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Inhibitory effect of 5-iodotubercidin on pigmentation
Biochemical and Biophysical Research Communications xxx (2017) 1e5
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Inhibitory effect of 5-iodotubercidin on pigmentation Kyung-Il Kim a, Hae Bong Jeong a, Hyunju Ro b, Jeung-Hoon Lee c, d, Chang Deok Kim c, *, Tae-Jin Yoon a, ** a
Department of Dermatology and Institute of Health Sciences, School of Medicine, Gyeongsang National University & Hospital, Jinju, Republic of Korea Department of Biological Sciences, College of Bioscience and Biotechnology, Chungnam National University, Daejeon, Republic of Korea c Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, Republic of Korea d Skin Med Co., Daejeon, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 June 2017 Accepted 2 July 2017 Available online xxx
Melanin pigments are the primary contributors for the skin color. They are produced in melanocytes and then transferred to keratinocytes, eventually giving various colors on skin surface. Although many depigmenting and/or skin-lightening agents have been developed, there is still a growing demand on materials for reducing pigmentation. We attempted to find materials for depigmentation and/or skinlightening using the small molecule compounds commercially available, and found that 5iodotubercidin had inhibitory potential on pigmentation. When HM3KO melanoma cells were treated with 5-iodotubercidin, pigmentation was dramatically reduced. The 5-iodotubercidin decreased the protein level for pigmentation-related molecules such as MITF, tyrosinase, and TRP1. In addition, 5iodotubercidin decreased the phosphorylation of CREB, while increased the phosphorylation of AKT and ERK. These data suggest that 5-iodotubercidin inhibits melanogenesis via the regulation of intracellular signaling related with pigmentation. Finally, 5-iodotubercidin markedly inhibited the melanogenesis of zebrafish embryos, an in vivo evaluation model for pigmentation. Together, these data suggest that 5-iodotubercidin can be developed as a depigmenting and/or skin-lightening agent. © 2017 Elsevier Inc. All rights reserved.
Keywords: 5-Iodotubercidin Pigmentation CREB AKT ERK Zebrafish
1. Introduction Skin color is determined by the deposition of melanin pigments in epidermal keratinocytes. Melanin pigments are produced by melanocytes that reside in the basal layer of epidermis, then transferred to neighboring keratinocytes via the mechanism including phagocytosis [1,2]. Because that skin is continuously exposed to ultraviolet (UV) light and UV can induce pigmentation, the primary role of melanocytes is believed to be the protection of epidermal cells by making melanin pigments that play a role as physical filters against UV light [3]. UV irradiation induces DNA damage in epidermal keratinocytes, leading to the activation of p53-dependent gene expression of
* Corresponding author. Department of Dermatology, School of Medicine, Chungnam National University, 266 Munhwa-ro, Jung-gu, Daejeon 35015, Republic of Korea. ** Corresponding author. Department of Dermatology and Institute of Health Sciences, School of Medicine, Gyeongsang National University & Hospital, 79 Gangnam-ro, Jinju 52727, Republic of Korea. E-mail addresses: [email protected] (C.D. Kim), [email protected] (T.-J. Yoon).
which targets include proopiomelanocortin (POMC) [4]. The POMC gene product is then cleaved into small peptide ligands such as amelanocyte stimulating hormone (a-MSH), and this important melanogenic inducer binds to melanocortin 1 receptor (MC1R) on the plasma membrane of melanocytes. Binding of a-MSH to MC1R leads to the increase of cyclic adenosine monophosphate (cAMP) and consequently activates protein kinase A (PKA) signaling pathway in melanocytes. As a result, the expression of pigmentation-related genes including microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related proteins (TRPs) is increased in melanocytes [5,6]. From dermatological and cosmetic viewpoints, the development of depigmenting and/or skin-lightening agents is still an important issue. To this end, many investigators are performing massive screening and validation tests using various experimental models including pigment producing melanoma cell line, guinea pig and zebrafish [7e9]. We previously attempted to screen the inhibitors for melanogenesis using the small molecule compounds commercially available, and found that adenosine kinase (ADK) inhibitor 5-iodotubercidin had prominent inhibitory potential on pigmentation in melanoma cells. In this study, we provide the
http://dx.doi.org/10.1016/j.bbrc.2017.07.008 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: K.-I. Kim, et al., Inhibitory effect of 5-iodotubercidin on pigmentation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.008
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evidence that 5-iodotubercidin inhibits pigmentation in melanoma cells and zebrafishes. 2. Materials and methods 2.1. Cell culture The HM3KO human melanoma cells were maintained in Minimum Essential Medium (MEM), supplemented with 10% fetal bovine serum (FBS) and antibiotics (Life Technologies Corporation, Grand Island, NY). The compound 5-iodotubercidin was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA) and dissolved in dimethyl sulfoxide (DMSO) and then diluted with culture medium (final concentrations of DMSO is 0.1%). 2.2. Cytotoxicity test For cytotoxicity test, cells were treated with 5-iodotubercidin for 24 h. Then the medium was replaced with fresh medium containing 0.5 mg/ml 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide (MTT) solution and cells were incubated for an additional 4 h. Finally, formazan crystal was dissolved with DMSO. Cell viability was determined by measuring optical density at 570 nm using an ELISA reader.
