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Citrus flavanone naringenin enhances melanogenesis through the activation of Wnt/[beta] -catenin signalling in mouse melanoma cells.


Citrus fruits are the major source of flavonoids for humans, and flavanones are the main flavonoids in the Citrus species. Among the Citrus flavanones, the glycoside derivatives of naringenin, naringin and narirutin, are the most abundant in grapefruit. The present study aimed to investigate the molecular events of melanogenesis induced by naringenin in murine B16-F10 melanoma cells. Melanin content, tyrosinase activity and Western blot analysis were performed to elucidate the possible underlying mechanisms. Exposure of melanoma cells to naringenin resulted in morphological changes accompanied by the induction of melanocyte differentiation-related markers, such as melanin synthesis, tyrosinase activity, and the expression of tyrosinase and microphthalmia-associated transcription factor (MITF). We also observed an increase in the intracellular accumulation of [beta] -catenin as well as the phosphorylation of glycogen synthase kinase-3[beta] (GSK3[beta]) protein after treatment with naringenin. Moreover, the activity of phosphatidylinositol 3-kinase (PI3K) was up-regulated by naringenin since the phosphorylated level of downstream Akt protein was enhanced. Based on these results, we concluded that naringenin induced melanogenesis through the Wnt [beta] -(3-catenin-signalling pathway.

[c] 2011 Elsevier GmbH. All rights reserved.


Keywords: Naringenin

Melanogenesis Phosphatidylinositol 3-kinase [beta] -Catenin Glycogen synthase kinase 3 [beta]


The colour of mammalian skin and hair is determined by a number of factors, the most obvious phenotypical characteristics of which is the distribution of the melanin pigment. Melanin synthesis is a complex process that occurs within specialized intracellular organelles named melanosomes in melanocytes. Melanogenesis can be stimulated by stress, including UV radiation, inflammation, and hormones (Costin and Hearing 2007). Furthermore, a-melanocyte-stimulating hormone (a-MSH) and cAMP-elevating agents, such as forskolin and IBMX, through the activation of protein kinase A (PKA) and cAMP-related element binding protein (CREB) transcription factor, promote an increase in the expression of microphthalmia-associated transcription factor (MITF), a master regulator of the development and differentiation of melanocytes. MITF transcriptional regulates the expression of tyrosinase and tyrosinase-related protein that control the conversion of tyrosine to melanin pigments (Vachtenheim and Borovansky 2010). Previous papers have linked up-regulated melanogenesis to melanoma, and this viewpoint enhances the importance of further melanogenetic studies.

The Wnt-signalling pathways play an important role in melanocyte development, melanoma genesis and pigment cell formation (O'Connell and Weeraratna 2009). Glycogen synthase kinase 3 (GSK3) is one of the few signalling mediators that play a central role in a diverse range of signalling pathways, including those activated by Wnts, growth factors, and G protein-coupled ligands (Wu and Pan 2010). When WNT proteins bind to their receptors, they inactivate GSK3[beta], an enzyme that phosphorylates [beta]-catenin and specifically targets its destruction in the proteasome (Bienz 2005). Then, p-catenin accumulates in the cytoplasm and translocates to the nucleus. Increased levels of nuclear [beta]-catenin increase the expression of MITF, and in turn increase the survival and proliferation of melanoma cells (Miller and Mihm 2006). However, GSK3[beta] which is a negative regulator of Wnt signalling, is capable of activating MITF function through phosphorylation at Ser 298 (Takeda et al. 2000a). A recent study has demonstrated that inhibiting GSK3[beta] increases melanogenesis both in murine B16 cells and human melanocytes (Bellei et al. 2008). It is therefore possible that GSK3[beta] contributes to maintaining the levels of MITF in melanogenesis.

