key: cord-0953193-rpkgu1iv
authors: Ito, Yusuke; Sato, Kazuomi
title: Salicylamide Enhances Melanin Synthesis in B16F1 Melanoma Cells
date: 2021-07-01
journal: Biomol Ther (Seoul)
DOI: 10.4062/biomolther.2020.222
sha: f6aa6dbbcc4bd12c6248439fc44b775272ca7084
doc_id: 953193
cord_uid: rpkgu1iv

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.

Non-steroidal anti-inflammatory drugs (NSAIDs) can directly suppress cyclooxygenase (COX) activity. COX catalyzes the conversion of arachidonic acid to prostaglandins (Weissmann, 1991; Desborough and Keeling, 2017) . Therefore, NSAIDs can reduce pain, decrease fever, and prevent inflammation. Moreover, many studies have demonstrated that NSAIDs can prevent the development of cancer in several tissues and can induce cell death and/or cell proliferation arrest in cancer cell lines (Piazuelo and Lanas, 2015; Yiannakopoulou, 2015; Seetha et al., 2020; Shi et al., 2020; Zappavigna et al., 2020) . Acetylsalicylic acid is one of the most commonly used and well-studied drugs globally. It not only exert the typical effects of NSAIDs but also inhibits platelet aggregation (Michalska-Malecka et al., 2016) . Therefore, acetylsalicylic acid is effective for prevention of heart attacks, stroke, and cerebral thrombosis (Weissmann, 1991; Butenas et al., 2001) .

Melanin is a key determinant of skin color, and melanin content is increased by exposure to UV radiation (Korner and Pawelek, 1982) . Melanin has an important role in the absorption of harmful UV radiation and in the reduction of UV-induced DNA damage. Tyrosinase is a key enzyme in melanogenesis that catalyzes two different chemical reactions: hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and oxidation of DOPA to DOPAquinone (Decher et al., 2006; Matoba et al., 2006) . DOPAquinone is converted to DOPAchrome and then to 5,6-dihydroxyindole (DHI) or indole 5,6-quione 2-carboxylic acid (DHICA). There are two other enzymes involved in melanin synthesis: TRP-2 catalyzes the conversion of DOPAchrome to DHICA, and TRP-1 catalyzes the oxidation of DHICA Tsukamoito et al., 1992; Kameyama et al., 1993) . Microphthalmia-associated transcription factor (Mitf) plays an important role in melanin synthesis and can activate the melanogenic gene transcription of tyrosinase, TRP-1 and TRP-2 (Bertolotto et al., 1998; Steingrimsson et al., 2004) .

Recently, we found that acetylsalicylic acid can also inhibit melanin synthesis in murine melanoma cells (Sato et al., 2008) . We demonstrated that several other NSAIDs could downregulated the expression of melanogenic genes, including Mitf and tyrosinase . Salicylamide is an over-the-counter drug used as an analgesic and antipyretic agent. Recently, Alhashimi et al. (2019) reported that salicyl-amide exerted antibacterial effects in multidrug-resistant gonorrhea. Moreover, Chowdhury et al. (2021) suggested that salicylamide is a promising drugs for the treatment of the novel coronavirus disease 2019 . The identification of new NSAIDs effects is very important for the discovery of additional opportunities for drug repositioning. To the best of our knowledge, there has been no study on the effect of salicylamide on melanin synthesis. In this study, we investigated the effect of salicylamide on melanogenesis in B16F1 melanoma cells and elucidated the underlying mechanism.

B16F1 melanoma cells (provided by the Riken BioResource Center, Ibaraki, Japan) were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 5% heat-inactivated fetal bovine serum (FBS), 50 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a humidified atmosphere containing 5% CO2.

The effect of salicylamide on cell viability and proliferation was assessed by the trypan blue exclusion test. Briefly, B16F1 melanoma cells were treated with or without salicylamide for 3 days at the indicated time points. After treatment, the cells were rinsed once with phosphate-buffered saline (PBS) and resuspended in trypsin/EDTA (Life Technologies, Carlsbad, CA, USA), and the number of viable and dead cells was determined using a Fuchs-Rosenthal cytometer under a light microscope. The percentage of viable cells was calculated as follows;

Number of viable cells/Total number of cells×100.

To determine the cell proliferation rate, the total number of viable cells was counted and compared to an untreated control.

