항산화 활성과 Melanoma 세포에서 멜라닌조절에 대한 Oenanthe javanica 에탄올 추출액의 효과

Effect of Oenanthe javanica Ethanolic Extracts on Antioxidant Activity and Melanogenesis in Melanoma Cells

Abstract

본 연구의 목적은 melanocyte (B16F1)에서 quercetin과 kaempferol을 포함하는 미나리 에탄올 추출물(OJE)의 멜라닌 합성효과에 미치는 영향을 조사한 것이다. OJE가 세포수준에서 멜라닌 합성을 억제하는지를 조사하기 위하여 여러 농도의 OJE 존재 하에서 B16F1세포를 배양하였다. 현재 연구에서 DPPH radical scavenging, reducing power, lipid peroxidation 및 DNA oxidation에 미치는 항산화 효과는 cell free system에서 평가되었다. 더욱이 멜라닌 생성에 대한 OJE 효과는 dopaquinone (DOPA) assay 및 tyrosinase 활성으로 결정되었다. 뿐만 아니라 superoxide dismutase (SOD)-1, -2, glutathione reductase (GSH)와 같은 항산화 효소 및 tyrosinase의 단백질발현이 western blot 분석을 이용하여 평가되었다. 본 연구에서 OJE는 지질과산화 억제효과를 나타내었고 fenton 반응에 의해서 생성되는 hydroxyl radical에 의하여 유발되는 DNA 산화를 보호하였다. OJE는 50 ${\mu}g/ml$ 이상에서 멜라닌 합성을 증가시켰고 tyrosinase 활성도 50 ${\mu}g/ml$에서 검출되었다. Western blot 분석에서는 OJE가 농도에 비례하여 tyrosinase SOD-1, -2 및 GSH의 발현 수준을 증가시켰다. 이러한 발견들은 항산화 효과를 가진 OJE가 melanocyte에서 tyrosinase 활성과 melanin 생성을 조절할 수 있어 피부를 산화스트레스로부터 보호할 수 있다는 것을 암시하고 있다.

The aim of this study is to investigate the melanogenic effect of Oenanthe javanica ethanolic extracts (OJE) containing quercetin and kaempferol in melanoma cells (B16F1). In order to determine whether OJE inhibits melanin synthesis at the cellular level, the melanoma cells were cultured in the presence of different concentrations of OJE. In the present study, the antioxidant effects of OJE on DPPH radical scavenging, power reduction, lipid peroxidation, and DNA oxidation were evaluated in a cell free system. Furthermore, the effect of OJE on the production of melanin was determined by dopaquinone (DOPA) assay and tyrosinase activity. In addition, the protein expression of tyrosinase, as well as antioxidant enzymes such as superoxide dismutase (SOD)-1, SOD-2 and glutathione reductase (GSH), were examined using Western blot analysis. In this study, it was observed that OJE exhibited an inhibitory effect on lipid peroxidation and blocked the DNA oxidation induced by the hydroxyl radical produced by Fenton's reagent. OJE increased melanin synthesis above 50 ${\mu}g/ml$ and tyrosinase activity was detected above 50 ${\mu}g/ml$. In Western blot analysis, OJE increased the expression levels of tyrosinase, SOD-1, SOD-2, and GSH in a dose-dependent manner. These findings indicate that OJE with antioxidant activity can regulate the tyrosinase activity and melanin production in melanocyte, suggesting that it could promote the development of black hair as well as protect skin from oxidative stress.

