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Andrographolide suppresses melanin synthesis through Akt/GSK3b/b-catenin signal pathway Ping-Ya Zhu, Wei-Han Yin, Meng-Ran Wang, Yong-Yan Dang, Xi-Yun Ye * Shanghai Key Laboratory of Regulatory Biology, School of Life Science, East China Normal University, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 September 2014 Received in revised form 18 March 2015 Accepted 19 March 2015
Background: Tyrosinase (TYR) is the key enzyme controlling the production of melanin. Very few papers have reported that andrographolide can inhibit melanin content. Objective: To investigate the effects of andrographolide on melanin synthesis. Methods: Cell viability, melanin content, TYR activity, transcriptional and protein expression levels of TYR family and other kinds of proteins involved in melanogenesis were measured after the treatments of andrographolide. Results: It was found that andrographolide decreased melanin content, TYR activity and transcriptional and protein expression of TYR family and microphthalmia-associated transcription factor (MITF) in B16F10 melanoma cells. Data showed andrographolide also decreased melanin content and TYR content in ultraviolet B (UVB) irradiation induced brown guinea pigs. Moreover, we found that melanin content and TYR activity were effectively inhibited in Human Epidermis Melanocyte (HEM) treated with andrographolide at the medium concentrations without apparent effect on cell viability. Results in experiments treated with MG-132 or cycloheximide (CHX) showed that andrographolide lowered the content of b-catenin in cell nucleus resulting from accelerating the degradation of b-catenin. Phosphorylation of glycogen synthase kinase 3b (GSK3b) and Akt decreased simultaneously. 6Bromoindirubin-30 -oxime (BIO, inhibitor of GSK3b) and insulin-like growth factors-1 (IGF-1, activator of Akt) could reverse the decline of b-catenin in B16F10 cells induced by andrographolide. Conclusion: These results demonstrate that andrographolide can effectively suppress melanin content and TYR activity in B16F10 cells, HEM cells and UVB-induced brown guinea pig skin by decreasing phosphorylation of GSK3b dependent on Akt, promoting the degradation of b-catenin, inhibiting bcatenin into the nucleus and decreasing the expression of MITF and TYR family. Data indicate that andrographolide may be a potential whiting agent which can have great market in cosmetics and in clinical such as curing hyperpigmentation disorders. ß 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Melanin synthesis Andrographolide TYR family b-Catenin
1. Introduction Melanin is synthesized in melanocyte which is located between epidermis and dermis and it can protect skin from harm brought by ultraviolet rays, such as aging and skin cancer. Melanin content in skin is influenced by melanin synthesis, melanin transportation, melanin degradation, thickness of stratum corneum, proliferation of the epidermis and other factors, which contribute to color diversity in skin. Melanin synthesis, the major contributor of skin
* Corresponding author at: School of Life Science, East China Normal University, No. 500, Dongchuan Road, 200241 Shanghai, China. Tel.: +86 21 54341038. E-mail address: [email protected] (X.-Y. Ye).
melanin content, depends on the number and the level of differentiation of melanocyte. Melanin synthesis happens in melanosome where the substrate tyrosine turns into intermediate product named DOPA quinone with the help of tyrosinase (TYR), and then the intermediate product turns into two kinds of melanin (eumelanin and pheomelanin) via other interrelated enzymatic reactions [1]. In this progress, TYR is the key enzyme controlling the production of melanin [2]. The expression of TYR family comprising of TYR, tyrosinase related protein 1 (TRP1) and tyrosinase related protein 2 (TRP2), is influenced by MITF. MITF can bind to Bbox and M-box motifs in the promoters of TYR family and then promotes the transcription of TYR family [3]. Recently, wnt/bcatenin signal pathway is known to play an important role in melanin synthesis [4].
http://dx.doi.org/10.1016/j.jdermsci.2015.03.013 0923-1811/ß 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Zhu P-Y, et al. Andrographolide suppresses melanin synthesis through Akt/GSK3b/b-catenin signal pathway. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.03.013
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Fig. 1. Effects of andrographolide on cell proliferation and melanin content in B16F10 murine melanoma cells and HEM cells. (A) The structure of andrographolide. (B) Cell viability of B16F10 cells was measured using MTS assay and shown by the absorbance at 490 nm after treatments with different concentrations of andrographolide for 48 h. (C) Melanin content in the supernatants from B16F10 cells cultured in red-free DMEM treated with different concentrations of andrographolide, positive whiting agents (kojic acid and arbutin) was determined by the absorbance at 490 nm. (D) The photos of supernatant color of samples from (C). (E) Representative morphology of B16F10 cells from (C) by inverted phase contrast microscope. (F) Cell viability of HEM cells was measured by counting
Please cite this article in press as: Zhu P-Y, et al. Andrographolide suppresses melanin synthesis through Akt/GSK3b/b-catenin signal pathway. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.03.013
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When wnt/b-catenin signal pathway is not activated, bcatenin is phosphorylated by a multiproteins complex comprising of axin, adenomatous polyposis coli (APC), glycogen synthase kinase 3b (GSK3b) and casein kinase 1 (CK1), and then affected by ubiquitin and degradated through proteasomes [5]. But when wnt/b-catenin signal pathway is activated by its wnt ligands such as wnt1, wnt3a and wnt8, GSK3b is negatively regulated resulting in the accumulation of b-catenin in cytoplasm which can be translocated into the nucleus and combines to the promoter of MITF together with LEF1 contributing to the transcription of MITF [6,7]. In addition, there are many kinds of stimulating factors which can stimulate melanin synthesis, such as a-melanocyte stimulating hormone (a-MSH) [8], UVB irradiation [9], pressure [10] and so on. In the field of cosmetics, there are several common whiting agents, such as kojic acid and arbutin. But it has been reported that kojic acid has the ability to induce cancer [11] and arbutin can promote melanin synthesis when its concentration is higher [12]. As a result, it is significative to search for new kinds of whiting agents. Some studies have indicated that several plant components, such as mulberry extract [13], can suppress melanin synthesis and have been added to skin care product and have not been found any damage on human skin so far. Therefore, it is feasible to search for new plant components which can suppress melanin synthesis and have enormous potential. Andrographolide is a kind of natural antibiotic and can inhibit the growth of tumor from a kind of plant named Andrographis paniculata [14]. However, there are very few data showing that andrographolide can inhibit melanin synthesis and TYR activity so far.
