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Anti-melanogenic effects of [delta]-tocotrienol are associated with tyrosinase-related proteins and MAPK signaling pathway in B16 melanoma cells.


Tocotrienols are known to possess potent antioxidant, anticancer, and cholesterol lowering activities. Being able to rapidly penetrate the skin, these vitamin E isoforms have been explored for potential treatment against melanoma. This study aimed to elucidate the mechanism involved in the anti-melanogenic effects of [delta]-tocotrienol ([delta]T3) in B16 melanoma cells. Results showed that at 20[micro]M of [delta]T3 significantly inhibited melanin formation and ROS generation. Treatment with [delta]T3 also effectively suppressed the expression of melanogenesis-related proteins, including MC1R, MITF, TYRP-1, and TYRP-2. More importantly, we observed that the mitogen-activated protein kinase (MAPK) pathway was involved in mediating [delta]T3's inhibitory effect against melanin production. Specifically, [delta]T3 treatment markedly induced the activation of extracellular signal-regulated kinases (ERK). The use of ERK activation inhibitor (PD98059) abrogated the [delta]T3-mediated downregulation expression melanogenesis-related proteins and restored melanin production. Furthermore, siRNA targeting ERK effectively blocked the [delta]T3-induced repression of tyrosinase and TYRP-1 expression. These results suggest that [delta]T3's inhibitory effect against melanogenesis is mediated by the activation of ERK signaling, thereby resulting in downstream repression of melanogenesis-related proteins and the subsequent melanin production. These data provide insight to [delta]T3's effect and the targeting of ERK signaling for treatment against melanogenesis.






ERK activation


Melanin, the biological pigment produced by melanocytes, forms the skin color and protects skin cells against radiation-induced damage (Costin and Hearing, 2007). However, its overproduction can darken and induce hyperpigmentation in the skin, such as chloasma, freckles, and solar lentigo (Briganti et al., 2003). Genetic influence is another important causal factor for skin pigmentation. Non-genetic factors such as aging, ultraviolet (UV) light, chronic inflammation, and hormonal changes are also involved in melanogenesis (Costin and Hearing, 2007). Regulatory enzymes such as tyrosinase and tyrosinase-related proteins (TYRP-1 and TYRP-2) are known to regulate skin pigmentation (Rondo and Hearing, 2011). The synthesis of melanin begins with the hydroxylation of L-tyrosine to L-dihydroxyphenyl-alanine (L-DOPA), and then the oxidation of DOPA to DOPAquinone. Both TYRP-1 and TYRP-2 (also called DOPAchrome tautomerase, DCT) are involved in the downstream reactions of the melanogenic pathway, aMelanocyte stimulating hormone ([alpha]-MSH), a physiological ligand that binds and acts on the G protein-coupled melanocortin 1 receptor (MC1R), potently activates the microphthalmia-associated transcription factor (MITF) to increase the expression of tyrosinase and the melanogenic enzymes TYRP-1 and TYRP-2, which ultimately induce melanin synthesis (Costin and Hearing, 2007; Park et al., 2009).

The mitogen-activated protein kinases (MAPKs) consist of three subtypes including the stress-activated protein kinases (SAPKs)/c-Jun N[H.sub.2]-terminal kinases (JNK), p38, and extracellular signal-regulated kinases (ERK) (Johnson and Lapadat, 2002). JNK and p38 kinases are stimulated by proinflammatory cytokines and environmental-induced stresses such as UV irradiation, heat, hydrogen peroxide, and DNA damage. MAPKs have been reported to control melanogenesis (Panka et al., 2006). Phosphorylated p38 can activate MITF, which then promotes melanin synthesis (Mizutani et al., 2010; Saha et al., 2006; Smalley and Eisen, 2000). In contrast, the activation of ERK reduces melanin synthesis by downregulating MITF expression (Kim et al., 2003; Wu et al., 2000; Xu et al., 2000). Suppression of the ERK signaling pathway in melanoma cells has been shown to induce cell differentiation and upregulation of tyrosinase activity, which leads to stimulation of melanogenesis (Englaro et al., 1998).