Flavanones occur almost exclusively in citrus fruits. Citrus flavanones exhibit wide ranges of biological activities, such as antioxidant, anti-inflammatory and anti-tumour activities, which indicate that these compounds may exert beneficial effects against cardiovascular diseases or cancers (Benavente-Garcia and Castillo 2008). The main flavonoids in grapefruit are naringin (naringenin-7-neohesperoside) (70%) and narirutin (naringenin-7-rutinoside) (20%) (Kawaii et al. 1999). Most studies on naringenin have reported its possible roles in grapefruit juicedrug interactions (Fuhr 1998). Recently, several reports have focused on the potential use of flavonoids for preventing oxidative skin damage (Mortimer 1997; Proteggente et al. 2003). Bioflavonoids with fla-vanone structures, such as hesperidin, have been found to inhibit tyrosinase activity in human primary melanocytes (Zhu and Gao 2008). In a previous study, it has been shown that naringenin increases the melanin content and tyrosinase activity by increasing the expression of melanogenic enzymes (Ohguchi et al. 2006). The mechanisms underlying the activities of naringenin and its glycosides on melanogenesis have not yet been well elucidated. The present study aimed to evaluate whether the flavanones in grapefruit juice affect melanogenesis in melanoma cells and to elucidate the possible underlying related signalling.

Materials and methods


Naringenin, l-DOPA, 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide (MTT), melanin and IBMX were obtained from Sigma-Aldrich (St. Louis, MO, USA). Naringin and narirutin were purchased from Acros Organics and ECHO Chemical Co., respectively. Antibodies for tyrosinase, [beta]-catenin, and phospho-Akt(Ser473) were obtained from Epitomics(Burlingame, CA, USA). Actin and phospho-GSK3[beta] (Ser 9) antibodies were supplied by Miliipore (Temecula, CA, USA). Anti-MITF antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Naringenin, narirutin, or naringin was dissolved in dimethyl-sulfoxide (DMSO) and further diluted in culture medium. The final DMSO concentration in the medium was 0.1% and did not affect cellular function or the assay systems used in this study.

Cell culture

The B16-F10 murine melanoma cells were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and maintained in Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel), 50U/ml penicillin, and 50 [micro]g/ml streptomycin in a humidified incubator at 37 [degrees]C in 5% CO2/air.

Cell viability assay

Briefly, cells were seeded at a density of 7 x [10.sup.4]/ml on 96-well plates and cultured overnight as described above. The medium was then replaced with fresh medium containing flavanones at various concentrations. After incubation for 48 h at 37 [degrees]C in 5% [C0.SUP.2]/air, MTT (final concentration, 0.5 mg/ml) was added, and the cells were then incubated at 37 [degrees]C for 2h. Finally, the cells were lysed and absorbance was detected at 550 nm. For cell number determination, a standard correlation between the known cell numbers and the absorbance density values was constructed for measuring the cell number from various detected absorbance density values.

Melanin content determination

The melanin content was measured by a previously described method (Kim et al. 2005) with slight modifications. Cells were seeded at a density of 9 x [10.SUP.5]/ml in 60-mm dishes and cultured as described above. After overnight incubation, cells were then cultured for 48 h with or without flavanones in either the absence or presence of LY294002. The medium was then removed, and the cells were washed twice with phosphate-buffered saline (PBS) and harvested by trypsinisation using 0.05% trypsin/0.02% EDTA. The harvested cells were centrifuged, and the pellet was dissolved by adding 1 N NaOH, followed by incubation at 60 [degrees]C for 1 h. The amount of melanin in the solution was determined by measuring the absorbance at 470 nm using the microplate reader (BioTek, Synergy HT). The total melanin content was estimated using the standard curve of synthetic melanin.

Tyrosinase assay

Tyrosinase enzyme activity was estimated spectrophotometri-cally as described earlier (Bellei et al. 2008), using l-DOPA as the substrate. B16-F10 cells cultured with or without naringenin for 48 h were solubilised with 0.1 M sodium phosphate buffer (pH 6.8) containing 1 % Triton X-l 00. The cells were then disrupted, and centrifuged at 10,000 x g for 30min. After protein quantification and adjustment, 90 [micro]lof cell lysate (each containing the same amount of protein) was incubated in duplicate with 10 [micro]l of 10 mMl-DOPA at 37 [degrees]C for 90 min. The absorbance was then monitored at 475 nm. In order to assess the direct activity of tyrosinase, naringenin was added to cell lysate at the highest concentration and incubated for 5 min at room temperature. The cell lysates were then mixed with l-DOPA solution and incubated at 37 [degrees]C for 2 h, as described above.