To assess the effect of salicylamide on the cell cycle, we performed flow cytometric analysis. First, B16F1 melanoma cells were seeded in a 60 mm dish and incubated for 24 h. After salicylamide treatment for 3 days, the cells were collected and centrifuged at 1,000 rpm for 10 min. Subsequently, the cells were washed with PBS and fixed with ice-cold 80% ethanol. The samples were stored at -20°C for at least 24 h. After permeabilization and fixation, ethanol was removed. Next, the cells were washed twice with PBS, treated with RNase A for 30 min at 37°C, and stained with propidium iodide (PI; Sigma) at 50 µg/mL. Before the analysis, the cells were filtered through a nylon mesh to remove cell debris. The fluorescence intensity of PI was measured using a BD FACS caliber (BD Biosciences, Franklin Lakes, NJ, USA).

The direct effect of salicylamide on monophenolase and diphenolase activities was determined with a cell-free assay system using mushroom tyrosinase Nishio et al., 2016) . In the monophenolase assay, L-tyrosine was used as substrate. First, 80 µL of 0.1 M phosphate buffer (pH 6.8), 80 µL of 1.25 mM L-tyrosine and 20 µL of DMSO with or without salicylamide were mixed. After adding 20 µL of 500 units/mL mushroom tyrosinase (Sigma) aqueous solution, the reaction was performed at 25°C. Enzyme activity was monitored every 30 seconds for 6.0 min at 475 nm using a microplate reader (SH-9000 Lab, Hitachi High-Tech Science Co., Tokyo, Japan). The percentage of monophenolase activity was calculated as follows:

100-[(A-B)/A×100], where A represents the difference in the absorbance of the control sample between an incubation time of 3.0 and 6.0 min, 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-3,4dihydroxyphenylalanine (L-DOPA, Sigma) as the substrate. First, 300 µL of 0.5 mg/mL L-DOPA solution, 50 µL of salicylamide in DMSO, and 1.1 mL of 0.1 M phosphate buffer (pH 6.8) were mixed. The mixture was preincubated at 25°C for 10 min before the addition of 50 µL of 1,000 units/mL mushroom tyrosinase (Sigma) in aqueous solution, and the reaction was monitored at 475 nm. The percentage of tyrosinase activity was calculated as follows:

100-[(A-B)/A×100], where A represents the difference in the absorbance of the control sample between an incubation time of 0.5 and 1.0 min, and B represents the difference in the absorbance of the test sample over the same incubation period.

To obtain intracellular tyrosinase, B16F1 melanoma cells were seeded on 90 mm dishes. After 24 h of incubation, the cells were treated with α-MSH (10 nM) for 3 days and then lysed with RIPA lysis buffer containing a protease inhibitor cocktail. After freezing and thawing, the cell lysates were centrifuged at 15,000 rpm for 5 min at 4°C, and the supernatants were collected. The protein concentrations in the lysate were evaluated using XL-Bradford protein assay kit (Integrale, Tokyo, Japan). Then, 80 µL of 0.1 M phosphate buffer (pH 6.8), 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 96-well plate and mixed well. The absorbance was measured at 475 nm using a microplate reader for 2 hours at 37°C. The percentage of tyrosinase activity was calculated as follows:

100-[(A-B)/A×100], where A represents the difference in the absorbance of the control sample between an incubation time of 60 and 120 min, and B represents the difference in the absorbance of the test sample over the same incubation period.

Melanin content was measured as previously described (Nishio et al., 2016) . B16F1 melanoma cells were treated with or without salicylamide (250-1,000 µM) for 3 days. After treatment, the cells were collected by trypsinization and counted using a cytometer. Subsequently, the cells were washed and lysed by boiling in 2 M NaOH. Melanin content was spectrophotometrically analyzed at 405 nm absorbance and normalized using synthetic melanin (Sigma).

The DOPA staining was carried out as described previously (Nishio et al., 2016) . B16F1 melanoma cells were treated with or without salicylamide (250-1,000 µM) for 3 days. After treatment, the cells were collected by trypsinization and lysed in 0.1 M sodium phosphate buffer (pH 6.8) containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, and 10 µg/mL leupeptin (but without mercaptoethanol or heating). After electrophoresis, SDS-PAGE gels were stained with 0.5 mg/mL L-DOPA for 2 h at 37°C. Gel images were collected with WSE-6100 LuminoGraph 1 (ATTO Corporation, Tokyo, Japan), and densitometric analysis was carried out using CS analyzer software (ATTO Corporation).