Rockford, IL). Samples (20e30 mg protein per lane) were run on SDS-polyacrylamide gels, transferred onto nitrocellulose membranes and incubated with appropriate antibodies for overnight at 4 C with gentle agitation. Blots were then incubated with peroxidase-conjugated secondary antibodies for 30 min at room temperature, and visualized by enhanced chemiluminescence (Intron). The following primary antibodies were used in this study: MITF, tyrosinase, TRP1, TRP2 (Santa Cruz Biotechnologies, Santa Cruz, CA); b-actin (Sigma-Aldrich, St. Louis, MO); phospho-AKT, phospho-ERK, phospho-CREB (Cell Signaling Technology, Danvers, MA). 2.5. Measurement of intracellular cAMP For measurement of intracellular cAMP, we used cAMP-Glo™ Assay kit purchased from Promega (Madison, WI). Cells were grown at 50% confluency in 12-well culture plate, then treated with 5-iodotubercidin for the indicated time points. After removing the medium, cells were lysed and incubated with reaction buffer and PKA solution for 30 min. Luminescence was then measured using Luminoskan™ Ascent Microplate Luminometer (Thermo Scientific, Rockford, IL). The amount of cAMP was expressed as a percentage to control. 2.6. Zebrafish model
2.3. Melanin content and tyrosinase activity For determination of pigmentation, cells were collected and pelleted by centrifugation. Melanin pigment was dissolved in 1 N NaOH at 100 C for 30 min, and quantified by measuring optical density at 405 nm. For determination of tyrosinase activity, cells were lysed in Pro-Prep protein extraction solution (Intron, Daejeon, Korea), then lysate was clarified by centrifugation. After quantification, 250 mg of total protein in 100 ml of lysis buffer was transferred into the 96-well plate, and 100 ml of 1 mM L-DOPA was added. After incubation for 30 min at 37 C, absorbance was measured at 405 nm. The tyrosinase activity was expressed as a percentage to control.
Evaluation using zebrafish was performed as previously reported [9]. Briefly, synchronized embryos were collected and treated with 5-iodotubercidin from 9 h post-fertilization (hpf) to 55 hpf. The effects on the pigmentation of zebrafish were observed under the stereomicroscope. Occasional stirring as well as replacement of the medium were done daily to ensure the even distribution of the compounds. In all experiments, 200 mM 1phenyl 2-thiourea (PTU) was used as a positive control [10]. For observation, embryos were dechorionated by forceps, anesthetized in tricaine methanesulfonate solution Sigma-Aldrich (St. Louis, MO), mounted in 3% methyl cellulose on a depression slide (Aquatic Eco-Systems, Apopka, FL), and photographed under the stereomicroscope MZ16 (Leica Microsystems, Ernst-Leitz-Strasse, Germany).
2.4. Western blot analysis 3. Results Cells were harvested by centrifugation and then lysed in ProPrep protein extraction solution (Intron). After vigorous pipetting, extracts were centrifuged for 15 min at 15,000 rpm. Total protein was measured using a BCA protein assay kit (Thermo Scientific,
The chemical structure of 5-iodotubercidin is very similar to the adenosine. The adenosine has nitrogen at position 7, while this nitrogen is replaced by carbon and then iodine is attached to this
Fig. 1. (A) Structure of 5-iodotubercidin. (B) Cytotoxicity of 5-iodotubercidin in HM3KO melanoma cells. Cells were treated with the indicated concentrations of 5-iodotubercidin for 72 h. Cytotoxicity was measured by MTT assay. There was no cytotoxicity of 5-iodotubercidin at the indicated concentrations. The data are represented as percent control (DMSOtreated group). The mean values ± SD are averages of triplicate measurements.
