Salicylamide Enhances Melanin Synthesis in B16F1 Melanoma Cells

Original Article Biomol Ther 29(4), 445-451 (2021) Salicylamide Enhances Melanin Synthesis in B16F1 Melanoma Cells Yusuke Ito1,ψ and Kazuomi Sato1,2,* 1Division of Animal Science, College of Agriculture, Tamagawa University, Tokyo 194-8610, 2Graduate School of Agriculture, Tamagawa University, Tokyo 194-8610, Japan Abstract Salicylamide, a non-steroidal anti-inflammatory drug (NSAID), is used as an analgesic and antipyretic agent. We have previously shown that several NSAIDs have anti-melanogenic properties in B16F1 melanoma cells. In contrast, we have found that salicylamide enhances melanin contents in B16F1 melanoma cells; however, the underlying mechanism is not known. Therefore, we investigated the mechanism through which salicylamide stimulates melanogenesis. Interestingly, salicylamide enhanced diphenolase activity in a cell-free assay. Western blotting and real-time RT-PCR revealed that salicylamide increased tyrosinase expression via transcriptional activation of the Mitf gene. Together, our results indicate that salicylamide could be used as an anti- hypopigmentation agent for skin and/or hair. Key Words: Salicylamide, Melanogenesis, Tyrosinase, Melanoma, Mitf INTRODUCTION of UV-induced DNA damage. Tyrosinase is a key enzyme in melanogenesis that catalyzes two different chemical reac- Non-steroidal anti-inflammatory drugs (NSAIDs) can di- tions: hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine rectly suppress cyclooxygenase (COX) activity. COX cata- (DOPA) and oxidation of DOPA to DOPAquinone (Decher et lyzes the conversion of arachidonic acid to prostaglandins al., 2006; Matoba et al., 2006). DOPAquinone is converted to (Weissmann, 1991; Desborough and Keeling, 2017). There- DOPAchrome and then to 5,6-dihydroxyindole (DHI) or indole fore, NSAIDs can reduce pain, decrease fever, and prevent in- 5,6-quione 2-carboxylic acid (DHICA). There are two other flammation. Moreover, many studies have demonstrated that enzymes involved in melanin synthesis: TRP-2 catalyzes the NSAIDs can prevent the development of cancer in several tis- conversion of DOPAchrome to DHICA, and TRP-1 catalyzes sues and can induce cell death and/or cell proliferation arrest the oxidation of DHICA (Jackson et al., 1992; Tsukamoito et in cancer cell lines (Piazuelo and Lanas, 2015; Yiannakopou- al., 1992; Kameyama et al., 1993). Microphthalmia-associated lou, 2015; Seetha et al., 2020; Shi et al., 2020; Zappavigna transcription factor (Mitf) plays an important role in melanin et al., 2020). Acetylsalicylic acid is one of the most commonly synthesis and can activate the melanogenic gene transcrip- used and well-studied drugs globally. It not only exert the typi- tion of tyrosinase, TRP-1 and TRP-2 (Bertolotto et al., 1998; cal effects of NSAIDs but also inhibits platelet aggregation Steingrimsson et al., 2004). (Michalska-Malecka et al., 2016). Therefore, acetylsalicylic Recently, we found that acetylsalicylic acid can also inhib- acid is effective for prevention of heart attacks, stroke, and it melanin synthesis in murine melanoma cells (Sato et al., cerebral thrombosis (Weissmann, 1991; Butenas et al., 2001). 2008). We demonstrated that several other NSAIDs could Melanin is a key determinant of skin color, and mela- downregulated the expression of melanogenic genes, includ- nin content is increased by exposure to UV radiation (Ko- ing Mitf and tyrosinase (Sato et al., 2011). Salicylamide is an rner and Pawelek, 1982). Melanin has an important role in over-the-counter drug used as an analgesic and antipyretic the absorption of harmful UV radiation and in the reduction agent. Recently, Alhashimi et al. (2019) reported that salicyl- Open Access https://doi.org/10.4062/biomolther.2020.222 Received Dec 9, 2020 Revised Feb 5, 2021 Accepted Feb 10, 2021 Published Online Mar 17, 2021 This is an Open Access article distributed under the terms of the Creative Com- mons Attribution Non-Commercial License (http://creativecommons.org/licens- *Corresponding Author es/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, E-mail: [email protected] and reproduction in any medium, provided the original work is properly cited. Tel: +81-42-739-8271, Fax: +81-42-739-8854 ψPresent address: Immunology Laboratory, Graduate School of Medical Life Science, Yokohama City University, Yokohama 230-0045, Japan Copyright © 2021 The Korean Society of Applied Pharmacology www.biomolther.org 445 Biomol Ther 29(4), 445-451 (2021) amide exerted antibacterial effects in multidrug-resistant gon- 20 µL of 500 units/mL mushroom tyrosinase (Sigma) aqueous orrhea. Moreover, Chowdhury et al. (2021) suggested that sal- solution, the reaction was performed at 25°C. Enzyme activity icylamide is a promising drugs for the treatment of the novel was monitored every 30 seconds for 6.0 min at 475 nm using coronavirus disease 2019 (COVID-19). The identification of