Introduction

Reactive oxygen species (ROS) are generated as by-products of cellular metabolism, primarily in the mitochondria [1]. Biomolecules such as lipids, proteins, and DNA are damaged by the ROS [11]. In particular, the unpaired electrons of oxygen including superoxide, hydrogen peroxide, hydroxyl radical, and peroxynitrite react to form partially reduced ROS [28]. In recent years, some ROS has been known to modulate enzyme activity as well as transcription factors [2]. ROS generated during melanogenesis have been implicated as cytotoxic molecules in the immune responses of insects against their internal metazoan parasites [15, 21]. Melanin produced by melanocyte exists in hair, eyes and skin [26]. Melanoma cell stretch swelling and transfer melanin to neighboring cells such as keratinocyte [27]. It function as to protect human body that melanin absorb UV [3]. Skin gets darken than normal one because melanin formation increases in melanocyte [18]. Melanocyte synthesizes melanin from L-tyrosine through stimulation of such as exposes to UV [17]. Tyrosinase which is rate-limiting enzyme, change from L-tyrosine to L-dihydroxy phenylalanine (L-DOPA) and finally to DOPA quinine [9]. DOPA chrome tautomerase (DCT) and tyrosinase-related protein-1 (TRP-1) involve synthesis of eumelain, black or brown pigment. Tyrosinase expression is raised when melanocyte is treated with melanocyte-stimulating hormone (α-MSH). The α-MSH augments cyclic AMP (cAMP) through melanocortin-1-receptor (MC1R) which increases the expression of microphthalmia-associated transcription factor (MITF) and activate cAMP responsive element binding protein (CREB). MITF which is transcription factor to involve expression of tyrosinase and tyrosinase-related protein-1 (TRP-1) enhances the gene expression of ty-rosinase and TRP-1 [16, 31]. Various reports have been pub-lished on Javan waterdropwort to prevent alcoholic liver dis-ease over a long period of time. Javan waterdropwort (Oenanthe javanica) inhibits a wetland in many Asia countries [14]. It was reported to contain flavonoids, choline, rutamin, γ-fagarine and coumarine [22, 33]. In addition, it has antioxidant ingredients such as vitamin E, eugenyl beta-D-glucopyranoside, pinoresinol beta-D-glucopyranoside, oenantho-side A and 2, 3-ethylenedioxy-5-allylphenyl beta-D-glucopyranoside [19]. Therefore, in this study it was examined that Oenanthe javanica ethanolic extracts with antioxidant activity could modulate melanin synthesis

 

Materials and Methods

Materials

Dulbecco’s Modified Eagle’s Medium (DMEM), Trypsin- EDTA, penicillin/ streptomycin/ amphotericin (10,000 U/ml, 10,000 μg/ml, and 2,500 μg/ml, respectively), fetal bovine serum (FBS) reagent were obtained from Gibco BRL, Life Technologies (Paisley, Scotland, UK). B16F1 cells were obtained from American Type of Culture Collection (Manassas, VA, USA), MTT reagent, gelatin, agarose, and PMA (phorbol 12-myristate 13-acetate) and other materials were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Extract preparation

For preparing Oenanthe javanica ethanolic extracts (OJE), the air-dried Oenanthe javanica ethanolic leaves and stems were homogenized using a grinder before extraction. The dried powder (1 kg) was extracted with 95% EtOH (1:10 w/v) and evaporated in vacuo. The concentrated OJE were freshly dissolved in DMSO before use.

Cell culture

Cell lines were separately grown as monolayers at 5% CO2 and 37℃ humidified atmosphere using appropriate media supplemented with 10% fetal bovine serum, 2 mM glutamine and 100 μg/ml penicillin-streptomycin. DMEM was used as the culture medium for B16F1 cells. Cells were passaged 3 times a week by treating with trypsin-EDTA and used for experiments after 5 passages.

MTT assay

Cytotoxic levels of OJE on B16F1 cells were measured using MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide) method as described by Hansen et al [13]. B16F1 cells were grown in 96-well plates at a density of 5×103 cells/well. After 24 h, cells were washed with fresh medium and were treated with different concentrations of OJE. After 48 h of incubation, cells were rewashed and 20 μl of MTT (5 mg/ml) was added and incubated for 4 h. Finally, DMSO (150 μl) was added to solubilize the formazan salt formed and amount of formazan salt was determined by measuring the OD at 540 nm using an GENios® microplate reader (Tecan Austria GmbH, Austria). Relative cell viability was determined by the amount of MTT converted into formazan salt. Viability of cells was quantified as a percentage compared to the blank and dose response curves were developed. The data were expressed as mean from at least three in-dependent experiments and p＜0.05 was considered significant.

DPPH radical scavenging assay

Freshly prepared 500 μl of DPPH (2, 2-diphenyl-1-picrylhydrazyl) solution was thoroughly mixed with 500 μl of OJE. The reaction mixture was incubated for 1 h at room temperature. Absorbance of the resultant mixture was recorded at 532 nm using UV-VIS spectrophotometer. Vitamin C at 10 μg/ml was used as a positive control in this study. The content of DPPH radical was calculated with absorbance and expressed as a percentage, compared to blank group.

Reducing power

Various concentrations of OJE were mixed with 200 μl of 200 mM sodium phosphate buffer (pH 6.6) and 1% potassium ferricynide. The mixture was incubated at 50℃ for 20 min. After added 200 μl of 10% trichloroacetic acid (TCA), the mixture was centrifuged at 3,000 rpm for 10min. The upper layer was mixed with 250 μl distilled water and 50 μl of 1% ferrichloride and the absorbance was measured at 700 nm using UV-VIS spectrophotometer. Vitamin C at 100 μg/ml was used as a positive control in this study. The level of reducing power was calculated with absorbance and ex-pressed as a percentage, compared to blank group.