Both tocopherols and tocotrienols (T3) belong to the family vitamin E, with each of them comprising four homologs ([alpha], [beta], [gamma], and [delta]); they differ structurally in that tocopherols contain a saturated phytyl chain whereas tocotrienols possess an unsaturated isoprenoid side chain attached to 2-position on the chroman ring (Sen et al., 2006) (Fig. 1). Studies have shown that T3 exert more potent effects on neuroprotection, antioxidation, anticancer, antiobesity and cholesterol lowering than tocopherols (Aggarwal et al., 2010). [delta]T3 has been found to decrease melanin levels in mouse B16 melanoma cells by inhibiting the oxidative reactions of tyrosinase (Michihara et al., 2009). It also caused a decrease in melanin content by downregulating TYRP-1, TYRP-2, and tyrosinase expression (Michihara et al., 2010). However, the precise role of [delta]T3 in controlling melanogenesis remains unclear and, furthermore, no study has been reported on the role of MAPK in the effects of T3 on melanocytes. Hence, this study aimed to evaluate the involvement of the MAPK pathway in [delta]T3-mediated inhibition on melanogenesis in B16 melanoma cells.

Materials and methods


DMEM medium, dimethyl sulfoxide (DMSO), acrylamide/bis TEMED (tetramethylene diamine), 3-(4, 5-dimethylthiazol-2-yl)2, 5-diphenyl-tertazolium bromide (MTT), penicillin, streptomycin, tryspin-EDTA, [alpha]-melanocyte stimulating hormone ([alpha]-MSH), arbutin, and anti-[beta]-actin were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) was obtained from GIBCO BRh (Gaithersburg, MD, USA). 2',7'-Dichlorodihydrofluorescein ([H.sub.2]DCFDA) was obtained from Molecular Probes (Eugene, OR, USA). ERK activation inhibitor (PD98059) was purchased from Calibiochem (San Diego, CA, USA). Anti-tyrosinase, anti-MC1R, anti-MITF, anti-TYRP-1, and anti-TYRP-2 antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). ERK-specific siRNA, anti-JNK, anti-p-JNK, anti-p38, anti-phosphop38, anti-ERK, anti-phospho-ERK, and anti-rabbit IgG antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-mouse IgG antibody was from Promega (Madison, WI, USA). Tocotrienol isomers ([alpha]T3, [gamma]T3, and [delta]T3) with a purity of at least 95% were kindly provided by Carotech Ltd. (Ipoh, Malaysia).

Cell culture and drug preparation

The B16 melanoma cell line (ATCC-CRh-6323) was obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were grown in 90% DMEM medium supplemented with 10% FBS, 100 units/ml penicillin, and 100 [micro]g/ml streptomycin. They were maintained at 37[degrees]C in a humidified atmosphere of 5% C[0.sub.2].

The stock solutions of various tocotrienol isomers were prepared in DMSO and were stored at -20[degrees]C until use. The concentrations used for the study were 1, 10, and 20 [micro]M, which were freshly prepared for each experiment with a final DMSO concentration of 0.1%. Controls were always treated with the same amount of DMSO (0.1%, v/v) as used in the corresponding experiments.

Cell viability assay

The percentage of survival cells was measured by MTT colorimetric assay according to the manufacturer's instructions. In brief, B16 melanoma cells were cultured at 5 x [10.sub.5] cells per well in 96-well plates containing 100 pi DMEM medium. After an overnight incubation, cells were treated with control alone (0.1% DMSO), different concentrations of various tocotrienol isomers ([alpha]T3, [gamma]T3, and [delta]T3) or arbutin for 24 h. Cells were harvested and washed with PBS before adding 50 [micro]l of FBS-free medium containing MTT(5 mg/ml). After 4 h of incubation at 37 [degrees]C, the medium was discarded and the formazan blue that formed in the cells was dissolved in DMSO. The optical density was measured at 550 nm using a plate reader.