Western blot analysis

Whole cell lysates were prepared using RIPA buffer (50 mM Tris-HCl, pH 7.4,150 mM NaCl, 1% NP-40,1 mM EDTA, 1 mM PMSF, 1 mM [Na.sub.3 ][ V0.sub.4],1 mM NaF, 1 [micro]/ml aprotinin, 1 ([micro]/ml pepstatin, and 1 [micro]/ml leupeptin). Aliquots of cell lysates were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes and then blotted with the appropriate antibodies. Finally, the proteins were detected using an enhanced chemiluminescence kit (Amersham Biosciences, Buckinghamshire, UK). Quantitative analysis was performed using ImageQuant analysis software (GE Healthcare).

Statistical evaluation

Data are expressed as mean[+ or -]S.E.M. of the indicated number of separate experiments. A one-way analysis of variance was performed for multiple comparisons, and if there was significant variation between treatment groups, the mean values were compared with the respective control using Student's r-test. P values less than 0.05 were considered significant.

Results and discussion

As a first step towards determining the effects of naringenin and its related glycosides on melanogenesis, we measured the cell viability and melanin content in B16-F10 melanoma cells. Cells treated with various concentrations of the flavanones (3-100 [micro]M) were estimated using the mitochondria MTT reduction assay. The results demonstrated that the three structure-related flavanones - naringenin, naringin and narirutin - had no cytotoxic effects at concentrations ranging from 3 to 50 [micro]M (data not shown). The cells were then exposed to the flavanones (50 [micro]M) for 48 h, and cellular melanin contents were examined. As shown in Fig. la, naringenin increased melanin synthesis apparently. Narirutin or naringin, the rutinose or neohesperidose glycoside of naringenin, did not show the melanogenic effects as potential as naringenin did. Although narirutin also enhanced the melanin production, it simultaneously increased the cell number, indicating that the effects of narirutin on melanogenesis may occur due to cell growth. The cell proliferation and membrane integrity of 50[micro]M naringenin was also evaluated by a trypan blue exclusion assay after 48 h treatment. Naringenin did not exert the proliferative effect in B16-F10 cells since no statistically significant differences were observed between naringenin-treated cells and DMSO-treated control cells (data not shown). Here, we confirmed that only naringenin enhanced melanin synthesis in Bl 6-F10 cells, but not its derivatives with the rutinose or neohesperidose glycone. The result observed from naringenin and its glycosides on melanogenesis was similar to quercetin and its glycoside. Quercetin rather induced melanogenesis in human melanoma cells and three-dimensional epidermal model, as has been reported (Nagata et al. 2004; Takeyama et al. 2004). However, the glycoside derived from quercetin, i.e., rutin, did not show the same activities (Nitoda et al. 2008). Naringenin was thus a more promising candidate for causing alterations in the mechanisms of melanogenesis, and was therefore investigated in later experiments.


Melanocyte differentiation is normally characterized as an increase in melanocyte dendrite production (Busca and Ballotti 2000). B16-F10 cells have short dendrites under normal culture conditions. However, cells treated with naringenin exhibited a fusiform shape associated with a parallel cell arrangement (Fig. 1 b). The number of elongated cells was notably increased. Therefore, we considered it worthy to elucidate the effects of naringenin carefully.

Upon naringenin treatment, the melanin content increased in a concentration-dependent manner. The effect of 50[micro]M naringenin on melanogenesis was comparable to that of 30 [micro]M IBMX (Fig. 2a). Since tyrosinase is known to play a key role in melanogenesis, the effect of naringenin on tyrosinase activity in cells was determined using an l-DOPA oxidation assay. The results obtained displayed that tyrosinase activity was up-regulated in a concentration-dependent manner by naringenin in B16-F10 cells (Fig. 2b). The increased tyrosinase activity caused by naringenin was similar to the increase in melanin content. To exclude a direct influence of naringenin on tyrosinase activity, we directly added naringenin to an untreated cell lysate and measured the in vitro tyrosinase activity (data not shown). It was found that naringenin did not directly regulate tyrosinase activity in an in vitro system, but did stimulate tyrosinase activity in B16-F10 cells.