Please cite this article in press as: K.-I. Kim, et al., Inhibitory effect of 5-iodotubercidin on pigmentation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.008
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Fig. 2. Effect of 5-iodotubercidin on pigmentation of HM3KO melanoma cells. (A) Cells were treated with 5-iodotubercidin at the indicated concentrations for 72 h. Cells were harvested by centrifugation, and pellet color was shown. The 5-iodotubercidin decreased the pigmentation markedly. (B) Cells were lysed and melanin was dissolved in 1 N NaOH at 100 C for 30 min. Melanin content was determined by spectrometer. Data are the means ± SD. *P < 0.01 vs. control. (C) Cell lysates were incubated with L-DOPA, then tyrosinase activity was determined by measuring the absorbance at 405 nm. Data are the means ± SD. *P < 0.01 vs. control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
carbon in 5-iodotubercidin. Thus, it is also called 7-iodo-7deazaadenosine (Fig. 1A). We first determined the cytotoxicity of 5-iodotubercidin on HM3KO melanoma cells. Up to 1.0 mM, 5iodotubercidin did not show significant cytotoxicity (Fig. 1B). Thus, we treated HM3KO melanoma cells with 5-iodotubercidin up to 1.0 mM in following experiments. Treatment of HM3KO melanoma cells with 5-iodotubercidin led to significant inhibition of pigmentation, evidenced by the cell pellet color (Fig. 2A). Consistent with this result, quantitation of melanin content showed significant reduction by 5-iodotubercidin in a dose-dependent manner (Fig. 2B). Since tyrosinase is the ratelimiting enzyme in melanogenesis, we then measured the tyrosinase activity using the cell lysates. As a result, treatment with 5iodotubercidin decreased tyrosinase activity markedly (Fig. 2C). As 5-iodotubercidin decreased tyrosinase activity in melanoma cells, we investigated whether 5-iodotubercidin affected gene expression for pigmentation. As anticipated, 5-iodotubercidin decreased protein level for pigmentation-related molecules such as MITF, tyrosinase and TRP1 in HM3KO melanoma cells, while TRP2 level was not affected significantly (Fig. 3A). Since melanogenesis is the process in which a variety of signaling pathways are coordinately involved [11,12], we next evaluated the effects of 5-iodotubercidin on intracellular signaling pathways. We first evaluated the effect of 5-iodotubercidin on the activation of cAMP response elementbinding protein (CREB), a well-established downstream of cAMP/ PKA signaling. Treatment with 5-iodotubercidin resulted in decrease of the phosphorylation of CREB (Fig. 3B). In contrast, the phosphorylation of AKT and extracellular signal-regulated kinase (ERK) was significantly increased by 5-iodotubercidin in HM3KO melanoma cells (Fig. 3B). These results suggest that 5iodotubercidin inhibits pigmentation of melanoma cells through the inhibition of PKA and activation of AKT/ERK pathways. We further determined the intracellular cAMP level after 5iodotubercidin treatment, however the intracellular cAMP level was not significantly affected by 5-iodotubercidin (Fig. 3C).
The zebrafish is a small tropical fish that can be used as an experimental model for pigmentation [9]. We treated zebrafish embryos with 5-iodotubercidin and determined its effect on pigmentation. A tyrosinase inhibitor PTU was included as a positive control, and PTU treatment resulted in remarkable inhibition of pigmentation at the dose of 200 mM. When zebrafish embryos were treated with 5-iodotubercidin, significant inhibition of pigmentation was observed at the dose of 50 mM, while its inhibitory potential on pigmentation was rarely detected at the dose of 5 mM. Furthermore, there were no noticeable side effects by 5iodotubercidin during the developmental stage of zebrafish embryo (Fig. 4). 4. Discussion Depigmentation and/or skin lightening are the important issues in cosmetic and dermatological aspects. Although the commercial products claiming the depigmenting and/or skin lightening effects are growing, there is still a need for the development of new materials that can be applied to such a purpose. To develop the depigmenting and/or skin lightening agents, many investigators are targeting the molecules involved in pigmentation process, such as tyrosinase, TRPs, and MITF. Also, the signaling pathways leading to pigmentation are the good targets for drug development. In this study, we found that a small molecule compound 5iodotubercidin decreased the pigmentation of melanoma cells. The 5-iodotubercidin decreased the protein level for pigmentationrelated molecules such as MITF, tyrosinase and TRP1. Furthermore, we demonstrated that the effect of 5-iodotubercidin was linked to the regulation of intracellular signaling: 5-iodotubercidin decreased the phosphorylation of CREB, while increased the phosphorylation of AKT and ERK. As previously mentioned, cAMP/PKA signaling molecules are the central players in the melanogenesis, which link the external stimuli such as a-MSH to the transcription factor MITF [5]. The
Please cite this article in press as: K.-I. Kim, et al., Inhibitory effect of 5-iodotubercidin on pigmentation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.008
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Fig. 3. Effect of 5-iodotubercidin on pigmentation-related molecules in HM3KO melanoma cells. (A) The protein level of the pigmentation-related molecules was assessed by Western blotting. b-actin was used as a loading control. The 5-iodotubercidin decreased the protein level for MITF, tyrosinase and TRP1. (B) Cells were then treated with 5iodotubercidin for the indicated time points. Phosphorylation of CREB, AKT and ERK was determined by Western blot. The 5-iodotubercidin decreased the phosphorylation of CREB, while increased the phosphorylation of CREB. (C) Intracellular cAMP level was not changed by 5-iodotubercidin. The data are represented as percent control (DMSO-treated group). The mean values ± SD are averages of triplicate measurements.
