Zerumbone, A Tropical Ginger Sesquiterpene Of Zingiber Officinale Roscoe, Attenuates α-MSH-Induced Melanogenesis in B16F10 Cells Part 1
Apr 25, 2023
Abstract: Zerumbone (ZER), an active constituent of the Zingiberaceae family, has been shown to exhibit several biological activities, such as anti-inflammatory, anti-allergic, anti-microbial, and anti-cancer; however, it has not been studied for anti-melanogenic properties. In the present study, we demonstrate that ZER and Zingiber are officinal (ZO) extracts that significantly attenuate melanin accumulation in α-melanocyte-stimulating hormone (α-MSH)-stimulated mouse melanogenic B16F10 cells. Further, to elucidate the molecular mechanism by which ZER suppresses melanin accumulation, we analyzed the expression of the melanogenesis-associated transcription factor, microphthalmia-associated transcription factor (MITF), and its target genes, such as tyrosinase, tyrosinase-related protein 1 (TYRP1), and tyrosinase-related protein 2 (TYRP2), in B16F10 cells that are stimulated by α-MSH. Here, we found that ZER inhibits the MITF-mediated expression of melanogenic genes upon α-MSH stimulation. Additionally, cells treated with different concentrations of zerumbone and ZO showed increased extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation, which are involved in the degradation mechanism of MITF. Pharmacological inhibition of ERK1/2 using U0126 sufficiently reversed the anti-melanogenic effect of ZER, suggesting that increased phosphorylation of ERK1/2 is required for its anti-melanogenic activity. Taken together, these results suggest that ZER and ZO extracts can be used as active ingredients in skin-whitening cosmetics because of their anti-melanogenic effect.
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david.deng@wecistanche.com WhatApp:86 13632399501
Keywords: zerumbone; Zingiber officinal Roscoe; melanogenesis; MITF; ERK1/2
1. Introduction
Melanogenesis, the production of melanin by epidermal melanocytes, is stimulated by the α-melanocyte stimulating hormone (α-MSH) that is secreted from keratinocytes upon exposure to ultraviolet (UV) radiation [1,2]. The stem cell factor (SCF) is another melanogenic factor that strictly controls melanocyte migration, proliferation, and differentiation for maintaining postnatal cutaneous melanogenesis [3]. Microphthalmia-associated transcription factor (MITF), which is a melanogenic transcription factor, is activated through the cAMP-PKA-CREB (cyclic adenosine monophosphate-protein kinase A-cAMP response element binding protein) signaling pathway upon α-MSH stimulation via melanocortin 1 receptor (MC1R) in cytoplasmic membranes of epidermal melanocytes [4]. Several chemicals, such as forskolin and IBMX (3-isobutyl-1-methylxanthine), are known to activate the cAMP-PKA-CREB signaling pathway, leading to the induction of melanogenesis [5]. Several studies have revealed that sustained the activation of extracellular-regulated kinases 1 and 2 (ERK1/2), which is involved in the molecular mechanism of oncogenesis, promotes MITF phosphorylation at Ser73 and its subsequent degradation via ubiquitin-dependent proteolysis [6,7]. Indeed, U0126, a selective ERK1/2 pathway inhibitor, has been reported to increase MITF expression and tyrosinase activity, leading to melanin production [6]. Stabilized and activated MITF increases the expression of melanogenic genes, such as tyrosinase, tyrosinase-related protein 1 (TYRP1), and tyrosinase-related protein 2 (TYRP2). The transformation of tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA), which is the initial step of melanogenesis, is catalyzed by tyrosinase. Tyrosinase-related proteins (TRPs) catalyze further oxidation and tautomerization reactions [8]. After melanin synthesis, mature melanin is transported from epidermal melanocytes into the cytoplasm of the basal keratinocytes to protect cells from UV radiation [2].
Abnormally increased melanogenesis causes multiple types of skin disorders, such as skin cancer, chloasma, and freckles [4]; thus, small molecules or natural products that target either the catalytic activity of tyrosinase, or regulators of signaling pathways in melanogenesis, including ERK1/2, or MITF-mediated transcription of melanogenic genes, have been identified as active ingredients to be exploited in the cosmetic industry. Several small molecules, such as kojic acid, arbutin, and niacinamide, are widely used as cosmetic ingredients due to their anti-melanogenic activities. However, reports have shown that kojic acid causes multiple side effects, including cytotoxicity, dermatitis, skin cancer, and hepatocellular carcinoma, because of its genotoxic activity [9]. Although arbutin which was isolated from the bearberry plant has been used to treat hyperpigmentation disorder, recently, its use as a cosmetic ingredient has been restricted due to its several side effects [9]. It has been reported that niacinamide has an anti-melanogenic activity and this activity is carried out by inhibiting melanosome transfer from melanocytes to surrounding keratinocytes [10]. Thus, it has been generally used as a skin-whitening compound in the cosmetic industry. To overcome the limitations of established skin whitening agents that have several side effects, it is important to develop safe skin whitening ingredients derived from natural sources. For example, α-thujaplicin, linderanolide B, 4-butyl resorcinol, and plumbagin have been identified as anti-melanogenic compounds due to their tyrosinase inhibitory effects [4,11]. Nobiletin, withaferin A, sesamol, and chaetocin exhibit anti-melanogenic properties by modulating signaling intermediates in the melanogenesis pathway [11].