Melanin content analysis

Melanin contents were measured according to the method as described by Tsuboi et al. (1998). In brief, after adding a-MSH (1 [micro]M) and treated the B16 melanoma cells with [delta]T3 or arbutin at various concentrations (1, 10, and 20 [micro]M) for 24h, cell pellets containing a known number of cells (5 x [10.sup.5]) were dissolved in 0.5 ml of 1 N NaOH at 100[degrees]C for 30 min, followed by centrifuging for 20 min at 16, 000 x g. The absorbance of supernatants was measured at 410 nm using a plate reader.

Intracellular ROS determination

Briefly, confluent B16 melanoma cells stimulated with [alpha]-MSH (1 [micro]M) were treated with different concentrations of [delta]T3 or arbutin (0, 1, 10, and 20 [micro]M) at 37 [degrees]C for 24 h. After washing the cultures with PBS twice, 10 [micro]M of [H.sub.2]DCFDA was applied to the cells, which were then incubated at 37[degrees]C for 30 min. A flow cytometer was used to detect the fluorescent dichlorofluoresein.

ERK activation inhibitor treatment

To determine whether the cell viability, melanin formation, and tyrosinase-related proteins were affected by the presence of the ERK activation inhibitor (PD98059), [alpha]-MSH (1 [micro]M)-stimulated B16 melanoma cells were treated with 20 [micro]M of PD98059 with or without [delta]T3 (20 [micro]M) for 24 h before analysis.

ERK siRNA transfection

Briefly, 2 x [10.sup.5] cells were plated on 60mm plates overnight, then stimulated with a-MSH (1 [micro]M), followed by transfection with ERK-targeting siRNA (final concentration = 100 nM) using DharmaFECT 1 transfection reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer's instructions. Non-specific scrambled siRNA was included as negative control. Cells were harvested at 48 h post-transfection for protein analysis by Western immunoblotting. For co-treatment with [delta]T3, 20 [micro]M of [delta]T3 was added to the cells at 24 h post-transfection and the samples were further incubated for 24 h before analysis.

Western blot analysis

Cells (1 x [10.sup.5] cells/ml) were stimulated with a-MSH (1 [micro]M) only (as control) or with a-MSH (1 [micro]M) plus the specific treatments for 24 h, followed by harvesting and lysing in ice-cold buffer (10 mM Tris-HCl, pH 7.5, 0.1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanate, and 120mM sodium chloride) containing 1 mM phenylmethylsulfonyl fluoride, 10 [micro]g/ml leupeptin, and 1 [micro]g/ml aprotonin. Lysates were centrifuged at 10, 000 xg for 10 min. Equal amounts of lysate protein (50 [micro]/lane) were loaded onto SDS-polyacrylamide gels and electrophoretically transferred to a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). After inhibiting the nonspecific binding sites with 5% (w/v) skim milk in 0.1% (v/v) Tween 20 containing PBS (PBST) for 1 h at room temperature, the membrane was incubated with the following specific primary antibodies in 5% (w/v) skim milk in PBST for 1 h at room temperature: anti-tyrosinase (1:1000), anti-MClR (1:500), anti-MITF (1:1000), anti-TYRP-1 (1:1000), anti-TYRP-2 (1:1000), anti-p38 (1:1000), anti-phospho-p38 (1:1000), anti-ERK (1:1000), anti-phospho-ERK (1:1000), and anti-[beta]-actin (1:5000) antibodies. Antibody recognition was detected with the respective secondary antibody, either anti-mouse IgG or anti-rabbit IgG antibodies linked to horseradish peroxidase. Antibody-bound proteins were detected by the ECL western blotting analysis system (Amersham, Aylesbury, UK). The expression of [beta]-actin was used as loading control. Relative fold change in protein expression is indicated in the figures below each band and was quantified densitometrically with the Alphalmager 2200 (Alpha Innotech Corp., San Leandro, CA, USA) and AlphaEaseFC software using the [beta]-actin as reference band. Representative data from three independent experiments are shown.