CREB is a well-established downstream of cAMP/PKA signaling that is involved in the gene expression for MITF. Thus, the inhibition of CREB activation by 5-iodotubercidin can explain the action mode of this chemical on pigmentation. Although the activation of CREB was significantly inhibited by 5-iodotubercidin, the intracellular cAMP level was not affected by 5-iodotubercidin. Thus, it can be speculated that inhibition of CREB activation by 5-iodotubercidin is directly on PKA level, not on the membrane receptor level such as MC1R which increases intracellular cAMP concentration. In melanogenesis, inhibition of AKT signaling leads to the activation of glycogen synthase kinase 3 b (GSK3b), and activated GSK3b facilitates MITF binding to the tyrosinase promoter. This event results in stimulation of melanogenesis. Conversely, activation of AKT leads to the prevention of GSK3b activation and consequently inhibits melanogenic process [13]. The mitogenactivated protein kinase ERK has been also implicated in melanogenesis. Many investigations reveal that activation of ERK is linked to the degradation of MITF, thereby showing phenotype of reduced pigmentation [14,15]. In this study, 5-iodotubercidin increased the phosphorylation of AKT and ERK. Based on previous reports, it is thought that phosphorylation of AKT and ERK by 5-iodotubercidin
is another mechanism underlying the inhibition of melanogenesis. We demonstrated that 5-iodotubercidin inhibited the melanogenesis in zebrafish model. Zebrafish is a highly advantageous vertebrate model organism, because of its similar organ systems and gene sequences to human being [16]. In previous study, we showed that melanogenic regulators can be screened and/or evaluated by simple incubation of zebrafish embryos in a small volume of media [9]. Besides its simplicity in manipulation, zebrafish model has additional advantage such as simultaneous determination of compound toxicity in an organismal basis. In this study, 5iodotubercidin showed prominent inhibitory potential on pigmentation of zebrafish at the dose of 50 mM without noticeable side effects. Thus, these data suggest that 5-iodotubercidin can be developed as a skin lightening and/or depigmenting agent that has no problem regarding the side effects. In summary, we demonstrate that 5-iodotubercidin has an inhibitory potential on pigmentation. It is thought that 5iodotubercidin affects several signaling events negatively and/or positively, of those regulation is coordinately integrated to culminate in reduction of pigmentation.
Please cite this article in press as: K.-I. Kim, et al., Inhibitory effect of 5-iodotubercidin on pigmentation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.008
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Fig. 4. Effect of 5-iodotubercidin on the pigmentation of zebrafish embryo. Synchronized embryos were treated with 5-iodotubercidin at the indicated concentrations. Test compounds were dissolved in 0.1% DMSO, then added to the embryo medium. The effect on the pigmentation of zebrafish were observed under the stereomicroscope. PTU was used as a positive control. Pictures are dorsal view of embryos at 55 hpf. The 5-iodotubercidin significantly inhibited pigmentation of zebrafish embryo.
Acknowledgement This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1A2B2005612). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.07.008. References [1] E. Bastonini, D. Kovacs, M. Picardo, Skin pigmentation and pigmentary disorders: focus on epidermal/dermal cross-talk, Ann. Dermatol. 28 (2016) 279e289. [2] H. Ando, Y. Niki, M. Ito, K. Akiyama, M.S. Matsui, D.B. Yarosh, M. Ichihashi, Melanosomes are transferred from melanocytes to keratinocytes through the processes of packaging, release, uptake, and dispersion, J. Invest. Dermatol. 132 (2012) 1222e1229. [3] V. Maresca, E. Flori, M. Picardo, Skin phototype: a new perspective, Pigment. Cell Melanoma Res. 28 (2015) 378e389. [4] R. Cui, H.R. Widlund, E. Feige, J.Y. Lin, D.L. Wilensky, V.E. Igras, J. D'Orazio, C.Y. Fung, C.F. Schanbacher, S.R. Granter, D.E. Fisher, Central role of p53 in the suntan response and pathologic hyperpigmentation, Cell 128 (2007) 853e864. [5] S. Im, O. Moro, F. Peng, E.E. Medrano, J. Cornelius, G. Babcock, J.J. Nordlund, Z.A. Abdel-Malek, Activation of the cyclic AMP pathway by alphamelanotropin mediates the response of human melanocytes to ultraviolet B