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2.3. Cell viability assay Viability of B16F10 cells was determined by MTS (Promega, Madison, USA) assay. After andrographolide treatments, the B16F10 cells were incubated for 48 h at 37 8C. Then 20 ml MTS was added to each well of a 96-well plate. After incubation for 0.5 h, the absorbance was measured at 490 nm using a SPECTRA MAX 190 spectrometer (Molecular Devices, CA, USA). The effect of andrographolide on the growth of HEM cells was assessed by counting cell number. After andrographolide treatments for 72 h, HEM cells were incubated with EDTA/trypsin (Cat. #0103 ScienCell, San Diego, USA) and added with trypsin/EDTA neutralization solution (TNS, Cat. #0113, ScienCell, San Diego, USA), then counted with blood counting chamber. 2.4. Melanin content measurement Extracellular melanin release of B16F10 cells was measured with a previously described method with slight modifications [15]. B16F10 cells (1.2 105/well in 6-well plates) were maintained in phenol red-free DMEM (Gibco, MA, USA) with andrographolide (20 mM), kojic acid (300 mM) or arbutin (100 mM) for 48 h. Controls were designed to put the same phenol red-free DMEM without cells or materials for 48 h. 200 ml of supernatant from all the wells was measured at 490 nm. HEM cells were harvested with EDTA/trypsin after treatments of andrographolide with different concentrations for 72 h. Then cells were centrifuged at 10,000 rpm for 15 min and dissolved in 1 M NaOH, whose absorbance was measured at 490 nm. 2.5. TYR activity assay
2. Materials and methods 2.1. Materials Tyrosinase from mushroom, L-3,4-dihydroxyphenylalanine (LDopa), 6-bromoindirubin-30 -oxime (BIO), cycloheximide (CHX) and kojic acid were purchased from Sigma–Aldrich (St Louis, USA). Protease inhibitor and phosphatase inhibitor were purchased from Roche (Manheim, Germany). Antibodies against TYR, TRP1, TRP2 and MITF were purchased from Abcam (Cambridge, UK). Antibody against b-catenin was from BD (Franklin Lake, USA). Antibodies against b-actin, GSK3b, phospho-GSK3b, Akt, phospho-Akt, histone H3.1 and hsp70 were from Cell Signaling Technology (Danvers, USA). MG-132 and IGF-1 were purchased from Cell Signaling Technology. Arbutin and andrographolide were purchased from Shanghai Winherb Medical Science Co., Ltd. (Shanghai, China). 2.2. Cell culture The murine B16F10 melanoma cell line (Shanghai Key Laboratory of Regulatory Biology, China) was maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, MA, USA) supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin (Wisent, Montreal, Canada). Human Epidermis Melanocyte (HEM, Cat. #2210, ScienCell, San Diego, USA) from human’s normal skin was maintained in melanocyte medium (ScienCell, San Diego, USA). Both of these two kinds of cells were in a humidified atmosphere with 5% CO2 at 37 8C.
The experiment of cellular TYR activity was conducted as described previously [4]. B16F10 cells were incubated with various concentrations of andrographolide for 36 h and then lysed with 0.1 M sodium phosphate buffer (pH 6.8) containing 1% Triton X100. Cell extracts were centrifuged at 12,000 rpm for 10 min at 4 8C, and the supernatants were used to measure TYR activity. 200 ml of 0.1 M sodium phosphate buffer (pH 6.8) containing 2 mM L-Dopa, the test supernatants or tyrosinase from mushroom was incubated in a 96-well plate for 0.5 h at 37 8C and then measured at 490 nm. 2.6. Semi-quantitative RT-PCR Total cellular RNA was prepared from B16F10 cells treated with andrographolide using RNAiso plus (TaKaRa, Dalian, China). cDNA was synthesized using PrimeScript RT Master Mix (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Semiquantitative PCR was conducted under the following conditions: 40 cycles of denaturation at 95 8C for 30 s, annealing at 55 8C for 30 s, and extension at 72 8C for 30 s. PCR primers were as follows: 50 -GAGAAGCGAGTCTTGATTAG-30 (forward) and 50 -TGGTGCTTCATGCGCAAAATC-30 (reverse) for TYR; 50 -GGCCTCTGAGGTTCTTTAAT-30 (forward) and 50 -AATGACAAATTGAGGGTGAG-30 (reverse) for TRP1; 50 -ATGAGAAACTGCCAACCTTA-30 (forward) and 50 AGGAGTGAGGCCAAGTTATGA-30 (reverse) for TRP2; 50 -AGTACAGGAGCTGGAGATG-30 (forward) and 50 -GTGAGATCCAGAGTTGTCGT-30 (reverse) for MITF; 50 -ATGAGAAGGAGATCACTGC-30 (forward) and 50 -CTGCGCAAGTTAGGTTTTGT-30 (reverse) for b-actin [16,17].
cell number after treatments with different concentrations of andrographolide for 72 h. (G) Melanin content in HEM cells treated with different concentrations of andrographolide was measured at 490 nm after dissolved in 1 M NaOH. (H) The photos of HEM cells color of samples from (G). (I) Representative morphology of HEM cells from (G). Data are representative of three independent experiments and expressed as means SEM. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control.
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Relative expression was determined by normalizing the data to bactin mRNA level.
Japan). All treatments on animals were in accordance with the ethical standards by Animal Ethics Committee of East China Normal University.
2.7. Western blot analysis 2.9. Schmorl staining Intracellular proteins were prepared from B16F10 cells treated with or without andrographolide. The protein concentration was measured through BCA kit assay. Proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk and exposed to appropriate antibodies at 4 8C overnight. Antibody binding was detected with goat anti-mouse or rabbit IgG with fluorescent monomer and measured by Odyssey. 2.8. UVB irradiation induced hyperpigmentation in brown guinea pigs Brown guinea pigs weighing 370.5–420.0 g obtained from Shaoxing Yue Feng Cage Equipment Co., Ltd. (Shengzhou City, China) were cut out four areas (2 cm 2 cm) evenly and throughly by using infant clipper (Codos, Shenzhen, China) and electric shaver (FLYCO, Shanghai, China). After UVB irradiation (2 J/cm2), guinea pigs were daubed with control cream, 5% andrographolide cream, 5% kojic acid cream and 0.5% andrographolide cream twice per day. When the color appeared different, all the guinea pigs were shaved and took photos by digital camera (Canon, Tokyo,
After dewaxing and hydration, skin tissues of the brown guinea pigs were incubated in the solution of 30 ml of 1% fresh ferric chloride, 4 ml of 1% fresh potassium ferrocyanide and 6 ml of distilled water for 10 min. Then samples were stained with eosin and observed from multiple randomly selected microscopic visual fields. 2.10. Immunohistochemistry Skin sections were treated with sodium citrate buffer and incubated with anti-TYR antibody at 4 8C overnight. Skin samples were covered with goat anti-rabbit IgG with horseradish peroxidase (HRP), stained with hematoxylin and then observed from multiple randomly selected microscopic visual fields. 2.11. Statistical analysis Data are presented as means standard error of mean (SEM) of three independent experiments and analyzed by Student’s t-test. A p value less than 0.05 is considered significant.
Fig. 2. Effects of andrographolide on TYR activity in B16F10 murine melanoma cells and HEM cells. (A) Supernatants were prepared from B16F10 cells treated with different concentrations of andrographolide for 36 h. TYR activity was detected after incubation at 37 8C for 0.5 h in the system containing 100 ml of 4 mM L-Dopa in 0.1 M sodium phosphate buffer (pH 6.8) and 100 ml of the test supernatant. (B) Supernatants were prepared from HEM cells treated with different concentrations of andrographolide for 72 h. TYR activity was detected after incubation at 37 8C for 0.5 h in the system containing 2 mM L-Dopa and the test supernatant. (C) Supernatants prepared from B16F10 cells treated with no andrographolide were mixed with different concentrations of andrographolide and L-Dopa, and then measured at 490 nm after incubation at 37 8C for 0.5 h. (D) Activity of tyrosinase from mushroom was measured at 490 nm in a 96-well plate where 25 U/ml tyrosinase from mushroom, 2 mM L-Dopa and different concentrations of andrographolide were incubated at 37 8C for 0.5 h. Data are representative of three independent experiments and expressed as means SEM. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control.