Statistical analysis

Data were presented as mean [+ or -] standard deviation (SD) from three independent experiments. Values were evaluated by one way ANOVA, followed by Duncan's multiple range tests using the Statistical Analysis System (SAS Institute, Cary, NC, USA). Difference was considered significant when p-value was <0.05.


Effect of [delta]T3 on B16 melanoma cell viability

To test the cytotoxicity of [delta]T3, various concentrations of the compound were used to treat B16 melanoma cells. T3 isomers such as [alpha]- and [gamma]-tocotrienols ([alpha]T3, [gamma]T3) and also arbutin, a commercial skin whitening agent, were included for comparison. Results demonstrated that [delta]T3 had a higher percentage of cell viability (>95%) than other T3 isomers (~66-86%) and arbutin (~82-95%) at concentrations of 1-20 [micro]M (Fig. 2). These results suggest that [delta]T3 is less toxic than other T3 isomers and arbutin on B16 melanoma cells, and a maximum concentration of 20 [micro]M for [delta]T3 was selected for the subsequent experiments.

[delta]T3 decreased melanin content and ROS production

To verify [delta]T3's effect on melanin production, B16 melanoma cells were treated with various concentrations of the compound. Since melanogenesis is known to induce ROS (Urabe et al., 1994), both the melanin content (Fig. 3A) and ROS (Fig. 3B) levels were analyzed. Results indicated that both arbutin and [delta]T3 dose-dependently decreased the melanin content and ROS generation. The potency of [delta]T3 was similar to arbutin, where both compounds at 20 [micro]M reduced the melanin content and ROS production by about 20% and 15%, respectively.

[delta]T3 inhibited the expression of melanogenesis-related proteins

To determine if the inhibition of melanin synthesis by [delta]T3 was due to interference with [alpha]-MSH induction of melanogenesis-related proteins, such as tyrosinase, MC1R, MITF, TYRP-1, and TYRP-2, B16 melanoma cells were treated with [delta]T3 at various concentrations for 24 h and then subjected to western immunoblotting analysis. As indicated in Fig. 4, [delta]T3 effectively downregulated the expression of tyrosinase, MC1R, MITF, TYRP-1, and TYRP-2 in a dose-dependent manner, suggesting that [delta]T3 was effective in inhibiting the expression of melanogenesis-related proteins.

[delta]T3-induced inhibition of melanogenesis is mediated through MAPI< signaling pathway

To investigate if the MAPK signaling pathway was involved in the inhibitory effect of [delta]T3 on melanogenesis, B16 melanoma cells were treated with [delta]T3 at various concentrations for 24 h. Results demonstrated that [delta]T3 significantly enhanced the phosphorylation of ERK (Fig. 5). Although the expression of p38 was not affected, the phosphorylation of p38 was partly inhibited in [delta]T3-treated cells. These results indicate that the MAPK signaling pathway is affected by treatment with [delta]T3, which suggests its involvement in the inhibitory effect of [delta]T3 on melanogenesis.

Use of ERK activation inhibitor relieves [delta]T3-mediated inhibition on melanin formation

To further substantiate the involvement of ERK in [delta]T3's inhibitory activity against melanogenesis, B16 melanoma cells were treated with the ERK activation inhibitor PD98059 in the presence or absence of [delta]T3 treatment. PD98059 prevents phosphorylation of ERK and hence inhibits its activation. Results showed that 20 [micro]M of PD98059 significantly increased melanin production in these cells by ~15% (Table 1), which was anticipated since activated ERK is known to negatively impact MITF and the subsequent melanin formation (Wu et al., 2000). More importantly, co-treatment of [delta]T3 with PD98059 could markedly alleviate the inhibition on melanin production due to [delta]T3. No apparent cytotoxic effect was observed in the B16 melanoma cells with PD98059 treatment even in the presence of [delta]T3 (Table 1). These results indicate that the inhibitory effect of [delta]T3 on melanization is in part due to ERK activation.