radiation, Cancer Res. 58 (1998) 47e54. [6] J.A. Lo, D.E. Fisher, The melanoma revolution: from UV carcinogenesis to a new era in therapeutics, Science 346 (2014) 945e949. [7] K. Murata, K. Takahashi, H. Nakamura, K. Itoh, H. Matsuda, Search for skinwhitening agent from Prunus plants and the molecular targets in melanogenesis pathway of active compounds, Nat. Prod. Commun. 9 (2014) 185e188. [8] T. Fujii, K. Ikeda, M. Saito, Inhibitory effect of rose hip (Rosa canina L.) on melanogenesis in mouse melanoma cells and on pigmentation in brown Guinea pigs, Biosci. Biotechnol. Biochem. 75 (2011) 489e495. [9] T.Y. Choi, J.H. Kim, D.H. Ko, C.H. Kim, J.S. Hwang, S. Ahn, S.Y. Kim, C.D. Kim, J.H. Lee, T.J. Yoon, Zebrafish as a new model for phenotype-based screening of melanogenic regulatory compounds, Pigment. Cell Res. 20 (2007) 120e127. [10] J. Karlsson, J. von Hofsten, P.E. Olsson, Generating transparent zebrafish: a refined method to improve detection of gene expression during embryonic development, Mar. Biotechnol. (N.Y.) 3 (2001) 522e527. [11] R. Speeckaert, M. Van Gele, M.M. Speeckaert, J. Lambert, N. van Geel, The biology of hyperpigmentation syndromes, Pigment. Cell Melanoma Res. 27 (2014) 512e524. [12] H. Chen, Q.Y. Weng, D.E. Fisher, UV signaling pathways within the skin, J. Invest. Dermatol. 134 (2014) 2080e2085. [13] C.F. Chan, C.C. Huang, M.Y. Lee, Y.S. Lin, Fermented broth in tyrosinase- and melanogenesis inhibition, Molecules 19 (2014) 13122e13135. [14] D.S. Kim, E.S. Hwang, J.E. Lee, S.Y. Kim, S.B. Kwon, K.C. Park, Sphingosine-1phosphate decreases melanin synthesis via sustained ERK activation and subsequent MITF degradation, J. Cell Sci. 116 (2003) 1699e1706. [15] Y.M. Kim, S.E. Cho, Y.K. Seo, The activation of melanogenesis by p-CREB and MITF signaling with extremely low-frequency electromagnetic fields on B16F10 melanoma, Life Sci. 162 (2016) 25e32. [16] D.R. Love, F.B. Pichler, A. Dodd, B.R. Copp, D.R. Greenwood, Technology for high-throughput screens: the present and future using zebrafish, Curr. Opin. Biotechnol. 15 (2004) 564e571.
Please cite this article in press as: K.-I. Kim, et al., Inhibitory effect of 5-iodotubercidin on pigmentation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.008
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Inhibitory effect of 5-iodotubercidin on pigmentation Kyung-Il Kim a, Hae Bong Jeong a, Hyunju Ro b, Jeung-Hoon Lee c, d, Chang Deok Kim c, *, Tae-Jin Yoon a, ** a
Department of Dermatology and Institute of Health Sciences, School of Medicine, Gyeongsang National University & Hospital, Jinju, Republic of Korea Department of Biological Sciences, College of Bioscience and Biotechnology, Chungnam National University, Daejeon, Republic of Korea c Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, Republic of Korea d Skin Med Co., Daejeon, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 June 2017 Accepted 2 July 2017 Available online xxx
Melanin pigments are the primary contributors for the skin color. They are produced in melanocytes and then transferred to keratinocytes, eventually giving various colors on skin surface. Although many depigmenting and/or skin-lightening agents have been developed, there is still a growing demand on materials for reducing pigmentation. We attempted to find materials for depigmentation and/or skinlightening using the small molecule compounds commercially available, and found that 5iodotubercidin had inhibitory potential on pigmentation. When HM3KO melanoma cells were treated with 5-iodotubercidin, pigmentation was dramatically reduced. The 5-iodotubercidin decreased the protein level for pigmentation-related molecules such as MITF, tyrosinase, and TRP1. In addition, 5iodotubercidin decreased the phosphorylation of CREB, while increased the phosphorylation of AKT and ERK. These data suggest that 5-iodotubercidin inhibits melanogenesis via the regulation of intracellular signaling related with pigmentation. Finally, 5-iodotubercidin markedly inhibited the melanogenesis of zebrafish embryos, an in vivo evaluation model for pigmentation. Together, these data suggest that 5-iodotubercidin can be developed as a depigmenting and/or skin-lightening agent. © 2017 Elsevier Inc. All rights reserved.
Keywords: 5-Iodotubercidin Pigmentation CREB AKT ERK Zebrafish
1. Introduction Skin color is determined by the deposition of melanin pigments in epidermal keratinocytes. Melanin pigments are produced by melanocytes that reside in the basal layer of epidermis, then transferred to neighboring keratinocytes via the mechanism including phagocytosis [1,2]. Because that skin is continuously exposed to ultraviolet (UV) light and UV can induce pigmentation, the primary role of melanocytes is believed to be the protection of epidermal cells by making melanin pigments that play a role as physical filters against UV light [3]. UV irradiation induces DNA damage in epidermal keratinocytes, leading to the activation of p53-dependent gene expression of
* Corresponding author. Department of Dermatology, School of Medicine, Chungnam National University, 266 Munhwa-ro, Jung-gu, Daejeon 35015, Republic of Korea. ** Corresponding author. Department of Dermatology and Institute of Health Sciences, School of Medicine, Gyeongsang National University & Hospital, 79 Gangnam-ro, Jinju 52727, Republic of Korea. E-mail addresses: [email protected] (C.D. Kim), [email protected] (T.-J. Yoon).