To elucidate protein levels, western blotting analyses were performed. Treated cells were lysed using a RIPA lysis buffer system (Santa Cruz Biotechnology, TX, USA). Whole cell lysates were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Merck Millipore, Burlington, MA, USA). After blocking with 5% skimmed milk in PBS containing 0.1% Tween 20, the membranes were probed overnight with specific primary antibodies at 4°C and were further incubated with horseradish peroxidase-conjugated secondary antibodies. Bound antibodies were detected by chemiluminescence using an ImmunoStar zeta (Fujifilm Wako Pure Chemical, Osaka, Japan) following the manufacturer's instructions. Antibodies to tyrosinase, TRP-1, TRP-2, Mitf, β-actin, antirabbit IgG, anti-goat IgG, and anti-mouse IgG were purchased from Santa Cruz Biotechnology.

To investigate the effects of salicylamide on melanogenic gene expression, quantitative RT-PCR analysis was performed. B16F1 melanoma cells were treated with or without salicylamide for 3 day. Total RNA was extracted from salicylamide-treated cells usig ISOGEN 2 (Nippongene, Tokyo, Japan). Reverse-transcription was performed utilizing the Ta-KaRa RT-PCR kit (TaKaRa, Kyoto, Japan).

Primers used for quantitative PCR were as follows: tyrosinase upstream 5'-TTG CCA CTT CAT GTC ATC ATA GAA TAT T-3', downstream 5'-TTT ATC AAA GGT GTG ACT GCT ATA CAA AT-3'; TRP-1 upstream 5'-ATG CGG TCT TTG ACG AAT GG-3', downstream 5'-CGT TTT CCA ACG GGA AGG T-3'; TRP-2 upstream 5'-CTC AGA GCT CGG GCT CAG TT-3', downstream 5'-TGT TCA GCA CGC CAT CCA-3'; Mitf upstream 5'-CGC CTG ATC TGG TGA ATC G-3', downstream 5'-CCT GGC TGC AGT TCT CAA GAA-3'; and GAPDH upstream 5'-CGT CCC GTA GAC AAA ATG GT-3', downstream 5'-TTG ATG GCA ACA ATC TCC AC-3'. PCR amplification was conducted using a 7500 fast real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA), THUNDERBIRD SYBR qPCR mix (TOYOBO, Osaka, Japan), synthesized cDNA, primers, and distilled water according to the manufacturer's instructions. The real-time PCR cycle conditions were as follows: 1 cycle at 95°C for 2 min, followed by 40 cycle at 95°C for 5 s and 60°C for 30 s. All samples were run in triplicates, and all data are expressed relative to the corresponding calculated GAPDH threshold cycle (ΔCt) values. The calibrated ΔCt values (ΔΔCt) for each sample and for the internal control (GAPDH) were calculated according to the 2 -ΔΔCt method (Livak and Schmittgen, 2001) , ΔΔCt=ΔCt treated sample-ΔCt control sample.

Results are expressed as the relative mRNA levels compared to controls.

Data are expressed as mean ± standard deviation (SD). The significance of differences between experimental conditions was determined using Dunnett's test.

First, we performed the trypan blue exclusion test to assess cell viability. In this assay, cells were treated with salicylamide at several concentrations for 3 days. As shown in Fig. 1A , salicylamide did not induce cell death. The same results were observed in time course studies (Fig. 1B) . We also assessed cell proliferation following salicylamide treatment. As shown in Fig. 1C , low concentrations of salicylamide (250 µM) did not inhibit cell proliferation. However, at high concentrations, salicylamide inhibited cell proliferation (approximately 70% and 55% at 500 µM and 1,000 µM, respectively). In time course experiments, salicylamide gradually inhibited cell proliferation (Fig. 1D ).

Next, we performed flow cytometric analysis to elucidate the effect of salicylamide on the cell cycle. As shown in Fig. 2 , the percentage of cells in the G1 phase increased in the pres- 

To elucidate the effect of salicylamide on melanin synthesis, we performed a melanin content assay. B16F1 melanoma cells were treated with or without salicylamide for 3 days and solubilized in 2 M NaOH. As shown in Fig. 3A , melanin content increased in the treated cells in a dose-dependent manner (approximately 15 pg/cell, 40 pg/cell, 70 pg/cell and 170 pg/cell for vehicle control, 250 µM, 500 µM, and 1,000 µM salicylamide, respectively). We also performed a time-course experiment at in the presence of 1,000 µM salicylamide. No pigmentation enhancement was observed after 24 h of treatment (Fig. 3B) . However, melanin content increased significantly after longer treatment periods (approximately 60 pg/cell at 48 h and 160 pg/cell at 72 h of treatment). These results show that melanin formation started at least 24 h after salicylamide treatment.