Please cite this article in press as: Zhu P-Y, et al. Andrographolide suppresses melanin synthesis through Akt/GSK3b/b-catenin signal pathway. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.03.013
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3. Results 3.1. Andrographolide inhibits melanin content in B16F10 cells and HEM cells Andrographolide could inhibit the proliferation of B16F10 cells when the concentration reached 25 mM even higher, while it (20 mM) had no effects on the growth of B16F10 cells meaning not increasing or reducing the number of cells (Fig. 1B). So the concentration of andrographolide less than 20 mM was used in the following experiments in cells. Non-toxic andrographolide treatments could suppress melanin content in B16F10 cells concentration in a dose dependent manner. Moreover, the inhibitory effects of andrographolide were better than those of the two kinds of common whiting agents named kojic acid (300 mM) and arbutin (100 mM) when the concentration of andrographolide reached 5 mM even higher (Fig. 1C and D). Meanwhile, andrographolide had effects on the morphology of B16F10 cells (Fig. 1E). Also, andrographolide had the ability to decrease melanin content in HEM cells in a dose dependent manner when its concentration is not higher than 20 mM (Fig. 1F and G). Importantly, HEM cell viability was not affected after treatments with andrographolide at the concentration less than 20 mM. However, it gave a decline to the quantity of HEM cells and inhibited the proliferation of cells when the concentration reached 25 mM (Fig. 1F and G). These results demonstrated that the inhibitory function of andrographolide on melanin content in HEM cells was not from suppressing HEM cell proliferation at the medium concentrations. The melanin content measured in HEM cells was prepared from the cell lysis and not from supernatant of cells as B16F10 cells did. The graph showed adrographolide could change the color of HEM
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cells and made them lighter, but could not change morphology of HEM cells apparently, simultaneously (Fig. 1H and I). Consequently, andrographolide probably inhibited melanin synthesis leading to the color of cells lighter. 3.2. Andrographolide inhibits melanin synthesis in B16F10 cells and HEM cells As shown in Fig. 2A and B, TYR activity was suppressed in B16F10 cells and HEM cells treated with andrographolide indicating that the decline to melanin content in B16F10 and HEM cells was dependent on the inhibition of TYR activity. Then TYR activity was also detected in B16F10 cells incubated with andrographolide after lysed with 1% Triton-X100 and the result showed no difference (Fig. 2B). Moreover, there were no obvious changes existing in activity of tyrosinase from mushroom whether treated with andrographolide or not (Fig. 2C). The data indicated that andrographolide probably inhibited the function of TYR by decreasing TYR content. So TYR expression was measured and it was found that the mRNA and protein levels of TYR family in B16F10 cells treated with andrographolide (different concentrations from 1 mM to 10 mM) both decreased (Fig. 3A–C). As TYR family is the downstream genes of MITF, the expression of MITF was measured. The graph demonstrated that the transcriptional and protein expression levels of MITF both reduced in B16F10 cells in the presence of andrographolide (Fig. 3A–C). 3.3. Andrographolide inhibits melanin synthesis in UVB irradiation induced brown guinea pigs As we know, UVB irradiation stimulates melanin synthesis in melanocyte [9,18] and it can be a slow progress in brown guinea
Fig. 3. Effects of andrographolide on transcriptional (A and B) and protein (C) levels of TYR family and MITF in B16F10 cells. (A)The mRNA levels of TYR family, MITF and bactin from B16F10 cells treated with andrographolide for 36 h were detected using semi-quantitative PCR. (B) Samples from (A) were analyzed by 2% agarose gel electrophoresis. (C) Intracellular proteins from B16F10 cells treated with different concentrations of andrographolide for 36 h were detected by Western blot.
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pigs. Brown guinea pigs were used in this study to determine whether andrographolide could inhibit melanin synthesis in animals. Data showed that 5% andrographolide could inhibit the increase in melanin and TYR content induced by UVB (Fig. 4A–C). Data also indicated andrographolide (5%) was strong as kojic acid (5%) in inhibiting melanin synthesis (Fig. 4A and B). The number of TYR-positive melanocyte increased markedly after UVB irradiation while andrographolide inhibited effectively the increase of TYRpositive melanocyte compared with the controls (Fig. 4D). 3.4. Andrographolide inhibits wnt signal pathway by regulating Akt signal molecule Wnt signal pathway was demonstrated to be closely related with melanin synthesis. In order to unravel the mechanism by which andrographolide inhibited melanin synthesis, we measured the changes of b-catenin in B16F10 cells treated with andrographolide. Data showed that andrographolide decreased intracellular b-catenin content in B16F10 cells (Fig. 5A). Importantly, b-catenin content in the nucleus (the active b-catenin) was reduced obviously while the content in the cytoplasm did not change. MG-132 is a kind of proteasome inhibitor. There was no obvious difference on b-catenin content in cells whether treated with andrographolide or not in the presence of MG-132 (Fig. 5B). Data
showed that the decline of total b-catenin induced by andrographolide probably resulted from promoting the degradation of b-catenin. Then CHX, a kind of material inhibiting the translation and blocking protein synthesis, was used in experiments to detect whether andrographolide could change the half-life of b-catenin. The results demonstrated that andrographolide could lower the half-life of b-catenin from 21.37 h to 15.14 h in the presence of CHX (Fig. 5C and D). GSK3b in the multiproteins complex can phosphorylate the ser/ thr residues of b-catenin, which contributes to the degradation of b-catenin through proteasome [7]. As expected, the level of phosphorylation of GSK3b decreased in B16F10 cells treated with andrographolide (Fig. 5A). BIO, a kind of GSK3b inhibitor promoting the phosphorylation of GSK3b, makes b-catenin accumulate in the cell resulting in an increase to TYR activity and melanin content [19]. The graph showed that BIO could reverse the decline of bcatenin content induced by andrographolide (Fig. 6A). Consistent with this, BIO also increased the TYR activity and melanin content in B16F10 cells in the presence of andrographolide evidently (Fig. 6B and C). These results demonstrated that andrographolide induced bcatenin degradation in a GSK3b-dependent manner. It has been reported that PI3K/Akt signal pathway has a close relationship to GSK3b [21,33]. Activated Akt can phosphorylate at ser 9 in GSK3b resulting in inactivating GSK3b and inhibiting the degradation of b-catenin [20]. Our data indicated that the content
Fig. 4. Effects of andrographolide on melanin content and TYR content in UVB-irradiation (2 J/cm2) induced brown guinea pigs. (A) Skin of area d and e was treated with different concentrations of andrographolide (0.05%, 5%) while area c was treated with kojic acid (5%). Area a is the negative control (no UVB irradiation or drug treatment) and area b is the control without drug treatment. When the color between four areas appeared different, digital camera was used to take photos. (B) Skin of area a, b, c, e was taken and melanin in skin was stained. (C) Skin samples from the same areas as (B) were marked with anti-TYR antibody. (D) The amount of the TYR-positive (TYR+) melanocyte from multiple randomly selected microscopic visual fields. Data are expressed as a ratio of the group only treated with UVB irradiation as mean SEM. #p < 0.01 compared with the second group.