a microplate reader (SH-9000 Lab, Hitachi High-Tech Science new NSAIDs effects is very important for the discovery of ad- Co., Tokyo, Japan). The percentage of monophenolase activ- ditional opportunities for drug repositioning. To the best of our ity was calculated as follows: knowledge, there has been no study on the effect of salicyl- 100–[(A–B)/A×100], amide on melanin synthesis. In this study, we investigated the where A represents the difference in the absorbance of the effect of salicylamide on melanogenesis in B16F1 melanoma control sample between an incubation time of 3.0 and 6.0 min, cells and elucidated the underlying mechanism. and B represents the difference in the absorbance of the test sample over the same incubation period. The diphenolase activity assay was performed using L-3,4- MATERIALS AND METHODS dihydroxyphenylalanine (L-DOPA, Sigma) as the substrate. First, 300 µL of 0.5 mg/mL L-DOPA solution, 50 µL of salicyl- Cell culture amide in DMSO, and 1.1 mL of 0.1 M phosphate buffer (pH B16F1 melanoma cells (provided by the Riken BioResource 6.8) were mixed. The mixture was preincubated at 25°C for 10 Center, Ibaraki, Japan) were cultured in Dulbecco’s modified min before the addition of 50 µL of 1,000 units/mL mushroom Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA) supple- tyrosinase (Sigma) in aqueous solution, and the reaction was mented with 5% heat-inactivated fetal bovine serum (FBS), monitored at 475 nm. The percentage of tyrosinase activity 50 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a was calculated as follows: humidified atmosphere containing 5% CO2. 100–[(A–B)/A×100], where A represents the difference in the absorbance of the Cell viability and proliferation control sample between an incubation time of 0.5 and 1.0 min, The effect of salicylamide on cell viability and proliferation and B represents the difference in the absorbance of the test was assessed by the trypan blue exclusion test. Briefly, B16F1 sample over the same incubation period. melanoma cells were treated with or without salicylamide for 3 days at the indicated time points. After treatment, the cells Measurement of tyrosinase activity in cell extract were rinsed once with phosphate-buffered saline (PBS) and To obtain intracellular tyrosinase, B16F1 melanoma cells resuspended in trypsin/EDTA (Life Technologies, Carlsbad, were seeded on 90 mm dishes. After 24 h of incubation, the CA, USA), and the number of viable and dead cells was de- cells were treated with α-MSH (10 nM) for 3 days and then termined using a Fuchs-Rosenthal cytometer under a light lysed with RIPA lysis buffer containing a protease inhibitor microscope. The percentage of viable cells was calculated as cocktail. After freezing and thawing, the cell lysates were cen- follows; trifuged at 15,000 rpm for 5 min at 4°C, and the supernatants Number of viable cells/Total number of cells×100. were collected. The protein concentrations in the lysate were To determine the cell proliferation rate, the total number of evaluated using XL-Bradford protein assay kit (Integrale, To- viable cells was counted and compared to an untreated con- kyo, Japan). Then, 80 µL of 0.1 M phosphate buffer (pH 6.8), trol. 80 µL of 0.5 mg/mL L-DOPA in 0.1 M phosphate buffer, 20 µL of sample in DMSO, and 20 µL of cell lysate were placed in a Flow cytometry for cell cycle analysis 96-well plate and mixed well. The absorbance was measured To assess the effect of salicylamide on the cell cycle, we at 475 nm using a microplate reader for 2 hours at 37°C. The performed flow cytometric analysis. First, B16F1 melanoma percentage of tyrosinase activity was calculated as follows: cells were seeded in a 60 mm dish and incubated for 24 h. Af- 100–[(A–B)/A×100], ter salicylamide treatment for 3 days, the cells were collected where A represents the difference in the absorbance of the and centrifuged at 1,000 rpm for 10 min. Subsequently, the control sample between an incubation time of 60 and 120 min, cells were washed with PBS and fixed with ice-cold 80% etha- and B represents the difference in the absorbance of the test nol. The samples were stored at –20°C for at least 24 h. After sample over the same incubation period. permeabilization and fixation, ethanol was removed. Next, the cells were washed twice with PBS, treated with RNase A for Melanin content 30 min at 37°C, and stained with propidium iodide (PI; Sig- Melanin content was measured as previously described ma) at 50 µg/mL. Before the analysis, the cells were filtered (Nishio et al., 2016). B16F1 melanoma cells were treated with through a nylon mesh to remove cell debris. The fluorescence or without salicylamide (250-1,000 µM) for 3 days. After treat- intensity of PI was measured using a BD FACS caliber (BD ment, the cells were collected by trypsinization and counted Biosciences, Franklin Lakes, NJ, USA). using a cytometer.

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