which targets include proopiomelanocortin (POMC) [4]. The POMC gene product is then cleaved into small peptide ligands such as amelanocyte stimulating hormone (a-MSH), and this important melanogenic inducer binds to melanocortin 1 receptor (MC1R) on the plasma membrane of melanocytes. Binding of a-MSH to MC1R leads to the increase of cyclic adenosine monophosphate (cAMP) and consequently activates protein kinase A (PKA) signaling pathway in melanocytes. As a result, the expression of pigmentation-related genes including microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related proteins (TRPs) is increased in melanocytes [5,6]. From dermatological and cosmetic viewpoints, the development of depigmenting and/or skin-lightening agents is still an important issue. To this end, many investigators are performing massive screening and validation tests using various experimental models including pigment producing melanoma cell line, guinea pig and zebrafish [7e9]. We previously attempted to screen the inhibitors for melanogenesis using the small molecule compounds commercially available, and found that adenosine kinase (ADK) inhibitor 5-iodotubercidin had prominent inhibitory potential on pigmentation in melanoma cells. In this study, we provide the
http://dx.doi.org/10.1016/j.bbrc.2017.07.008 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: K.-I. Kim, et al., Inhibitory effect of 5-iodotubercidin on pigmentation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.008
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evidence that 5-iodotubercidin inhibits pigmentation in melanoma cells and zebrafishes. 2. Materials and methods 2.1. Cell culture The HM3KO human melanoma cells were maintained in Minimum Essential Medium (MEM), supplemented with 10% fetal bovine serum (FBS) and antibiotics (Life Technologies Corporation, Grand Island, NY). The compound 5-iodotubercidin was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA) and dissolved in dimethyl sulfoxide (DMSO) and then diluted with culture medium (final concentrations of DMSO is 0.1%). 2.2. Cytotoxicity test For cytotoxicity test, cells were treated with 5-iodotubercidin for 24 h. Then the medium was replaced with fresh medium containing 0.5 mg/ml 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide (MTT) solution and cells were incubated for an additional 4 h. Finally, formazan crystal was dissolved with DMSO. Cell viability was determined by measuring optical density at 570 nm using an ELISA reader.
Rockford, IL). Samples (20e30 mg protein per lane) were run on SDS-polyacrylamide gels, transferred onto nitrocellulose membranes and incubated with appropriate antibodies for overnight at 4 C with gentle agitation. Blots were then incubated with peroxidase-conjugated secondary antibodies for 30 min at room temperature, and visualized by enhanced chemiluminescence (Intron). The following primary antibodies were used in this study: MITF, tyrosinase, TRP1, TRP2 (Santa Cruz Biotechnologies, Santa Cruz, CA); b-actin (Sigma-Aldrich, St. Louis, MO); phospho-AKT, phospho-ERK, phospho-CREB (Cell Signaling Technology, Danvers, MA). 2.5. Measurement of intracellular cAMP For measurement of intracellular cAMP, we used cAMP-Glo™ Assay kit purchased from Promega (Madison, WI). Cells were grown at 50% confluency in 12-well culture plate, then treated with 5-iodotubercidin for the indicated time points. After removing the medium, cells were lysed and incubated with reaction buffer and PKA solution for 30 min. Luminescence was then measured using Luminoskan™ Ascent Microplate Luminometer (Thermo Scientific, Rockford, IL). The amount of cAMP was expressed as a percentage to control. 2.6. Zebrafish model
2.3. Melanin content and tyrosinase activity For determination of pigmentation, cells were collected and pelleted by centrifugation. Melanin pigment was dissolved in 1 N NaOH at 100 C for 30 min, and quantified by measuring optical density at 405 nm. For determination of tyrosinase activity, cells were lysed in Pro-Prep protein extraction solution (Intron, Daejeon, Korea), then lysate was clarified by centrifugation. After quantification, 250 mg of total protein in 100 ml of lysis buffer was transferred into the 96-well plate, and 100 ml of 1 mM L-DOPA was added. After incubation for 30 min at 37 C, absorbance was measured at 405 nm. The tyrosinase activity was expressed as a percentage to control.
Evaluation using zebrafish was performed as previously reported [9]. Briefly, synchronized embryos were collected and treated with 5-iodotubercidin from 9 h post-fertilization (hpf) to 55 hpf. The effects on the pigmentation of zebrafish were observed under the stereomicroscope. Occasional stirring as well as replacement of the medium were done daily to ensure the even distribution of the compounds. In all experiments, 200 mM 1phenyl 2-thiourea (PTU) was used as a positive control [10]. For observation, embryos were dechorionated by forceps, anesthetized in tricaine methanesulfonate solution Sigma-Aldrich (St. Louis, MO), mounted in 3% methyl cellulose on a depression slide (Aquatic Eco-Systems, Apopka, FL), and photographed under the stereomicroscope MZ16 (Leica Microsystems, Ernst-Leitz-Strasse, Germany).