Lipid peroxidation in-vitro using TBARS assay

The inhibitory effect of OJE on lipid peroxidation was investigated using TBARS assay. OJE was added to 9 ml of 0.04 M phosphate buffer and 2.88 ml of 2.51% linolenic acid and then incubated at 40℃. This solution was added to 0.75% TBA and 35% trichloroacetic acid which was then added to the reaction solution, heated for 15 min at 95℃ in a water bath, and cooled immediately. The absorbance at 517 nm was measured using UV-VIS spectrophotometer. The level of lipid peroxidation was calculated with absorbance and expressed as a percentage, compared to blank group.

Genomic DNA isolation

Genomic high molecular weight DNA was extracted from B16F1 cells using standard phenol/proteinase K procedure with slight modifications [30]. Briefly, cells culturing in 10 cm dishes were washed twice with PBS and scraped into 1 ml of PBS containing 10 mM EDTA. After centrifugation cells were dissolved in RNase (0.03 mg/ml), sodium acetate (0.175 M), proteinase K (0.25 mg/ml) and SDS (0.6%). The mixture was then incubated for 30 min at 37℃ and 1 h at 55℃. Following incubation, phenol:chloroform:isoamylalcohol was added at 1:1 ratio and mixture was centrifuged at 6,000× g for 5 min at 4℃. Following centrifugation, supernatant was mixed with 100% ice cold ethanolic at 1:1.5 ratio and kept for 15 min at -20℃. After centrifugation at 14,000× g for 5 min, the pellet was dissolved in TE buffer and purity of DNA was spectrophotometrically determined at 260/280 nm. Further, the quality of isolated DNA was evaluated with 1% agarose gel electrophoresis in 0.04 M Tris–acetate 0.001 M EDTA buffer.

Determination of radical mediated DNA damage

H2O2 mediated DNA oxidation was determined by a method described previously [25]. For that, 100 μl of DNA reaction mixture was prepared by adding pre-determined concentrations of OJE (or same volume of distilled water as control), 200 μM final concentration of FeSO4, 1 mM final concentration of H2O2 and 50 μg/ml final concentration of genomic DNA in the same order. Then the mixture was in-cubated at room temperature for 30 min and reaction was terminated by adding 10 mM final concentration of EDTA. Aliquot (20 μl) of reaction mixture containing about 1 μg of DNA was electrophorased on a 1% agarose gel for 30 min at 100 V. Gels were then stained with 1 mg/ml ethidium bromide and visualized by UV light using AlphaEase® gel image analysis software (Alpha Innotech, CA, USA). The level of DNA protection was calculated with absorbance and expressed as a percentage, compared to blank group.

Assay of cellular tyrosinase activity

Cellular tyrosinase activity using L-DOPA as the substrate was assayed by the method of Maeda and Fukuda [20]. About 1×106 cells were washed with 10 mM phosphate-buffered saline (PBS) and lysed with 45 μl of 1% Triton X-100-PBS. After sonication, 5 μl of 20mML-DOPA were added to each well. The 96 well plates were incubated at 37℃ for 1 h, and the absorbance was measured at 475 nm using an UV-VIS spectrophotometer. The absorbance values were compared with a standard curve obtained with purified mushroom tyrosinase. The standard curve was linear within the range of experimental values [23].

Melanin synthesis assay

Melanin contents were measured as previously described [29], but with a slight modification. Briefly, the cells were treated with the samples in DMEM containing 10% FBS for 3 d. After concentration by centrifugation, the cell pellets were dissolved in 1 ml of 1 N NaOH at 100℃ for 30 min and centrifuged again for 20 min at 16,000× g. The optical densities of the supernatants were measured at 400 nm using an UV-VIS spectrophotometer [6]. The level of DNA protection was calculated with absorbance and expressed as a percentage, compared to blank group.

Western blot analysis

Western blotting was performed according to standard procedures. Briefly, B16F1 cells were lysed in lysis buffer (50 mM Tris-HCl (pH 7.5), 0.4% Nonidet P-40, 120 mM NaCl, 1.5 mM MgCl2, 2 mM phenylmethylsulfonyl fluoride, 80 μ g/ml leupeptin, 3 mM NaF and 1mM DTT at 4℃ for 30 min. 10 μg of cell lysates were resolved on gradient 4-20% Tris-HCl gels (Novex), electrotransferred onto a nitrocellulose membrane, and blocked in 5% BSA. The following primary antibodies were used: SOD-1, SOD-2, SOD-3, glutathione reductase, tyrosinase and β-actin. Primary antibodies were detected by chemiluminescent ECL kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Image of bands on Western blots was obtained using LAS3000® Luminescent image analyzer (Fujifilm Life Science, Tokyo, Japan).