Zerumbone (ZER) is a natural product, which is isolated from the volatile essential oils of plants belonging to the Zingiberaceae family, such as Zingiber zerumbet Smith and Zingiber officinal Roscoe [12]. Several studies have shown that ZER has a broad range of biological activities, including antimicrobial, antioxidant, anti-diabetic, anticancer, anti-inflammatory, antiallergenic, and anti-angiogenic activities [12]. Interestingly, ZER has been shown to exert a protective effect against ultraviolet A (UVA)-induced oxidative damage in skin keratinocytes, owing to its ability to scavenge reactive oxygen species (ROS) via the activation of nuclear-factor-E2-related factor-2 (Nrf2) [1,13]. This suggests that ZER can be used as a functional additive in skin care cosmetics. However, the detailed mechanism of the anti-melanogenic properties of ZER action is yet to be studied.
In the present study, the inhibitory effects of ZER and Zingiber officinal (ZO) extract on α-MSH-stimulated melanogenesis, and their underlying mechanisms were studied. Here, we show that ZER significantly suppresses α-MSH induced melanogenesis by upregulating the phosphorylation of ERK1/2 and inhibiting MITF-mediated expression of melanogenic genes.
2. Results
2.1. Zerumbone (ZER) Suppresses α-MSH Induced Melanogenesis in B16F10 Mouse Melanoma Cells
To study the anti-melanogenic effect of zerumbone (ZER), we initially evaluated its cytotoxicity in both B16F10 and HaCaT cells. The chemical structure of ZER is shown in Figure 1A. It was observed that ZER at concentrations above 20 µM exhibited a strong cytotoxic effect, whereas, at those below 20 µM, ZER did not show cytotoxicity in both cell lines (Figure 1B). Next, we investigated the inhibitory effects of ZER on α-melanocytes stimulating hormone (α-MSH)-induced melanin accumulation and secretion in B16F10 cells. ZER was shown to strongly suppress α-MSH-induced intracellular accumulation of melanin and its secretion into the cultured medium (Figure 1C, D). Moreover, we found that ZER attenuates melanogenesis more effectively than 1 mM arbutin or 0.2 mM kojic acid, the well-known active constituents of skin-whitening cosmetics (Figure 1D). To elucidate whether ZER is sufficient to suppress melanogenesis in human cells, we used melanin-producing G361 human melanoma cells. As shown in Figure 1E, ZER significantly decreases stem cell factor (SCF)-induced extra- and intracellular melanin contents. These results confirm the anti-melanogenic activity of ZER in melanogenic mouse B16F10 and human G361 cells.
2.2. Zerumbone Suppresses Gene Expression of Melanogenesis Transcription Factor, MITF, and Its Target Genes in G361 Human Melanoma Cells
Endothelin-1 and SCF signaling have been reported to play essential roles in melanogenesis in human melanocytes and several subtypes of melanoma [3,14–16]. In this study, we found that ZER suppresses melanogenesis upon melanogenic stimuli, α-MSH and SCF, in mouse B16F10 and human G361 melanoma cells (Figure 1).
Thus, we measured alterations in the expression levels of melanogenesis-related genes and proteins upon SCF stimulation in G361 human melanoma cells. We found that ZER attenuates SCF-induced MITF and tyrosinase protein expression 2–4 h and 24–48 h after SCF stimulation, respectively (Figure 2A). In addition, gene expression of melanogenesis-related genes, such as MITF, tyrosinase, and tyrosinase-related protein 1 (TYRP1) was suppressed in ZER-treated G361 cells (Figure 2B, C). These results revealed the peak time for the expression of melanogenesis-related genes upon SCF stimulation; maximal MITF mRNA induction was observed within 1–2 h, and mRNA levels of tyrosinase and TYRP1 were observed to reach their maximum 24–48 h after SCF stimulation.