2.4. Western blot analysis 3. Results Cells were harvested by centrifugation and then lysed in ProPrep protein extraction solution (Intron). After vigorous pipetting, extracts were centrifuged for 15 min at 15,000 rpm. Total protein was measured using a BCA protein assay kit (Thermo Scientific,
The chemical structure of 5-iodotubercidin is very similar to the adenosine. The adenosine has nitrogen at position 7, while this nitrogen is replaced by carbon and then iodine is attached to this
Fig. 1. (A) Structure of 5-iodotubercidin. (B) Cytotoxicity of 5-iodotubercidin in HM3KO melanoma cells. Cells were treated with the indicated concentrations of 5-iodotubercidin for 72 h. Cytotoxicity was measured by MTT assay. There was no cytotoxicity of 5-iodotubercidin at the indicated concentrations. The data are represented as percent control (DMSOtreated group). The mean values ± SD are averages of triplicate measurements.
Please cite this article in press as: K.-I. Kim, et al., Inhibitory effect of 5-iodotubercidin on pigmentation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.008
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Fig. 2. Effect of 5-iodotubercidin on pigmentation of HM3KO melanoma cells. (A) Cells were treated with 5-iodotubercidin at the indicated concentrations for 72 h. Cells were harvested by centrifugation, and pellet color was shown. The 5-iodotubercidin decreased the pigmentation markedly. (B) Cells were lysed and melanin was dissolved in 1 N NaOH at 100 C for 30 min. Melanin content was determined by spectrometer. Data are the means ± SD. *P < 0.01 vs. control. (C) Cell lysates were incubated with L-DOPA, then tyrosinase activity was determined by measuring the absorbance at 405 nm. Data are the means ± SD. *P < 0.01 vs. control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
carbon in 5-iodotubercidin. Thus, it is also called 7-iodo-7deazaadenosine (Fig. 1A). We first determined the cytotoxicity of 5-iodotubercidin on HM3KO melanoma cells. Up to 1.0 mM, 5iodotubercidin did not show significant cytotoxicity (Fig. 1B). Thus, we treated HM3KO melanoma cells with 5-iodotubercidin up to 1.0 mM in following experiments. Treatment of HM3KO melanoma cells with 5-iodotubercidin led to significant inhibition of pigmentation, evidenced by the cell pellet color (Fig. 2A). Consistent with this result, quantitation of melanin content showed significant reduction by 5-iodotubercidin in a dose-dependent manner (Fig. 2B). Since tyrosinase is the ratelimiting enzyme in melanogenesis, we then measured the tyrosinase activity using the cell lysates. As a result, treatment with 5iodotubercidin decreased tyrosinase activity markedly (Fig. 2C). As 5-iodotubercidin decreased tyrosinase activity in melanoma cells, we investigated whether 5-iodotubercidin affected gene expression for pigmentation. As anticipated, 5-iodotubercidin decreased protein level for pigmentation-related molecules such as MITF, tyrosinase and TRP1 in HM3KO melanoma cells, while TRP2 level was not affected significantly (Fig. 3A). Since melanogenesis is the process in which a variety of signaling pathways are coordinately involved [11,12], we next evaluated the effects of 5-iodotubercidin on intracellular signaling pathways. We first evaluated the effect of 5-iodotubercidin on the activation of cAMP response elementbinding protein (CREB), a well-established downstream of cAMP/ PKA signaling. Treatment with 5-iodotubercidin resulted in decrease of the phosphorylation of CREB (Fig. 3B). In contrast, the phosphorylation of AKT and extracellular signal-regulated kinase (ERK) was significantly increased by 5-iodotubercidin in HM3KO melanoma cells (Fig. 3B). These results suggest that 5iodotubercidin inhibits pigmentation of melanoma cells through the inhibition of PKA and activation of AKT/ERK pathways. We further determined the intracellular cAMP level after 5iodotubercidin treatment, however the intracellular cAMP level was not significantly affected by 5-iodotubercidin (Fig. 3C).