 

Results

DPPH radical scavenging assay of OJE

DPPH radical scavenging assay is the simplest method to measure the ability of antioxidants to remove free radicals. DPPH is converted from purple into yellow color when exposed to antioxidant. After DPPH was reacted with OJE, the absorbance at 517 nm was measured. As depected in Fig. 1, vitamin C at 10 μg/ml used as a positive control in this experiment clearly showed antioxidant effect on DPPH radical. But OJE is not showed DPPH radical scavenging activity.

Fig. 1.Scavenging effect of DPPH radical. Vitamin C (Vit C) at 10 μg/ml was used as a positive control. Data are given as means of values ± S.D. from three independent experiments (***p＜0.001).

Inhibitory effect of OJE on lipid peroxidation

TBARS assay was performed to investigate inhibitory effect of OJE on lipid peroxidation. Vitamin E at 100 μg/ml was used as a positive control in this experiment. As shown in Fig. 2, OJE significantly inhibited lipid peroxidation at 50 μg/ml compared with blank (p＜0.05). It was observed that OJE reduced the lipid peroxidation by 27%.

Fig. 2.Inhibitory effect of OJE on lipid peroxidation. Vitamin E (Vit E) at 100 μg/ml was used as a positive control. Lipid peroxidation was determined by TBARS. Data are given as means of values ± S.D. from three independent experiments (*p＜0.05, **p＜0.01).

Reducing Power of OJE

The reducing ability of a compound generally depends on the power of reductants, which exhibit antioxidative potential by breaking the free radical chain, donating a hydrogen atom. Vitamin C at 10 μg/ml was used as a positive control in this experiment. As shown in Fig. 3, OJE at 10 μg/ ml or higher significantly exerted reducing power compared with blank group.

Fig. 3.Reducing power of OJE. Vitamin C (Vit C) at 10 μg/ml was used as a positive control. Data are given as means of values ± S.D. from three independent experiments (**p＜0.01, ***p＜0.001).

Protective effect of OJE on DNA oxidative damage induced by hydroxyl radical

In a subsequent experiment, genomic DNA was isolated from B16F1 cells to study the protective effect of OJE against DNA oxidative damage induced by hydroxyl radical. It was clearly observed that the genomic DNA of blank group was almost degraded by hydroxyl radical produced by fenton reaction, as shown in Fig. 4. Treatment with OJE significantly inhibited the oxidative damage of DNA at 50 μg/ml or higher compared with blank group.

Fig. 4.Protective effect of OJE on DNA oxidative damage induced by hydroxyl radical. Genomic DNA purified from B16F1 cells was pre-treated with OJE for 1 h and exposed to OH· using fenton reaction. After 30 min, reaction mixture containing 5 μg of DNA was electrophoresed on a 1% agarose gel for 30 min at 100 V and visualized by UV light after stained with 1 mg/ml ethidium bromide. Data are given as means of values ± S.D. from three independent experiments (**p＜0.01, ***p＜0.001).

Effect of OJE on cell viability

In order to investigate the cytotoxic effect of OJE on B16F1 cells, MTT assay was carried out following treatment with OJE at the indicated concentration. Fig. 5 showed that OJE at all concentrations did not exert any cytotoxic effect on melanoma cells. On that ground, this result demonstrates that OJE below 100 μg/ml has no cytotoxicity in B16F1 cells used in this study.

Fig. 5.Viability of B16F1 cells treated with OJE. Data are given as means of values ± S.D. from three independent experiments.

Effect of OJE on tyrosinase activity

In order to investigate effect of OJE on tyrosinase activity in B16F1, this experiment was carried out following treatment with OJE at the indicated concentration. As shown in Fig. 6. vitamin C at 1,000 μg/ml used as a positive control inhibited tyrosinase activity by 24%. However, OJE did not inhibit tyrosiase activity. In contrast, it increase tyrosiase activity by 12% at 1,000 μg/ml in B16F1 cells.

Fig. 6.Effect of OJE on tyrosinase activity in B16F1. Vitamin C (Vit C) at 1,000 μg/ml was used as a positive control. Data are given as means of values ± S.D. from three independent experiments (*p＜0.05 **p＜0.01).

Effect of OJE on melanin generated by L-DOPA

In order to investigate effect of OJE on melanin synthesis B16F1, L-DOPA was used as a substrate following treatment with OJE at the indicated concentration. As shown in Fig. 7. vitamin C at 1,000 μg/ml used as a positive control inhibited melanin synthesis by 30%. Similar to the result of tyrosinase, OJE did not inhibit melanin synthesis. In contrast, it increase melanin synthesis promoted melanin synthesis B16F1 cells.

