang KH, Ryu B, Kim SK, Je JY, Heo SJ, Oh C, Kang DH, Park WS. In Vitro antioxidant activities of the fermented marine microalga Palova lutheri (Haptophyta) with the yeast Hansenula polymorpha. Journal of Phycology 2012;48:475–82. C¸elik E, C¸alık P. Production of recombinant proteins by yeast cells. Biotechnology Advances 2012;30:1108–18. Park H, Kosmadaki M, Yaar M, Gilchrest B. Cellular mechanisms regulating human melanogenesis. Cellular and Molecular Life Sciences 2009;66:1493–506. Ortonne JP. Photoprotective properties of skin melanin. British Journal of Dermatology 2002;146:7–10. Briganti S, Camera E, Picardo M. Chemical and instrumental approaches to treat hyperpigmentation. Pigment Cell Research 2003;16:101–10. Hearing VJ. Biochemical control of melanogenesis and melanosomal organization. Journal of Investigative Dermatology Symposium Proceedings 1999;4:24–8 [Nature Publishing Group]. Yamaguchi Y, Hearing VJ. Physiological factors that regulate skin pigmentation. Biofactors 2009;35:193–9. Ito S, Wakamatsu K. Quantitative analysis of eumelanin and pheomelanin in humans, mice, and other animals: a comparative review. Pigment Cell Research 2003;16:523–31. Feissner RF, Skalska J, Gaum WE, Sheu SS. Crosstalk signaling between mitochondrial Ca2+ and ROS. Frontiers in Bioscience: A Journal and Virtual Library 2009;14:1197–218. Yoshimura M, Watanabe Y, Kasai K, Yamakoshi J, Koga T. Inhibitory effect of an ellagic acid-rich pomegranate extract on tyrosinase activity and ultraviolet-induced pigmentation. Bioscience, Biotechnology, and Biochemistry 2005;69:2368–73. Yamakoshi J, Otsuka F, Sano A, Tokutake S, Saito M, Kikuchi M, Kubota Y. Lightening effect on ultraviolet-induced pigmentation of guinea pig skin by oral administration of a proanthocyanidin-rich extract from grape seeds. Pigment Cell Research 2003;16:629–38. Quevedo Jr WC, Holstein TJ, Dyckman JACOB, McDonald CJ, Isaacson EL. Inhibition of UVR-induced tanning and immunosuppression by topical applications of vitamins C and E to the skin of hairless (hr/hr) mice. Pigment Cell Research/Sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society 2000;13:89–98. Nanjo F, Goto K, Seto R, Suzuki M, Sakai M, Hara Y. Scavenging effects of tea catechins and their derivatives on 1,1-diphenyl-2-picrylhydrazyl radical. Free Radical Biology and Medicine 1996;21:895–902. Rosen GM, Rauckman EJ. Spin trapping of superoxide and hydroxyl radicals. Methods in Enzymology 1984;105:198–209. Müller HE. Detection of hydrogen peroxide produced by microorganisms on an ABTS peroxidase medium. Zentralblatt für Bakteriologie, Mikrobiologie und Hygiene Series A: Medical Microbiology, Infectious Diseases, Virology, Parasitology 1985;259:151–4. Sambrook J, Russell DW, Russell DW. Molecular cloning: a laboratory manual (3-volumeset). Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2001. Ngo DN, Kim MM, Qian ZJ, Jung WK, Lee SH, Kim SK. Free radical-scavenging activities of low molecular weight chitin oligosaccharides lead to antioxidant effect in live cells. Journal of Food Biochemistry 2010;34:161–77. Mendis E, Kim MM, Rajapakse N, Kim SK. An in vitro cellular analysis of the radical scavenging efficacy of chitooligosaccharides. Life Sciences 2007;80:2118–27. Tsuboi T, Kondoh H, Hiratsuka J, Mishima Y. Enhanced melanogenesis induced by tyrosinase gene-transfer increases boron-uptake and killing effect of boron neutron capture therapy for amelanotic melanoma. Pigment Cell Research 1998;11:275–82. Shen T, Heo SI, Wang MH. Involvement of the p38 MAPK and ERK signaling pathway in the anti-melanogenic effect of methyl 3, 5-dicaffeoyl quinate in B16F10 mouse melanoma cells. Chemico-Biological Interactions 2012;199: 106–11. Ryu B, Kang KH, Ngo DH, Qian ZJ, Kim SK. Statistical optimization of microalgaePavlova lutheri cultivation conditions and its fermentation conditions by yeast, Candida rugopelliculosa. Bioresource Technology 2012;107:307–13. Kim YJ. Antimelanogenic and antioxidant properties of gallic acid. Biological and Pharmaceutical Bulletin 2007;30:1052–5. Chou TH, Ding HY, Hung WJ, Liang CH. Antioxidative characteristics and inhibition of ␣-melanocyte-stimulating hormone-stimulated melanogenesis of vanillin and vanillic acid from Origanum vulgare. Experimental Dermatology 2010;19:742–50.