We observed that naringenin enhanced melanogenesis by activating tyrosinase. We hypothesized that the modulation of the melanogenic signalling pathway may be responsible for the stimulatory activity of naringenin on the melanin synthesis. Therefore, the cell lysates obtained after treatment with different doses of naringenin were subjected to Western blot analysis in order to determine the expression of melanogenesis-related proteins. It was noted that the levels of tyrosinase and MITF protein was augmented by naringenin (Fig. 3a). Quantification of the signal detected using the ImageQuant program revealed a 1.4-fold increase in the level of tyrosinase following treatment with 50 [micro]M naringenin (P< 0.01, n = 4) as compared to the DMSO-treated control value. Moreover, the expression of [beta]-catenin, one of the factors that control MITF transcription, was increased gradually by the increasing concentrations of naringenin (Fig. 3a). After a period of 6h, naringenin augmented the expression p-catenin to about 1.3-fold, and this phenomenon was sustained until the end of the treatment (Fig. 3b). According to the above data, naringenin induced the accumulation of intracellular [beta]-catenin while promoting melanogenesis by up-regulating the expression of MITF and tyrosinase, resulting in increased melanin content.


[beta]-Catenin is known to directly interact with the MITF protein and then activate MITF-specific target genes (Schepsky et al. 2006). Both MITF and [beta]-catenin are mediators of Wnt signals during melanocyte differentiation (O'Connell and Weeraratna 2009; Takeda et al. 2000b). The inhibition of GSK3-mediated [beta]-catenin phosphorylation is known to be the key event in Wnt-[beta]-catenin signalling (Wu and Pan 2010). The amount of phospho-GSK3[beta] in response to naringenin was also up-regulated, thus causing the inactivation of GSK3[beta] (Fig. 3a).

GSK3 has constitutive kinase activity; further, its activity is significantly reduced by phosphorylation at Ser 9 in GSK3[beta] and Ser 21 in GSK3[alpha]. Several kinases can phosphorylate these serine residues, such as Akt, PKA, protein kinase C, and MAPK-activated protein kinase-1 (MAPKAP-K1, also called [p90.SUP.rsk])(Jope and Johnson 2004; Stambolic and Woodgett 1994). Phosphatidylinositol 3-kinase (PI3K/Akt) signalling has been suggested to be involved in the regulation of melanogenesis. Therefore, we estimated the protein expression of phospho-Akt. The results showed that the activation of Akt was induced by naringenin from early time points (Fig. 3b). After treated with naringenin for 1 h, the levels of phospho-Akt (Ser 473) were increased. The Akt activation occurred prior to p-catenin accumulation. These results indicated that in naringenin-induced melanogenesis stimulation, the activity of tyrosinase increased along with up-regulation of the upstream signalling pathway related to its activity and expression.


In this respect, we observed a naringenin-induced increase in phospho-Akt prior to the elevation in the level of [beta]-catenin. It was reasonable to speculate that the activation down-stream target of PI3K by naringenin was associated with the protein stabilization of [beta]-catenin. In addition, the increase in phospho-GSK3p levels corresponded to that in [beta]-catenin levels. In other words, the phosphorylation of GSK3p by naringenin inactivated the kinase activity of GSK3p, resulting in the intracellular accumulation of [beta]-catenin. Therefore, naringenin elicited cellular responses associated with the activation of the Wnt pathway (Fig. 4). To the best of our knowledge, this is the first time that a Citrus flavonoid has been demonstrated to induce melanogenesis by activating the Wnt signalling pathway.

Naringenin via the direct activation of PI3K has been proved to have an insulin-like effect on apolipoprotein B secretion by HepG2 cells (Borradaile et al. 2003). In the present study, it was unclear whether naringenin activated PI3K directly to stimulate melanogenesis since the increase in melanin content following naringenin treatment was not abolished by the P13K inhibitor LY294002. On the contrary, the melanin production induced by naringenin was potentiated by LY294002 (data dot shown). LY294002 has been noted to up-regulate the expression of melanogenic enzymes through a transcriptional mechanism (Khaled et al. 2003). In a previous report, it was also shown that the phosphorylation of Akt as well as of GSK3[beta] was attenuated by forskolin, a cAMP-elevating agent. Correspondingly, the activation of GSK3[beta] is known to stimulate the expression of melanogenic enzymes (Khaled et al. 2002). These data possibly indicate that PI3K/MAPK pathways are consti-tutively active and negatively regulate melanogenesis in B16-F10 cells (Singh et al. 2005). Since naringenin did not repress the activation of Akt nor GSK3[beta] as did forskolin, we hypothesize that cAMP-involved regulatory activity in melanogenesis did not contribute to the effects of naringenin.