We previously reported that some NSAIDs could inhibit the monophenolase and/or diphenolase activities of mushroom tyrosinase . Therefore, in the cur-rent study, we assessed the effect of salicylamide on monophenolase and diphenolase activities. As shown in Fig. 4A , salicylamide slightly inhibited enzyme activity in a monophenolase assay (approximately 82% at 1,000 µM). However, salicylamide surprisingly enhanced diphenolase activity. As shown in Fig. 4B , diphenolase activity increased in a dose-dependent manner with up to 1,000 µM salicylamide treatment (approximately 145% at 1,000 µM). However, diphenolase activity peaked in the presence of 2,000 µM salicylamide (approximately 135% at 2,000 µM).

Next, we elucidated the direct effect of salicylamide on cellular tyrosinase. In this assay, we used the cell lysate of α-MSH-stimulated B16F1 melanoma cells. As shown in Fig.  4C , salicylamide did not affect mouse melanoma-derived tyrosinase activity.

To elucidate whether salicylamide affects intracellular tyrosinase activity, we performed a DOPA staining assay. In this Biomol Ther 29 (4) assay, we treated B16F1 cells with several concentrations of salicylamide for the indicated time periods. As shown in Fig. 5 , salicylamide enhanced tyrosinase activity in a time-and dosedependent manner. Interestingly, 24 h treatment with salicylamide enhanced tyrosinase activity only at the 1,000 µM dose.

These results indicate that salicylamide enhances intracellular tyrosinase activity directly and/or via transactivation of melanogenesis-related genes.

We speculated that the enhancement of melanogenesis caused by salicylamide results in the activation of gene transcription and/or protein expression. Therefore, we carried out western blotting analysis. B16F1 melanoma cells were treated with or without salicylamide for 3 days. As shown in Fig. 6 , salicylamide treatment significantly enhanced tyrosinase protein levels. However, the levels of other melanogenic proteins, such as TRP-1, TRP-2, and Mitf, were not changed in the presence of salicylamide.

To clarify the effect of salicylamide on melanogenic gene transcription, we carried out real-time RT-PCR. As shown in Fig. 7A , tyrosinase mRNA level was dramatically enhanced after 72 h of salicylamide treatment whereas the level of other melanogenic genes remained unchanged. We also evaluated the effect of salicylamide on Mitf mRNA level during shorter time periods (0.5-6 h). The results indicated that Mitf mRNA enhancement started at 2 h of salicylamide treatment (Fig.  7B ).

We previously reported that several NSAIDs could inhibit melanin synthesis. Kumar et al. (2018) also investigated the effect of aspirin on melanoma metabolism and indicated that the depigmenting effect of aspirin results in PGE2 suppression. Unpredictably, we found that salicylamide activated melanin synthesis in B16 melanoma cells. Therefore, in this study, we investigated the mechanism through which salicylamide activates melanogenesis. Salicylamide did not show any cytotoxicity in B16F1 melanoma cells. However, salicylamide treatment arrested cell proliferation. To investigate the underlying mechanism, we evaluated the cell cycle of treated cells.

In the presence of salicylamide, the percentage of cells in the G1 phase increased, whereas the percentages of cells in the S and G2/M phases decreased. Generally, melanogenesis inducers, such as α-MSH and IBMX, inhibit cell proliferation; thus, we speculate that salicylamide-induced cell cycle arrest results in the activation of melanogenesis. In the melanin content assay, salicylamide induced melanogenesis in a dosedependent manner. This result indicates that salicylamide is a potential strong inducer of melanogenesis.