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Fig. 5. Effects of andrographolide on wnt/b-catenin and Akt signal molecular in B16F10 cells. (A) B16F10 cells were treated with andrographolide of 10 mM for 6 h, the expression levels of proteins including b-catenin, p-GSK3b and p-Akt were detected by using western blot. (B) b-Catenin was detected in B16F10 cells pre-treated with or without MG-132 (20 mM) for 2 h and incubated with andrographolide (10 mM) or not for 6 h. (C) b-Catenin was detected from B16F10 cells treated with CHX for different time in the presence or absence of andrographolide. (D) Quantitation from (C) by using software. b-Actin, hsp70, histone H3.1 were included as protein-loading controls. Data are expressed as a ratio of the group treated with CHX for 0 h as mean SEM. **p < 0.01, ***p < 0.001 compared with the control.
of p-Akt and p-GSK3b decreased in B16F10 cells in the presence of andrographolide simultaneously (Fig. 5A). IGF-1, a kind of molecular like insulin in structure and function, can activate PI3K/Akt signal pathway and increase the phosphorylation level of Akt positively regulating wnt/b-catenin signal pathway [21]. We observed that there was no mark distinction on b-catenin content in B16F10 cells treated with or without andrographolide in the presence of IGF-1 (Fig. 6D). We assumed that IGF-1 could reverse the decline of total b-catenin content in B16F10 cells induced by andrographolide, which depended on phosphorylating Akt and GSK3b. But IGF-1 did not change TYR activity and melanin content in the presence or absent of andrographolide (Fig. 6E and F).
4. Discussion Andrographolide, a kind of plant extract from A. paniculata, belongs to diterpenoid lactone. It has been reported that andrographolide has hepatoprotective activity against paracetamol and carbontetrachloride intoxication in rats [22,23], stands up against influenza virus [24], plays a role in protecting from HIV [25]. It can also decrease plasma glucose and resist hyperglycaemia through increasing glucose utilization [26]. In addition, it is involved in immunity and inflammation via inhibition of the nuclear factor-kB signaling [27]. Yuh-Chiang Shen et al. [28] reported that andrographolide has the ability to prevent production of oxygen radical. Meanwhile, oxygen radical (usually called reactive oxygen species, ROS) has the ability to stimulate pigmentation and melanin has property of free radical scavenging [29,30]. Therefore, we assumed that maybe andrographolide had
the inhibitory effects on melanin synthesis, which was proven in this study. Signal pathways involved in melanin synthesis include mitogen-activated protein kinases (MAPKs) signal pathway, cAMP signal pathway, PI3K/Akt signal pathway, wnt/b-catenin signal pathway and so on [4,17,31]. In wnt/b-catenin signal pathway, bcatenin accumulating in cytoplasm up-regulates MITF content through promoting transcription with LEF1 after coming into nucleus [4]. In the present study, we found that b-catenin can be phosphorylated with several methods including axin-mediated CKI phosphorylation at ser 45 [5], GSK3b phosphorylation and PKCa phosphorylation at N-terminal ser/thr residues [7]. Siah-1 recruits the ubiquitination complex resulting in the degradation of b-catenin through proteasome in a ubiquitin-dependent way [32,33]. Here, decreased p-GSK3b (inactive form of GSK3b) leads to the degradation of b-catenin. Activation of GSK3b can be mediated by some kinases including Akt, PKA, PKC and p90rsk (also called MAPK-activated protein kinase-1) via phosphorylation at ser 9 in GSK3b [34]. It has been reported that andrographolide can affect PI3K/Akt signal pathway through inhibition of PI3K and Akt phosphorylation [14]. Here, andrographolide inhibited the phosphorylation level of Akt. We assumed that IGF-1, a potential Akt activator, could improve TYR activity and melanin content by increasing the phosphorylation of Akt and GSK3b, inhibiting the degradation of b-catenin and promoting the transcriptional and protein expression of MITF and TYR family. But results show no apparent change existing in TYR activity and melanin content treated with IGF-1. It has been reported that GSK3b can promote MITF bind to TYR family promoters and increase the transcription of TYR family [35].
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Fig. 6. Effects of andrographolide on b-catenin, TYR activity and melanin content in B16F10 cells treated with BIO or IGF-1. (A) b-Catenin was detected in B16F10 cells pretreated with or without BIO (2 mM) and incubated with andrographolide (10 mM) or not. (B) TYR activity was measured using samples from (A). (C) Melanin content was measured at 490 nm using samples from (A). (D) The phosphorylation of Akt and GSK3b and the content of b-catenin were determined by western blot in B16F10 cells treated with andrographolide or not in the presence or absence of IGF-1 (200 ng/ml) for 24 h. (E) TYR activity was measured using samples from (D). (F) Melanin content was measured at 490 nm using samples from (D). Data are representative of three independent experiments and expressed as means SEM. ***p < 0.001 compared with the control. ###p < 0.001 compared with the group only treated with andrographolide.
Probably, IGF-1 has potential to decrease TYR activity and melanin content by increasing the inactivation of GSK3b and inhibiting the binding of MITF to its target genes. But IGF-1 also could promote the accumulation of b-catenin which could increase the transcription of MITF in this study. As a result, these two different effects of IGF-1 on melanin synthesis probably explain the consequent results seen in Fig. 6E and F. Interestingly, B16F10 cells maintained in DMEM for 72 h could release melanin, while HEM cells could not even in melanocyte medium for 96 h. It was found that when the culture of B16F10 cells reached 100% confluency and still maintained in DMEM, the medium became dark due to melanin release. It was also observed that the mass of B16F10 cells was grayish-black before the color of medium changed. However, the color of melanocyte medium incubated with HEM cells always had little change, indicating no melanin released from HEM cells. As a result, melanin content in B16F10 cells can be assessed by measuring the absorbance of the medium, which is convenient but not suitable for measure of melanin content in HEM cells. Before melanin content in HEM cells is measured, HEM cells should be harvested and dissolved in 1 M NaOH.
It has been reported that melanosomes, where melanin is synthesized, are located in melanocyte, and then transported to adjacent keratinocytes leading to pigmentation [36,37]. B16F10 murine melanoma cells is a kind of carcinoma cell line, where gene mutations have happened and some features have changed, such as melanin released from cells immediately without keratinocytes. That may explain why B16F10 cells can release melanin to the culture medium but not for HEM cells. In this study, we also found that andrographolide could change the morphology of B16F10 cells but had little effects on HEM cells, which may resulted from the difference between carcinoma cells and normal cells. In summary, andrographolide inhibits TYR activity and melanin content probably via down-regulating MITF expression depending on the inhibition of Akt phosphorylation and GSK3b phosphorylation and induction of b-catenin degradation in B16F10 cells (see Fig. 7). In vivo, andrographolide also inhibits TYR content and melanin content in UVB irradiation induced brown guinea pig skin. Importantly, we found that andrographolide can effectively reduce melanin content and TYR activity in HEM cells with no apparent change of morphology. Thus, andrographolide probably can be a whiting agent widely used in cosmetics in the future.
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Fig. 7. The possible mechanism of andrographolide inhibiting melanin content in B16F10 cells. Andrographolide decreases phosphorylation of Akt and GSK3b and thus probably increases the activated GSK3b, which induces the phosphorylation of b-catenin and causes its degradation via proteasome. The reduction of b-catenin probably leads to the inhibition of transcription of MITF and TYR family and the subsequent reduction of melanin synthesis.