Fig. 7.Effect of OJE on melanin synthesis in B16F1. Vitamin C (Vit C) at 1,000 μg/ml was used as a positive control. Data are given as means of values ± S.D. from three independent experiments (*p＜0.05 , **p＜0.01).

Effect of OJE on protein expressions in melanoma cells

In order to clarify how the activity of tyrosinase is increased by OJE, the expression of proteins involved in signaling pathways of melanin synthesis was investigated using western blot analysis. After B16F1 cells were treated with OJE at 1, 5, 10 and 50 μg/ml, cell lysates was collected and their protein expressions were evaluated using antibodies including tyrosinase, SOD-1, SOD-2 and glutathione reductase (GSH). As shown in Fig. 8, OJE treatment at 10 μg/ml enhanced the expression level of SOD-1and SOD-2 in B16F1 cells. In addition, the expression level of GSH was increased in the presence of OJE at 10 μg/ml. These results indicate that it could induce the expression of antioxidant enzymes capable of suppressing oxidative stress.

Fig. 8.Effect of OJE on protein expressions of tyrosinase, SOD-1, SOD-2 and GSH in B16F1 cells. The cells were treated with OJE at 1, 5, 10 and 50 μg/ml. Western blot analysis of cell lysates was performed using the antibodies as indicated.

 

Discussion

Reactive oxygen species (ROS) produced by from mitochondria and other cellular organelle have been traditionally regarded as toxic by-products of metabolism leading to damage lipids, proteins, and DNA [10, 28]. For this reason, cells possess several antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase to protect themselves from against the potentially damaging effects of ROS. Thus oxidative stress may be broadly defined as an imbalance between oxidant production and the antioxidant capacity of the cell to prevent oxidative injury. These Oxidative stresses have been implicated in a large number of human diseases including atherosclerosis, pulmonary fibrosis, cancer, neurodegenerative diseases, and aging [8, 12, 28]. Previous studies have reported that these ROS were eliminated by antioxidant like vitamin C, vitamin E, BTH and BTA [4]. In this study we investigated the effect of OJE on melanin production related to antioxidant activity in melanocyte, B16F1. OJE exhibited inhibitory effect of lipid peroxidation and reducing power in in vitro. These results suggest that OJE can reacts radical and eliminate free radical. Next, DNA oxidation assay was carried out using the genomic DNA isolated from melanocyte in order to look into protective effect of OJE on DNA oxidative damage. It was observed that OJE exert the protective effect on DNA damage caused by hydroxyl radical. These findings support the assumption that components of OJE strongly reacts phosphate group with negative charge in nucleic acid, so that they protect DNA [5]. Our results are consistent with previous report that natural compounds with antioxidant activity can inhibit DNA damage induced by hydroxyl radical. Previous reports suggested that ROS play a key role in melanin synthesis. Melanin synthesized by tyrosinase has been widely known to be involved in not only formation of melasma and freckle but also generation of skin, hair and pupil color [21]. Thus, tyrosinase divided from B16F1 was used to investigate effect of OJE on tyrosinase activity. For that, B16F1 cells were exposed to L-DOPA to stimulate melanin synthesis. Our result confirmed that OJE increased tyrosinase activity. In general tyrosinase oxidizes 3,4-dihydroxyphenylalanine (DOPA) lead to production of melanin [21, 24]. Unlike our expectation that antioxidant inhibits melanin systhesis, even though OJE has antioxidant activity, it could not inhibit oxidation of L-DOPA. In this study it was found that OJE increases the synthesis of melanin in DOPA-induced melanocyte. Therefore, it was suggested that OJE augment melanin synthesis by tyrosinase activation. These results are in a good agreement with that component of OJE, quercetin, stimulates melanogenesis by increasing tyrosinase activity [23]. In addition, the effect of OJE on expression of antioxidant enzymes and tyrosinase were exa-mined in B16F1 cells. In this study, OJE increased the ex-pression of antioxidant enzyme such as SOD-1, -2 and GSH. Previous study demonstrated that quercetin has protective effect in diabetes by decreasing oxidative stress and preservation of pancreatic cell integrity [7]. The expression of tyrosinase was enhanced in the presence of OJE. Therefore, this result reveals that the increase in melanin synthesis is due to the enhanced expression level of tyrosinase [32]. In conclusion, these findings suggest that OJE with antioxidant activity can regulate the tyrosinase activity and melanin production in melanocyte, suggesting that it could not only protect skin from oxidative stress but also promote development of black hair.