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[30] Ko SC, Kim D, Jeon YJ. Protective effect of a novel antioxidative peptide purified from a marine Chlorella ellipsoidea protein against free radical-induced oxidative stress. Food and Chemical Toxicology 2012;50:2294–302. [31] Rokka T, Syvaoja EL, Tuominen J, Korhonen H. Release of bioactive peptides by enzymatic proteolysis of Lactobacillus GG fermented UHT milk. Milchwissenschaft 1997;52:675–8. [32] Ashar MN, Chand R. Fermented milk containing ACE-inhibitory peptides reduces blood pressure in middle aged hypertensive subjects. Milchwissenschaft 2004;59:363–6. [33] Sheih I, Wu TK, Fang TJ. Antioxidant properties of a new antioxidative peptide from algae protein waste hydrolysate in different oxidation systems. Bioresource Technology 2009;100:3419–25. [34] Samaranayaka AG, Lichan EC. Food-derived peptidic antioxidants: a review of their production, assessment, and potential applications. Journal of Functional Foods 2011;3:229–54. [35] Qian ZJ, Jung WK, Kim SK. Free radical scavenging activity of a novel antioxidativepeptide purified from hydrolysate of bullfrog skin, Rana catesbeiana Shaw. Bioresource Technology 2008;99:1690–8. [36] Heo SJ, Jeon YJ. Radical scavenging capacity and cytoprotective effect of enzymatic digests of Ishige okamurae. Journal of Applied Phycology 2008;20:1087–95. [37] Aruoma OI, Kaur H, Halliwell B. Oxygen free radicals and human diseases. The Journal of the Royal Society for the Promotion of Health 1991;111:172–7. [38] Ngo DH, Qian ZJ, Ryu B, Park JW, Kim SK. In vitro antioxidant activity of a peptide isolated from Nile tilapia (Oreochromis niloticus) scale gelatin in free radicalmediated oxidative systems. Journal of Functional Foods 2010;2:107–17. [39] Heo SJ, Ko SC, Kang SM, Cha SH, Lee SH, Kang DH, Jung WK, Affan A, Oh C, Jeon YJ. Inhibitory effect of diphlorethohydroxycarmalol on melanogenesis and its protective effect against UV-B radiation-induced cell damage. Food and Chemical Toxicology 2010;48:1355–61. [40] Heo SJ, Ko SC, Cha SH, Kang DH, Park HS, Choi YU, Kim D, Jung WK, Jeon YJ. Effect of phlorotannins isolated from Ecklonia cava on melanogenesis and their protective effect against photo-oxidative stress induced by UV-B radiation. Toxicology In Vitro 2009;23:1123–30.
9
[41] Kang SM, Heo SJ, Kim KN, Lee SH, Yang HM, Kim AD, Jeon YJ. Molecular docking studies of a phlorotannin, dieckol isolated from Ecklonia cava with tyrosinase inhibitory activity. Bioorganic & Medicinal Chemistry 2012;20:311–6. [42] Kim KN, Yang HM, Kang SM, Kim D, Ahn G, Jeon YJ. Octaphlorethol A isolated from Ishige foliacea inhibits ␣-MSH-stimulated induced melanogenesis via ERK pathway in B16F10 melanoma cells. Food and Chemical Toxicology 2013;59:521–6. [43] Kim DS, Park SH, Park KC. Transforming growth factor-1 decreases melanin synthesis via delayed extracellular signal-regulated kinase activation. The International Journal of Biochemistry & Cell Biology 2004;36:1482–91. [44] Levy C, Khaled M, Fisher DE. MITF: master regulator of melanocyte development and melanoma oncogene. Trends in Molecular Medicine 2006;12:406–14. [45] Jeong HS, Lee SH, Yun HY, Baek KJ, Kwon NS, Park KC, Kim DS. Involvement of mTOR signaling in sphingosylphosphorylcholine-induced hypopigmentation effects. Journal of Biomedical Science 2011;8:55. [46] Jiang Z, Xu J, Long M, Tu Z, Yang G, He G. 2,3,5,4 -tetrahydroxystilbene-2-O--dglucoside (THSG) induces melanogenesis in B16 cells by MAP kinase activation and tyrosinase upregulation. Life Sciences 2009;85:345–50. [47] Singh SK, Sarkar C, Mallick S, Saha B, Bera R, Bhadra R. Human placental lipid induces melanogenesis through p38 MAPK in B16F10 mouse melanoma. Pigment Cell Research 2005;18:113–21. [48] Li X, Guo L, Sun Y, Zhou J, Gu Y, Li Y. Baicalein inhibits melanogenesis through activation of the ERK signaling pathway. International Journal of Molecular Medicine 2010;25:923–7. [49] Ahn JH, Jin SH, Kang HY. LPS induces melanogenesis through p38 MAPK activation in human melanocytes. Archives of Dermatological Research 2008;300:325–9. [50] Liu WS, Kuan YD, Chiu KH, Wang WK, Chang FH, Liu CH, Lee CH. The extract of Rhodobacter sphaeroides inhibits melanogenesis through the MEK/ERK signaling pathway. Marine Drugs 2013;11:1899–908. [51] Kim EH, Jeong HS, Yun HY, Baek KJ, Kwon NS, Park KC, Kim DS. Geranylgeranylacetone inhibits melanin synthesis via ERK activation in Mel-Ab cells. Life Sciences 2013;93:226–32.
Please cite this article in press as: Oh G-W, et al. A novel peptide purified from the fermented microalga Pavlova lutheri attenuates oxidative stress and melanogenesis in B16F10 melanoma cells. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.05.007