Funding disclosures This work was supported by the grants from National Natural Science Foundation of China (No. 81272226, No. 81271742), the grants from the Key Science and Technology Projects from the Science and Technology Commission of Shanghai Municipality (13JC1405102).
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Andrographolide suppresses melanin synthesis through Akt/GSK3b/b-catenin signal pathway Ping-Ya Zhu, Wei-Han Yin, Meng-Ran Wang, Yong-Yan Dang, Xi-Yun Ye * Shanghai Key Laboratory of Regulatory Biology, School of Life Science, East China Normal University, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 September 2014 Received in revised form 18 March 2015 Accepted 19 March 2015
Background: Tyrosinase (TYR) is the key enzyme controlling the production of melanin. Very few papers have reported that andrographolide can inhibit melanin content. Objective: To investigate the effects of andrographolide on melanin synthesis. Methods: Cell viability, melanin content, TYR activity, transcriptional and protein expression levels of TYR family and other kinds of proteins involved in melanogenesis were measured after the treatments of andrographolide. Results: It was found that andrographolide decreased melanin content, TYR activity and transcriptional and protein expression of TYR family and microphthalmia-associated transcription factor (MITF) in B16F10 melanoma cells. Data showed andrographolide also decreased melanin content and TYR content in ultraviolet B (UVB) irradiation induced brown guinea pigs. Moreover, we found that melanin content and TYR activity were effectively inhibited in Human Epidermis Melanocyte (HEM) treated with andrographolide at the medium concentrations without apparent effect on cell viability. Results in experiments treated with MG-132 or cycloheximide (CHX) showed that andrographolide lowered the content of b-catenin in cell nucleus resulting from accelerating the degradation of b-catenin. Phosphorylation of glycogen synthase kinase 3b (GSK3b) and Akt decreased simultaneously. 6Bromoindirubin-30 -oxime (BIO, inhibitor of GSK3b) and insulin-like growth factors-1 (IGF-1, activator of Akt) could reverse the decline of b-catenin in B16F10 cells induced by andrographolide. Conclusion: These results demonstrate that andrographolide can effectively suppress melanin content and TYR activity in B16F10 cells, HEM cells and UVB-induced brown guinea pig skin by decreasing phosphorylation of GSK3b dependent on Akt, promoting the degradation of b-catenin, inhibiting bcatenin into the nucleus and decreasing the expression of MITF and TYR family. Data indicate that andrographolide may be a potential whiting agent which can have great market in cosmetics and in clinical such as curing hyperpigmentation disorders. ß 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Melanin synthesis Andrographolide TYR family b-Catenin
1. Introduction Melanin is synthesized in melanocyte which is located between epidermis and dermis and it can protect skin from harm brought by ultraviolet rays, such as aging and skin cancer. Melanin content in skin is influenced by melanin synthesis, melanin transportation, melanin degradation, thickness of stratum corneum, proliferation of the epidermis and other factors, which contribute to color diversity in skin. Melanin synthesis, the major contributor of skin
* Corresponding author at: School of Life Science, East China Normal University, No. 500, Dongchuan Road, 200241 Shanghai, China. Tel.: +86 21 54341038. E-mail address: [email protected] (X.-Y. Ye).
melanin content, depends on the number and the level of differentiation of melanocyte. Melanin synthesis happens in melanosome where the substrate tyrosine turns into intermediate product named DOPA quinone with the help of tyrosinase (TYR), and then the intermediate product turns into two kinds of melanin (eumelanin and pheomelanin) via other interrelated enzymatic reactions [1]. In this progress, TYR is the key enzyme controlling the production of melanin [2]. The expression of TYR family comprising of TYR, tyrosinase related protein 1 (TRP1) and tyrosinase related protein 2 (TRP2), is influenced by MITF. MITF can bind to Bbox and M-box motifs in the promoters of TYR family and then promotes the transcription of TYR family [3]. Recently, wnt/bcatenin signal pathway is known to play an important role in melanin synthesis [4].
http://dx.doi.org/10.1016/j.jdermsci.2015.03.013 0923-1811/ß 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
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Fig. 1. Effects of andrographolide on cell proliferation and melanin content in B16F10 murine melanoma cells and HEM cells. (A) The structure of andrographolide. (B) Cell viability of B16F10 cells was measured using MTS assay and shown by the absorbance at 490 nm after treatments with different concentrations of andrographolide for 48 h. (C) Melanin content in the supernatants from B16F10 cells cultured in red-free DMEM treated with different concentrations of andrographolide, positive whiting agents (kojic acid and arbutin) was determined by the absorbance at 490 nm. (D) The photos of supernatant color of samples from (C). (E) Representative morphology of B16F10 cells from (C) by inverted phase contrast microscope. (F) Cell viability of HEM cells was measured by counting
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When wnt/b-catenin signal pathway is not activated, bcatenin is phosphorylated by a multiproteins complex comprising of axin, adenomatous polyposis coli (APC), glycogen synthase kinase 3b (GSK3b) and casein kinase 1 (CK1), and then affected by ubiquitin and degradated through proteasomes [5]. But when wnt/b-catenin signal pathway is activated by its wnt ligands such as wnt1, wnt3a and wnt8, GSK3b is negatively regulated resulting in the accumulation of b-catenin in cytoplasm which can be translocated into the nucleus and combines to the promoter of MITF together with LEF1 contributing to the transcription of MITF [6,7]. In addition, there are many kinds of stimulating factors which can stimulate melanin synthesis, such as a-melanocyte stimulating hormone (a-MSH) [8], UVB irradiation [9], pressure [10] and so on. In the field of cosmetics, there are several common whiting agents, such as kojic acid and arbutin. But it has been reported that kojic acid has the ability to induce cancer [11] and arbutin can promote melanin synthesis when its concentration is higher [12]. As a result, it is significative to search for new kinds of whiting agents. Some studies have indicated that several plant components, such as mulberry extract [13], can suppress melanin synthesis and have been added to skin care product and have not been found any damage on human skin so far. Therefore, it is feasible to search for new plant components which can suppress melanin synthesis and have enormous potential. Andrographolide is a kind of natural antibiotic and can inhibit the growth of tumor from a kind of plant named Andrographis paniculata [14]. However, there are very few data showing that andrographolide can inhibit melanin synthesis and TYR activity so far.