ERK inhibition through the use of ERK activation inhibitor or ERK-specific siRNA in the presence of [delta]T3 moderately relieves the inhibitory effect of [delta]T3 on melanogenesis-related proteins

To further elucidate the role of ERK activation in [delta]T3-induced suppression of melanogenesis, B16 melanoma cells were treated with the ERK activation inhibitor PD98059 or siRNA targeting ERK in the presence or absence of [delta]T3, and analyzed for expression of ERK and the melanogenesis-related proteins. ERK expression was downregulated by siRNA directed against ERK whereas the non-specific scrambled siRNA had no effect (data not shown). Upon treatment with 20 [micro]M of [delta]T3, ERK was effectively activated (increase in expression of phospho-ERK) which could be blocked when PD98059 was added (Fig. 6A). Likewise, the use of siRNA to downregulate ERK expression also decreased the amount of ERK phosphorylation induced by [delta]T3 (Fig. 6B). In agreement with our earlier observation, [delta]T3 treatment caused a dramatic reduction in tyrosinase, MC1R, MITF, TYRP-1, and TYRP-2 expression (Fig. 7A). In contrast, treatment with the ERK activation inhibitor PD98059 significantly upregulated the expression of these melanogenesisrelated proteins and could partially rescue their suppression due to [delta]T3. A similar trend was noted with the siRNA experiment, whereby co-treatment with ERK-specific siRNA could moderately relieve the [delta]T3-triggered inhibition on tyrosinase and TYRP-1 expression (Fig. 7B). Together, these results indicate that the [delta]T3-induced inhibition on melanogenesis is mediated through the suppression of tyrosinase, MC1R, MITF, TYRP-1, and TYRP-2 expression, and was achieved at least in part due to activation of ERK signaling.


In recent years, the inhibition of melanogenesis has been the focus of medicinal and cosmetic treatments for skin pigmentation. ROS induces melanin biosynthesis and DNA damage, which could then stimulate the proliferation and/or apoptosis of melanocytes. Several antioxidants and ROS scavengers have been reported to inhibit or delay hyperpigmentation including protocatechuic acid (Chou et al., 2010), proanthocyanidin from grape seed extract (Shoji et al., 2005), and the vitamins ascorbic acid (Panich et al., 2011) and a-tocopherol (Hachinohe and Matsumoto, 2005). Consistent with previous studies (Michihara et al., 2009,2010; Yap et al., 2010), our results also showed that [delta]T3 significantly inhibited melanin synthesis in B16 melanoma cells. It displayed potent inhibitory effects on melanin formation and ROS production, suggesting that this potency could be related to its high antioxidative efficiency. Therefore, the free radical scavenging effect of [delta]T3 may play an important role in inhibiting the melanogenic action.

Mechanistically, [delta]T3 appears to downregulate the expression of tyrosinase, TYRP-1, and TYRP-2, which could be mediated by the suppression of transcription factors such as MITF. This is in contrast to the depigmentation mechanism of the commercial whitening agent, arbutin, which involves the inhibition of melanosomal tyrosinase activity but not the suppression of the enzyme's synthesis and expression (Maeda and Fukuda, 1996). More importantly, treatment with [delta]T3 led to phosphorylation of ERK with a concomitant decrease in melanogenesis-related proteins including MITF, and these results are reversed by inhibition of ERK using ERK activation inhibitor or siRNA targeting ERK. Since inhibition of ERK in melanocytes stimulates melanogenesis (Englaro et al., 1998), whereas the activation of ERK causes MITF degradation and leads to the reduction of melanogenesis (Kim et al., 2003; Wu et al., 2000), our data therefore suggest that [delta]T3 may depress MITF expression by activating the ERK cascade and, in turn, results in the inhibition of melanogenesis. In addition, [delta]T3 also partially blocked the phosphorylation of p38, whose activation is known to promote melanogenesis (Smalley and Eisen, 2000). Altogether, our results highlight the importance of MAPK cascades, particularly the ERK signaling pathway in inhibiting melanogenesis by [delta]T3 treatment. The mechanism of inhibiting melanin production via ERK activation has also been observed with several other metabolites including sphingosine-1 phosphate (Kim et al., 2003), sulforaphane (Shirasugi et al., 2010), and curcumin (Tu et al., 2012).