Naringenin is known to play an inhibitory role on cyclic nucleotide phosphodiesterases (PDEs), such as PDE1, PDE4, and PDE5, to increase the production of cGMP and cAMP (Orallo et al. 2005). Therefore, we evaluated whether naringenin induces alterations in cAMP levels in B16-F10 cells. In our unpublished data, cell incubation with 50|xM naringenin for 30min did not alter the cAMP levels, while a 4.2-fold increase was observed in cAMP levels following IBMX treatment. In addition, naringenin-induced a fusiform shape rather than the dendritic phenotype as forskolin did. Hence, our results suggest that naringenin mediates its melanogenic effects through a cAMP-independent pathway. However, our present data are insufficient for excluding the possibility of cGMP-mediated signalling participation in naringenin-induced melanogenesis. Moreover, PKC-related signalling has also been implicated in melanocyte differentiation (Park et al. 2009). Whether naringenin acts via the activation of PKC for executing its melanogenic effects requires further clarification.


The most abundant Citrus flavonoids are flavanones(Benavente-Garcia and Castillo 2008). Citrus flavanones are present in the glycoside or aglycoside forms. Grapefruit juice contains quite high amounts of these compounds, and therefore the intake from diet can be relatively high. Erlund et al. (2000) studied the bioavailability and pharmacokinetics of flavanones after single ingestion of grapefruit juice (8 ml/kg). The resulting plasma concentrations of naringenin were comparatively high (up to 14.8[micro]ml/l). When single oral administration of 135 mg of naringenin, the mean peak plasma concentration was 7.39 [+ or -]2.83 [micro]l/l (Kanaze et al. 2007). Upon the limited information, the concentrations that were required for obtaining the melanogenetic effects in vitro in the B16-F10 cell model were comparatively higher than the plasma concentrations obtained after oral ingestion of naringenin in human.

Topically applied dihydroxyacetone and melanins have been shown to provide some photoprotection. The skin pigmentation enhancers have the potential to reduce both photodamage and skin cancer incidence. Bicyclic monoterpenes (BMPs) are abundant in plants and foods, and BMP diols when applied to human skin have been found to be efficacious in the induction of pigmentation (Brown 2001). Naringenin possesses a flavanone backbone and belongs to polyphenols structure. GSK3 inhibitors stimulated melanin synthesis both in murine melanoma B16 and normal human melanocytes (Bellei et al. 2008). Further studies are needed to determine if naringenin have the resembling effects in human melanocytes.

In conclusion, naringenin enhances melanin synthesis and tyrosinase activity in B16-F10 cells by promoting the expression of tyrosinase, M1TF, and [beta]-catenin along with the enhanced phosphorylation of Akt or GSK3[beta]. These results suggest that naringenin induces melanogenic signalling by activating the P13K/Akt or Wnt/[beta]-catenin pathways. This is of great cosmeceutical importance for designing tanning products with potential to reduce skin cancer risks.


This study was supported by grants from the National Science Council of the Republic of China (NSC98-2320-B-126-001-MY3) and Providence University (PU97-11100-B15).


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Yu-Chun Huang *, Chao-Hsun Yang, Yi-Ling Chiou

Department of Cosmetic Science, Providence University, 200 Chung-Chi Rd., Shalu, Taichung 43301. Taiwan, ROC

* Corresponding author. Tel.: +886 4 2631 1167; fax: +886 4 2631 1167.

E-mail address: (Y.-C. Huang).

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Author:Yu-Chun, Huang; Chao-Hsun, Yang; Yi-Ling, Chiou
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Date:Nov 15, 2011
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