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2.3. Cell viability assay Viability of B16F10 cells was determined by MTS (Promega, Madison, USA) assay. After andrographolide treatments, the B16F10 cells were incubated for 48 h at 37 8C. Then 20 ml MTS was added to each well of a 96-well plate. After incubation for 0.5 h, the absorbance was measured at 490 nm using a SPECTRA MAX 190 spectrometer (Molecular Devices, CA, USA). The effect of andrographolide on the growth of HEM cells was assessed by counting cell number. After andrographolide treatments for 72 h, HEM cells were incubated with EDTA/trypsin (Cat. #0103 ScienCell, San Diego, USA) and added with trypsin/EDTA neutralization solution (TNS, Cat. #0113, ScienCell, San Diego, USA), then counted with blood counting chamber. 2.4. Melanin content measurement Extracellular melanin release of B16F10 cells was measured with a previously described method with slight modifications [15]. B16F10 cells (1.2 105/well in 6-well plates) were maintained in phenol red-free DMEM (Gibco, MA, USA) with andrographolide (20 mM), kojic acid (300 mM) or arbutin (100 mM) for 48 h. Controls were designed to put the same phenol red-free DMEM without cells or materials for 48 h. 200 ml of supernatant from all the wells was measured at 490 nm. HEM cells were harvested with EDTA/trypsin after treatments of andrographolide with different concentrations for 72 h. Then cells were centrifuged at 10,000 rpm for 15 min and dissolved in 1 M NaOH, whose absorbance was measured at 490 nm. 2.5. TYR activity assay
2. Materials and methods 2.1. Materials Tyrosinase from mushroom, L-3,4-dihydroxyphenylalanine (LDopa), 6-bromoindirubin-30 -oxime (BIO), cycloheximide (CHX) and kojic acid were purchased from Sigma–Aldrich (St Louis, USA). Protease inhibitor and phosphatase inhibitor were purchased from Roche (Manheim, Germany). Antibodies against TYR, TRP1, TRP2 and MITF were purchased from Abcam (Cambridge, UK). Antibody against b-catenin was from BD (Franklin Lake, USA). Antibodies against b-actin, GSK3b, phospho-GSK3b, Akt, phospho-Akt, histone H3.1 and hsp70 were from Cell Signaling Technology (Danvers, USA). MG-132 and IGF-1 were purchased from Cell Signaling Technology. Arbutin and andrographolide were purchased from Shanghai Winherb Medical Science Co., Ltd. (Shanghai, China). 2.2. Cell culture The murine B16F10 melanoma cell line (Shanghai Key Laboratory of Regulatory Biology, China) was maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, MA, USA) supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin (Wisent, Montreal, Canada). Human Epidermis Melanocyte (HEM, Cat. #2210, ScienCell, San Diego, USA) from human’s normal skin was maintained in melanocyte medium (ScienCell, San Diego, USA). Both of these two kinds of cells were in a humidified atmosphere with 5% CO2 at 37 8C.
The experiment of cellular TYR activity was conducted as described previously [4]. B16F10 cells were incubated with various concentrations of andrographolide for 36 h and then lysed with 0.1 M sodium phosphate buffer (pH 6.8) containing 1% Triton X100. Cell extracts were centrifuged at 12,000 rpm for 10 min at 4 8C, and the supernatants were used to measure TYR activity. 200 ml of 0.1 M sodium phosphate buffer (pH 6.8) containing 2 mM L-Dopa, the test supernatants or tyrosinase from mushroom was incubated in a 96-well plate for 0.5 h at 37 8C and then measured at 490 nm. 2.6. Semi-quantitative RT-PCR Total cellular RNA was prepared from B16F10 cells treated with andrographolide using RNAiso plus (TaKaRa, Dalian, China). cDNA was synthesized using PrimeScript RT Master Mix (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Semiquantitative PCR was conducted under the following conditions: 40 cycles of denaturation at 95 8C for 30 s, annealing at 55 8C for 30 s, and extension at 72 8C for 30 s. PCR primers were as follows: 50 -GAGAAGCGAGTCTTGATTAG-30 (forward) and 50 -TGGTGCTTCATGCGCAAAATC-30 (reverse) for TYR; 50 -GGCCTCTGAGGTTCTTTAAT-30 (forward) and 50 -AATGACAAATTGAGGGTGAG-30 (reverse) for TRP1; 50 -ATGAGAAACTGCCAACCTTA-30 (forward) and 50 AGGAGTGAGGCCAAGTTATGA-30 (reverse) for TRP2; 50 -AGTACAGGAGCTGGAGATG-30 (forward) and 50 -GTGAGATCCAGAGTTGTCGT-30 (reverse) for MITF; 50 -ATGAGAAGGAGATCACTGC-30 (forward) and 50 -CTGCGCAAGTTAGGTTTTGT-30 (reverse) for b-actin [16,17].
cell number after treatments with different concentrations of andrographolide for 72 h. (G) Melanin content in HEM cells treated with different concentrations of andrographolide was measured at 490 nm after dissolved in 1 M NaOH. (H) The photos of HEM cells color of samples from (G). (I) Representative morphology of HEM cells from (G). Data are representative of three independent experiments and expressed as means SEM. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control.
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Relative expression was determined by normalizing the data to bactin mRNA level.
Japan). All treatments on animals were in accordance with the ethical standards by Animal Ethics Committee of East China Normal University.
2.7. Western blot analysis 2.9. Schmorl staining Intracellular proteins were prepared from B16F10 cells treated with or without andrographolide. The protein concentration was measured through BCA kit assay. Proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk and exposed to appropriate antibodies at 4 8C overnight. Antibody binding was detected with goat anti-mouse or rabbit IgG with fluorescent monomer and measured by Odyssey. 2.8. UVB irradiation induced hyperpigmentation in brown guinea pigs Brown guinea pigs weighing 370.5–420.0 g obtained from Shaoxing Yue Feng Cage Equipment Co., Ltd. (Shengzhou City, China) were cut out four areas (2 cm 2 cm) evenly and throughly by using infant clipper (Codos, Shenzhen, China) and electric shaver (FLYCO, Shanghai, China). After UVB irradiation (2 J/cm2), guinea pigs were daubed with control cream, 5% andrographolide cream, 5% kojic acid cream and 0.5% andrographolide cream twice per day. When the color appeared different, all the guinea pigs were shaved and took photos by digital camera (Canon, Tokyo,
After dewaxing and hydration, skin tissues of the brown guinea pigs were incubated in the solution of 30 ml of 1% fresh ferric chloride, 4 ml of 1% fresh potassium ferrocyanide and 6 ml of distilled water for 10 min. Then samples were stained with eosin and observed from multiple randomly selected microscopic visual fields. 2.10. Immunohistochemistry Skin sections were treated with sodium citrate buffer and incubated with anti-TYR antibody at 4 8C overnight. Skin samples were covered with goat anti-rabbit IgG with horseradish peroxidase (HRP), stained with hematoxylin and then observed from multiple randomly selected microscopic visual fields. 2.11. Statistical analysis Data are presented as means standard error of mean (SEM) of three independent experiments and analyzed by Student’s t-test. A p value less than 0.05 is considered significant.
Fig. 2. Effects of andrographolide on TYR activity in B16F10 murine melanoma cells and HEM cells. (A) Supernatants were prepared from B16F10 cells treated with different concentrations of andrographolide for 36 h. TYR activity was detected after incubation at 37 8C for 0.5 h in the system containing 100 ml of 4 mM L-Dopa in 0.1 M sodium phosphate buffer (pH 6.8) and 100 ml of the test supernatant. (B) Supernatants were prepared from HEM cells treated with different concentrations of andrographolide for 72 h. TYR activity was detected after incubation at 37 8C for 0.5 h in the system containing 2 mM L-Dopa and the test supernatant. (C) Supernatants prepared from B16F10 cells treated with no andrographolide were mixed with different concentrations of andrographolide and L-Dopa, and then measured at 490 nm after incubation at 37 8C for 0.5 h. (D) Activity of tyrosinase from mushroom was measured at 490 nm in a 96-well plate where 25 U/ml tyrosinase from mushroom, 2 mM L-Dopa and different concentrations of andrographolide were incubated at 37 8C for 0.5 h. Data are representative of three independent experiments and expressed as means SEM. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control.