The zebrafish is a small tropical fish that can be used as an experimental model for pigmentation [9]. We treated zebrafish embryos with 5-iodotubercidin and determined its effect on pigmentation. A tyrosinase inhibitor PTU was included as a positive control, and PTU treatment resulted in remarkable inhibition of pigmentation at the dose of 200 mM. When zebrafish embryos were treated with 5-iodotubercidin, significant inhibition of pigmentation was observed at the dose of 50 mM, while its inhibitory potential on pigmentation was rarely detected at the dose of 5 mM. Furthermore, there were no noticeable side effects by 5iodotubercidin during the developmental stage of zebrafish embryo (Fig. 4). 4. Discussion Depigmentation and/or skin lightening are the important issues in cosmetic and dermatological aspects. Although the commercial products claiming the depigmenting and/or skin lightening effects are growing, there is still a need for the development of new materials that can be applied to such a purpose. To develop the depigmenting and/or skin lightening agents, many investigators are targeting the molecules involved in pigmentation process, such as tyrosinase, TRPs, and MITF. Also, the signaling pathways leading to pigmentation are the good targets for drug development. In this study, we found that a small molecule compound 5iodotubercidin decreased the pigmentation of melanoma cells. The 5-iodotubercidin decreased the protein level for pigmentationrelated molecules such as MITF, tyrosinase and TRP1. Furthermore, we demonstrated that the effect of 5-iodotubercidin was linked to the regulation of intracellular signaling: 5-iodotubercidin decreased the phosphorylation of CREB, while increased the phosphorylation of AKT and ERK. As previously mentioned, cAMP/PKA signaling molecules are the central players in the melanogenesis, which link the external stimuli such as a-MSH to the transcription factor MITF [5]. The
Please cite this article in press as: K.-I. Kim, et al., Inhibitory effect of 5-iodotubercidin on pigmentation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.008
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Fig. 3. Effect of 5-iodotubercidin on pigmentation-related molecules in HM3KO melanoma cells. (A) The protein level of the pigmentation-related molecules was assessed by Western blotting. b-actin was used as a loading control. The 5-iodotubercidin decreased the protein level for MITF, tyrosinase and TRP1. (B) Cells were then treated with 5iodotubercidin for the indicated time points. Phosphorylation of CREB, AKT and ERK was determined by Western blot. The 5-iodotubercidin decreased the phosphorylation of CREB, while increased the phosphorylation of CREB. (C) Intracellular cAMP level was not changed by 5-iodotubercidin. The data are represented as percent control (DMSO-treated group). The mean values ± SD are averages of triplicate measurements.
CREB is a well-established downstream of cAMP/PKA signaling that is involved in the gene expression for MITF. Thus, the inhibition of CREB activation by 5-iodotubercidin can explain the action mode of this chemical on pigmentation. Although the activation of CREB was significantly inhibited by 5-iodotubercidin, the intracellular cAMP level was not affected by 5-iodotubercidin. Thus, it can be speculated that inhibition of CREB activation by 5-iodotubercidin is directly on PKA level, not on the membrane receptor level such as MC1R which increases intracellular cAMP concentration. In melanogenesis, inhibition of AKT signaling leads to the activation of glycogen synthase kinase 3 b (GSK3b), and activated GSK3b facilitates MITF binding to the tyrosinase promoter. This event results in stimulation of melanogenesis. Conversely, activation of AKT leads to the prevention of GSK3b activation and consequently inhibits melanogenic process [13]. The mitogenactivated protein kinase ERK has been also implicated in melanogenesis. Many investigations reveal that activation of ERK is linked to the degradation of MITF, thereby showing phenotype of reduced pigmentation [14,15]. In this study, 5-iodotubercidin increased the phosphorylation of AKT and ERK. Based on previous reports, it is thought that phosphorylation of AKT and ERK by 5-iodotubercidin
is another mechanism underlying the inhibition of melanogenesis. We demonstrated that 5-iodotubercidin inhibited the melanogenesis in zebrafish model. Zebrafish is a highly advantageous vertebrate model organism, because of its similar organ systems and gene sequences to human being [16]. In previous study, we showed that melanogenic regulators can be screened and/or evaluated by simple incubation of zebrafish embryos in a small volume of media [9]. Besides its simplicity in manipulation, zebrafish model has additional advantage such as simultaneous determination of compound toxicity in an organismal basis. In this study, 5iodotubercidin showed prominent inhibitory potential on pigmentation of zebrafish at the dose of 50 mM without noticeable side effects. Thus, these data suggest that 5-iodotubercidin can be developed as a skin lightening and/or depigmenting agent that has no problem regarding the side effects. In summary, we demonstrate that 5-iodotubercidin has an inhibitory potential on pigmentation. It is thought that 5iodotubercidin affects several signaling events negatively and/or positively, of those regulation is coordinately integrated to culminate in reduction of pigmentation.
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Fig. 4. Effect of 5-iodotubercidin on the pigmentation of zebrafish embryo. Synchronized embryos were treated with 5-iodotubercidin at the indicated concentrations. Test compounds were dissolved in 0.1% DMSO, then added to the embryo medium. The effect on the pigmentation of zebrafish were observed under the stereomicroscope. PTU was used as a positive control. Pictures are dorsal view of embryos at 55 hpf. The 5-iodotubercidin significantly inhibited pigmentation of zebrafish embryo.
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Please cite this article in press as: K.-I. Kim, et al., Inhibitory effect of 5-iodotubercidin on pigmentation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.008