To investigate the melanism of salicylamide-enhanced melanin synthesis, we performed several assays. First, we assessed the effect of salicylamide on mushroom tyrosinase activity. Interestingly, salicylamide enhanced diphenolase activity directly. In contrast, monophenolase activity was slightly inhibited in the presence of salicylamide. Furthermore, we elucidated the direct effect of salicylamide on mouse tyrosinase using B16F1 melanoma cell lysate. However, salicylamide did not affect cellular tyrosinase activity. The influence of salicylamide on tyrosinase activities does not apparently contribute to the enhancement of melanin synthesis in B16F1 melanoma cells. Previously, we assessed several NSAIDs for their effects on monophenolase and diphenolase activities . We found that diflunisal and indomethacin inhibited tyrosinase activity. However, several NSAIDs, such as acetylsalicylic acid, ibuprofen, and diclofenac, did not show any inhibitory activity. Based on these results, we hypothesize that the effect of NSAIDs as cyclooxygenase inhibitors is not related to tyrosinase activity.

Next, we performed a DOPA staining assay in gels. In this assay, melanoma cells were treated with or without salicylamide for the indicated time periods. Thus, we can estimate the amount of active tyrosinase generated and removed by de novo synthesis and proteolysis, respectively. The intracellular active form of tyrosinase was enhanced in a time-and dosedependent manner. In the melanin content assay, the cells did not show any significant enhancement 24 h after treatment. However, we confirmed that the active form of intracellular tyrosinase was up-regulated only after 24 h of salicylamide treatment. These results show that salicylamide not only increases tyrosinase enzyme activity but also induces tyrosinase expression.

To investigate the effect of salicylamide on intracellular melanogenic protein levels and gene transcription, we carried out western blotting analysis and quantitative RT-PCR, respectively. Tyrosinase is a critical enzyme that can enhance melanogenesis; therefore, this enzyme is the main target for screening of melanogenic regulatory agents. Our results indicate that salicylamide treatment increased tyrosinase mRNA and protein levels. However, other melanogenic proteins, such as Mitf, TRP-1, and TRP-2, were not affected after 72 h of salicylamide treatment. Mitf plays a pivotal role in tyrosinase gene transcription (Bertolotto et al., 1998; Steingrimsson et al., 2004) . To solve this inconsistency, we assessed Mitf mRNA levels after salicylamide treatment for shorter periods of time. After treatment for only 2 h, Mitf mRNA level was increased. Therefore, salicylamide can enhance Mitf mRNA level and subsequent tyrosinase transcription. Salicylamide is widely used as an analgesic antipyretic drug, for which the peak blood concentration after oral co-administration with acetylsalicylic acid is 24.8 µM (Abdel-Rahman et al., 1991) . Under our experimental conditions, the concentrations of salicylamide were much higher; however, we assume the drug would be administered topically. Moreover, the fact that salicylamide enhances melanin synthesis is an important finding with regard to drug repurposing.

Skin and hair color are directly related to beauty and at-tractiveness. Abnormal melanin synthesis leads to several skin disorders. For example, melanin overproduction leads to melasma, freckles, and post-inflammatory hyperpigmentation. In contrast, melanin underproduction leads to vitiligo and leukoderma (Nicolaidou and Katsambas, 2014) . Vitiligo is one of the most common depigmentation disorders that affects an estimated 0.5%-1% of the world population (Ezzedine et al., 2012; Niu and Aisa, 2017) . Several hypotheses have been proposed to explain the causes of this disorder, the most prominent being accumulation of oxidative stress, an autoimmune disorder, and genetic aspects (Alikhan et al., 2011) . To date, several compounds and plant extracts have been reported to stimulate melanin synthesis. For example, Kim et al. (2020) reported that an ethanolic extract of the root of Polygonum multiflorum Thunb. stimulated melanin synthesis by inducing Mitf expression in B16F10 melanoma cells. Ullah et al. (2020) demonstrated that the antibiotic fosfomycin enhanced melanogenesis through the upregulation of c-Jun N-terminal kinases and p38 pathways, which are involved in the regulation of Mitf. Currently, the common treatment for vitiligo is skin exposure to UVB (Scherschun et al., 2001) . However, UV is harmful to the skin. Thus, screening for new compounds with an anti-whitening effect is very important.

In conclusion, our results indicate that salicylamide can induce melanin synthesis rapidly, mainly by upregulating tyrosinase expression via Mitf transcription. Therefore, this study demonstrates that salicylamide may be a useful inducer of melanogenesis in cosmetics (including for hair graying) or for the medical treatment of hypopigmentation disorders.

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We are grateful to Professor Keiko Ohashi-Kaneko of Tamagawa University and Mr. Takashi Nishio for their contributions to our work. We also thank Mr. Yuto Seki and Mr. Ryosuke Izumi for their technical assistance.