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3. Results 3.1. Andrographolide inhibits melanin content in B16F10 cells and HEM cells Andrographolide could inhibit the proliferation of B16F10 cells when the concentration reached 25 mM even higher, while it (20 mM) had no effects on the growth of B16F10 cells meaning not increasing or reducing the number of cells (Fig. 1B). So the concentration of andrographolide less than 20 mM was used in the following experiments in cells. Non-toxic andrographolide treatments could suppress melanin content in B16F10 cells concentration in a dose dependent manner. Moreover, the inhibitory effects of andrographolide were better than those of the two kinds of common whiting agents named kojic acid (300 mM) and arbutin (100 mM) when the concentration of andrographolide reached 5 mM even higher (Fig. 1C and D). Meanwhile, andrographolide had effects on the morphology of B16F10 cells (Fig. 1E). Also, andrographolide had the ability to decrease melanin content in HEM cells in a dose dependent manner when its concentration is not higher than 20 mM (Fig. 1F and G). Importantly, HEM cell viability was not affected after treatments with andrographolide at the concentration less than 20 mM. However, it gave a decline to the quantity of HEM cells and inhibited the proliferation of cells when the concentration reached 25 mM (Fig. 1F and G). These results demonstrated that the inhibitory function of andrographolide on melanin content in HEM cells was not from suppressing HEM cell proliferation at the medium concentrations. The melanin content measured in HEM cells was prepared from the cell lysis and not from supernatant of cells as B16F10 cells did. The graph showed adrographolide could change the color of HEM
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cells and made them lighter, but could not change morphology of HEM cells apparently, simultaneously (Fig. 1H and I). Consequently, andrographolide probably inhibited melanin synthesis leading to the color of cells lighter. 3.2. Andrographolide inhibits melanin synthesis in B16F10 cells and HEM cells As shown in Fig. 2A and B, TYR activity was suppressed in B16F10 cells and HEM cells treated with andrographolide indicating that the decline to melanin content in B16F10 and HEM cells was dependent on the inhibition of TYR activity. Then TYR activity was also detected in B16F10 cells incubated with andrographolide after lysed with 1% Triton-X100 and the result showed no difference (Fig. 2B). Moreover, there were no obvious changes existing in activity of tyrosinase from mushroom whether treated with andrographolide or not (Fig. 2C). The data indicated that andrographolide probably inhibited the function of TYR by decreasing TYR content. So TYR expression was measured and it was found that the mRNA and protein levels of TYR family in B16F10 cells treated with andrographolide (different concentrations from 1 mM to 10 mM) both decreased (Fig. 3A–C). As TYR family is the downstream genes of MITF, the expression of MITF was measured. The graph demonstrated that the transcriptional and protein expression levels of MITF both reduced in B16F10 cells in the presence of andrographolide (Fig. 3A–C). 3.3. Andrographolide inhibits melanin synthesis in UVB irradiation induced brown guinea pigs As we know, UVB irradiation stimulates melanin synthesis in melanocyte [9,18] and it can be a slow progress in brown guinea
Fig. 3. Effects of andrographolide on transcriptional (A and B) and protein (C) levels of TYR family and MITF in B16F10 cells. (A)The mRNA levels of TYR family, MITF and bactin from B16F10 cells treated with andrographolide for 36 h were detected using semi-quantitative PCR. (B) Samples from (A) were analyzed by 2% agarose gel electrophoresis. (C) Intracellular proteins from B16F10 cells treated with different concentrations of andrographolide for 36 h were detected by Western blot.
Please cite this article in press as: Zhu P-Y, et al. Andrographolide suppresses melanin synthesis through Akt/GSK3b/b-catenin signal pathway. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.03.013
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pigs. Brown guinea pigs were used in this study to determine whether andrographolide could inhibit melanin synthesis in animals. Data showed that 5% andrographolide could inhibit the increase in melanin and TYR content induced by UVB (Fig. 4A–C). Data also indicated andrographolide (5%) was strong as kojic acid (5%) in inhibiting melanin synthesis (Fig. 4A and B). The number of TYR-positive melanocyte increased markedly after UVB irradiation while andrographolide inhibited effectively the increase of TYRpositive melanocyte compared with the controls (Fig. 4D). 3.4. Andrographolide inhibits wnt signal pathway by regulating Akt signal molecule Wnt signal pathway was demonstrated to be closely related with melanin synthesis. In order to unravel the mechanism by which andrographolide inhibited melanin synthesis, we measured the changes of b-catenin in B16F10 cells treated with andrographolide. Data showed that andrographolide decreased intracellular b-catenin content in B16F10 cells (Fig. 5A). Importantly, b-catenin content in the nucleus (the active b-catenin) was reduced obviously while the content in the cytoplasm did not change. MG-132 is a kind of proteasome inhibitor. There was no obvious difference on b-catenin content in cells whether treated with andrographolide or not in the presence of MG-132 (Fig. 5B). Data
showed that the decline of total b-catenin induced by andrographolide probably resulted from promoting the degradation of b-catenin. Then CHX, a kind of material inhibiting the translation and blocking protein synthesis, was used in experiments to detect whether andrographolide could change the half-life of b-catenin. The results demonstrated that andrographolide could lower the half-life of b-catenin from 21.37 h to 15.14 h in the presence of CHX (Fig. 5C and D). GSK3b in the multiproteins complex can phosphorylate the ser/ thr residues of b-catenin, which contributes to the degradation of b-catenin through proteasome [7]. As expected, the level of phosphorylation of GSK3b decreased in B16F10 cells treated with andrographolide (Fig. 5A). BIO, a kind of GSK3b inhibitor promoting the phosphorylation of GSK3b, makes b-catenin accumulate in the cell resulting in an increase to TYR activity and melanin content [19]. The graph showed that BIO could reverse the decline of bcatenin content induced by andrographolide (Fig. 6A). Consistent with this, BIO also increased the TYR activity and melanin content in B16F10 cells in the presence of andrographolide evidently (Fig. 6B and C). These results demonstrated that andrographolide induced bcatenin degradation in a GSK3b-dependent manner. It has been reported that PI3K/Akt signal pathway has a close relationship to GSK3b [21,33]. Activated Akt can phosphorylate at ser 9 in GSK3b resulting in inactivating GSK3b and inhibiting the degradation of b-catenin [20]. Our data indicated that the content
Fig. 4. Effects of andrographolide on melanin content and TYR content in UVB-irradiation (2 J/cm2) induced brown guinea pigs. (A) Skin of area d and e was treated with different concentrations of andrographolide (0.05%, 5%) while area c was treated with kojic acid (5%). Area a is the negative control (no UVB irradiation or drug treatment) and area b is the control without drug treatment. When the color between four areas appeared different, digital camera was used to take photos. (B) Skin of area a, b, c, e was taken and melanin in skin was stained. (C) Skin samples from the same areas as (B) were marked with anti-TYR antibody. (D) The amount of the TYR-positive (TYR+) melanocyte from multiple randomly selected microscopic visual fields. Data are expressed as a ratio of the group only treated with UVB irradiation as mean SEM. #p < 0.01 compared with the second group.