In conclusion, we have identified [delta]T3 as a potent inhibitor of melanogenesis that targets melanogenic proteins by activating ERK signaling. As the presently available tyrosinase inhibitors such as arbutin and kojic acid have been reported to possess adverse effects (Nakagawa and Kawai, 1995; Parvez et al., 2006), [delta]T3 may be a good candidate to be explored as whitening agent for photoprotection on melanocytes. Moreover, tocotrienols, which possess higher fluidity compared to tocopherols (Serbinova et al., 1991), have been shown to efficiently penetrate the skin (Traber et al., 1998), thus highlighting its potential applicability. We suggest that [delta]T3 may be of value for further development as therapeutics or complementary agent against hyperpigmentation.


Article history:

Received 28 August 2013

Received in revised form 21 January 2014

Accepted 2 March 2014

Conflicts of interest

The authors declare that no conflicts of interest exist. Acknowledgements

The authors would like to thank Wen-Chan Hsu and members at the Research Center of Biotechnology of Chia Nan University of Pharmacy and Science, Tainan, Taiwan, for their assistance.


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Lean-Teik Ng (a,1), Liang-Tzung Lin (b,1), Chiu-Lan Chenc, Hsiu-Wen Chend, Shu-Jing Wu d,**, Chun-Ching Lin (e),*

(a) Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan

(b) Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

(c) Department of Pharmacy, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan

(d) Department of Nutritional Health, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan

(e) Faculty of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan

* Corresponding author at: School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan. Tel.: +886 7 3121101x2122: fax: +886 7 3135215.

** Corresponding author at: Department of Nutritional Health, Chia-Nan University of Pharmacy and Science, 60 Erh-Jen 1st Road, Jen-Te Hsiang, Tainan 717, Taiwan. Tel.: +886 6 2668409; fax: +886 6 2668409.

E-mail addresses: (S.-J. Wu), (C.-C. Lin).

(1) These authors contributed equally to this study.

Table 1
Effect of [delta]T3 with and without ERK activation inhibitor (PD98059)
on cell viability and melanin formation in B16 melanoma cells.

Treatments        Cell viability             Melanin content
                  (% of control)             (% of control)

Control           100.00 [+ or -] 0.57 (a)   100.00 [+ or -] 0.98 (b)
  (0.1% DMSO)
[delta]T3          96.00 [+ or -] 2.06 (a)    76.37 [+ or -] 1.50 (d)
  (20 [micro]M)
PD98059           100.00 [+ or -] 4.99 (a)   114.36 [+ or -] 1.83 (a)
  (20 [micro]M)
PD98059           100.00 [+ or -] 0.39 (a)    91.61 [+ or -] 1.10 (c)
  (20 [micro]M)
  + [delta]T3
  (20 [micro]M)

Values are mean [+ or -] SD of three independent experiments. Means
within the same column with the different superscript letters are
significantly different from each other at p < 0.05 as analyzed by
Duncan's multiple range tests.
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Title Annotation:mitogen activated protein kinases
Author:Ng, Lean-Teik; Lin, Liang-Tzung; Chen, Chiu-Lan; Chen, Hsiu-Wen; Wud, Shu-Jing; Lin, Chun-Ching
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Jun 15, 2014
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