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Fig. 5. Effects of andrographolide on wnt/b-catenin and Akt signal molecular in B16F10 cells. (A) B16F10 cells were treated with andrographolide of 10 mM for 6 h, the expression levels of proteins including b-catenin, p-GSK3b and p-Akt were detected by using western blot. (B) b-Catenin was detected in B16F10 cells pre-treated with or without MG-132 (20 mM) for 2 h and incubated with andrographolide (10 mM) or not for 6 h. (C) b-Catenin was detected from B16F10 cells treated with CHX for different time in the presence or absence of andrographolide. (D) Quantitation from (C) by using software. b-Actin, hsp70, histone H3.1 were included as protein-loading controls. Data are expressed as a ratio of the group treated with CHX for 0 h as mean SEM. **p < 0.01, ***p < 0.001 compared with the control.
of p-Akt and p-GSK3b decreased in B16F10 cells in the presence of andrographolide simultaneously (Fig. 5A). IGF-1, a kind of molecular like insulin in structure and function, can activate PI3K/Akt signal pathway and increase the phosphorylation level of Akt positively regulating wnt/b-catenin signal pathway [21]. We observed that there was no mark distinction on b-catenin content in B16F10 cells treated with or without andrographolide in the presence of IGF-1 (Fig. 6D). We assumed that IGF-1 could reverse the decline of total b-catenin content in B16F10 cells induced by andrographolide, which depended on phosphorylating Akt and GSK3b. But IGF-1 did not change TYR activity and melanin content in the presence or absent of andrographolide (Fig. 6E and F).
4. Discussion Andrographolide, a kind of plant extract from A. paniculata, belongs to diterpenoid lactone. It has been reported that andrographolide has hepatoprotective activity against paracetamol and carbontetrachloride intoxication in rats [22,23], stands up against influenza virus [24], plays a role in protecting from HIV [25]. It can also decrease plasma glucose and resist hyperglycaemia through increasing glucose utilization [26]. In addition, it is involved in immunity and inflammation via inhibition of the nuclear factor-kB signaling [27]. Yuh-Chiang Shen et al. [28] reported that andrographolide has the ability to prevent production of oxygen radical. Meanwhile, oxygen radical (usually called reactive oxygen species, ROS) has the ability to stimulate pigmentation and melanin has property of free radical scavenging [29,30]. Therefore, we assumed that maybe andrographolide had
the inhibitory effects on melanin synthesis, which was proven in this study. Signal pathways involved in melanin synthesis include mitogen-activated protein kinases (MAPKs) signal pathway, cAMP signal pathway, PI3K/Akt signal pathway, wnt/b-catenin signal pathway and so on [4,17,31]. In wnt/b-catenin signal pathway, bcatenin accumulating in cytoplasm up-regulates MITF content through promoting transcription with LEF1 after coming into nucleus [4]. In the present study, we found that b-catenin can be phosphorylated with several methods including axin-mediated CKI phosphorylation at ser 45 [5], GSK3b phosphorylation and PKCa phosphorylation at N-terminal ser/thr residues [7]. Siah-1 recruits the ubiquitination complex resulting in the degradation of b-catenin through proteasome in a ubiquitin-dependent way [32,33]. Here, decreased p-GSK3b (inactive form of GSK3b) leads to the degradation of b-catenin. Activation of GSK3b can be mediated by some kinases including Akt, PKA, PKC and p90rsk (also called MAPK-activated protein kinase-1) via phosphorylation at ser 9 in GSK3b [34]. It has been reported that andrographolide can affect PI3K/Akt signal pathway through inhibition of PI3K and Akt phosphorylation [14]. Here, andrographolide inhibited the phosphorylation level of Akt. We assumed that IGF-1, a potential Akt activator, could improve TYR activity and melanin content by increasing the phosphorylation of Akt and GSK3b, inhibiting the degradation of b-catenin and promoting the transcriptional and protein expression of MITF and TYR family. But results show no apparent change existing in TYR activity and melanin content treated with IGF-1. It has been reported that GSK3b can promote MITF bind to TYR family promoters and increase the transcription of TYR family [35].
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Fig. 6. Effects of andrographolide on b-catenin, TYR activity and melanin content in B16F10 cells treated with BIO or IGF-1. (A) b-Catenin was detected in B16F10 cells pretreated with or without BIO (2 mM) and incubated with andrographolide (10 mM) or not. (B) TYR activity was measured using samples from (A). (C) Melanin content was measured at 490 nm using samples from (A). (D) The phosphorylation of Akt and GSK3b and the content of b-catenin were determined by western blot in B16F10 cells treated with andrographolide or not in the presence or absence of IGF-1 (200 ng/ml) for 24 h. (E) TYR activity was measured using samples from (D). (F) Melanin content was measured at 490 nm using samples from (D). Data are representative of three independent experiments and expressed as means SEM. ***p < 0.001 compared with the control. ###p < 0.001 compared with the group only treated with andrographolide.
Probably, IGF-1 has potential to decrease TYR activity and melanin content by increasing the inactivation of GSK3b and inhibiting the binding of MITF to its target genes. But IGF-1 also could promote the accumulation of b-catenin which could increase the transcription of MITF in this study. As a result, these two different effects of IGF-1 on melanin synthesis probably explain the consequent results seen in Fig. 6E and F. Interestingly, B16F10 cells maintained in DMEM for 72 h could release melanin, while HEM cells could not even in melanocyte medium for 96 h. It was found that when the culture of B16F10 cells reached 100% confluency and still maintained in DMEM, the medium became dark due to melanin release. It was also observed that the mass of B16F10 cells was grayish-black before the color of medium changed. However, the color of melanocyte medium incubated with HEM cells always had little change, indicating no melanin released from HEM cells. As a result, melanin content in B16F10 cells can be assessed by measuring the absorbance of the medium, which is convenient but not suitable for measure of melanin content in HEM cells. Before melanin content in HEM cells is measured, HEM cells should be harvested and dissolved in 1 M NaOH.
It has been reported that melanosomes, where melanin is synthesized, are located in melanocyte, and then transported to adjacent keratinocytes leading to pigmentation [36,37]. B16F10 murine melanoma cells is a kind of carcinoma cell line, where gene mutations have happened and some features have changed, such as melanin released from cells immediately without keratinocytes. That may explain why B16F10 cells can release melanin to the culture medium but not for HEM cells. In this study, we also found that andrographolide could change the morphology of B16F10 cells but had little effects on HEM cells, which may resulted from the difference between carcinoma cells and normal cells. In summary, andrographolide inhibits TYR activity and melanin content probably via down-regulating MITF expression depending on the inhibition of Akt phosphorylation and GSK3b phosphorylation and induction of b-catenin degradation in B16F10 cells (see Fig. 7). In vivo, andrographolide also inhibits TYR content and melanin content in UVB irradiation induced brown guinea pig skin. Importantly, we found that andrographolide can effectively reduce melanin content and TYR activity in HEM cells with no apparent change of morphology. Thus, andrographolide probably can be a whiting agent widely used in cosmetics in the future.
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Fig. 7. The possible mechanism of andrographolide inhibiting melanin content in B16F10 cells. Andrographolide decreases phosphorylation of Akt and GSK3b and thus probably increases the activated GSK3b, which induces the phosphorylation of b-catenin and causes its degradation via proteasome. The reduction of b-catenin probably leads to the inhibition of transcription of MITF and TYR family and the subsequent reduction of melanin synthesis.
Funding disclosures This work was supported by the grants from National Natural Science Foundation of China (No. 81272226, No. 81271742), the grants from the Key Science and Technology Projects from the Science and Technology Commission of Shanghai Municipality (13JC1405102).
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