2.3. Zerumbone Suppresses Melanogenic Genes and Enzymes Expression in Mouse B16F10 Cells
To investigate the molecular mechanism by which ZER suppresses melanogenesis, we measured gene expression of the melanogenesis transcription factor, MITF, and its target genes, such as tyrosinase-related protein 1 (TYRP1), tyrosinase, and tyrosinase-related protein 2 (TYRP2) in the absence or presence of ZER. Since the peak time for melanogenesis-related genes expression upon SCF and α-MSH stimulation is known for human G361 (Figure 2) and mouse B16F10 melanoma cells [4], respectively, we further measured the expression of MITF, tyrosinase, TYRP1, and TYRP2 in ZER treated mouse B16F10 melanoma cells after 2 h or 48 h of incubation with α-MSH. Figure 3A shows that ZER is sufficient to attenuate α-MSH-induced MITF, TYRP1, TYRP2, and tyrosinase expression in B16F10 cells. Similarly, MITF, tyrosinase, and TYRP2 protein expression levels were decreased upon ZER treatment. Protein kinase A (PKA)-mediated phosphorylation of CREB is a major signaling pathway increasing MITF at the transcriptional level upon α-MSH stimulation [4]. Thus, here, we analyzed whether decreased phosphorylation of CREB by ZER could mediate the suppression of MITF. We found that α-MSH-induced CREB phosphorylation was not affected by ZER treatment, suggesting that ZER suppresses α-MSH-induced MITF expression independent of the PKA-CREB signaling pathway axis (Figure 3B). Because tyrosinase is a rate-limiting enzyme, that regulates melanin synthesis [2,4,11], we further analyzed intracellular tyrosinase protein levels and its enzymatic activity upon arbutin, kojic acid, and ZER treatment. Figure 3C shows that ZER sufficiently reduces α-MSH-induced tyrosinase protein levels in B16F10 cells. In addition, Figure 3D reveals that 10 µM ZER more effectively suppresses L-DOPA oxidation than tyrosinase inhibitors, arbutin, and kojic acid, suggesting that the oxidation of L-DOPA to dopaquinone through the enzymatic activity of tyrosinase is suppressed in ZER-treated cells. These results demonstrate that ZER attenuates α-MSH-mediated melanogenesis by suppressing gene expression of MITF, a melanogenesis-associated transcription factor, and its target genes.
2.4. Anti-Melanogenic Effect of Zerumbone Is through Phosphorylation of ERK1/2
Activation of protein kinase B (AKT) and extracellular signal-regulated kinases (ERK1/2) by growth factors or melanogenic stimuli is known to suppress melanogenesis through the decreased expression of MITF and its target genes [11]. Phosphorylation of MITF at Ser73 and Ser409 in response to the ERK1/2 signaling pathway promotes its proteasome-dependent degradation [17,18]. Therefore, we investigated whether ZER regulates ERK1/2 phosphorylation in B16F10 mouse and G361 human melanoma cells. Increased phosphorylation of ERK1/2 (p-ERK1/2) but not total ERK1/2 (T-ERK1/2) was observed upon ZER treatment in a time- and dose-dependent manner (Figure 4A, B). Interestingly, the phosphorylation of ERK1/2 was observed to be rapidly increased within 10–30 min and 1–2 h post ZER treatment in mouse B16F10 and human G361 melanoma cells (Figure 4A). However, the phosphorylation of AKT and MEK was not observed upon ZER treatment, suggesting that ZER-induced ERK1/2 phosphorylation may suppress MITF (Figure 4B). Because ERK1/2 phosphorylation promotes the phosphorylation and proteasomal degradation of MITF [17,18], we investigated whether proteasome inhibitor prevents the reduction of MITF by ZER. Suppression of MITF by ZER was not observed in the presence of MG132, confirming that ZER decreases MITF expression through proteasomal degradation (Figure 4C). Next, we investigated whether ZER phosphorylates MITF at Ser73, which is the target residue of ERK1/2. To detect phosphorylated MITF, cells were exposed to α-MSH and MG132. Here, we found that phosphorylated MITF (Ser73) was significantly increased by ZER in the presence of α-MSH and MG132 (Figure 4D). U0126, a selective mitogen-activated protein kinase (MAPK) inhibitor [19], abolished ZER-induced MITF phosphorylation (Figure 4D). Moreover, total MITF protein levels in whole cell lysates (WCL) were not altered by ZER and U0126 in the presence of MG132 (Figure 4D). These results indicate that ERK1/2-mediated MITF (Ser73) phosphorylation and proteasomal degradation is a critical mechanism in the ZER-mediated suppression of MITF. In addition, Figure 4E shows that U0126 significantly restored reduced MITF protein levels in ZER-treated cells. Consistently, decreased intracellular melanin content by ZER was restored by U0126 to approximately 40% (Figure 4F), suggesting that the activation of ERK1/2 is required for the anti-melanogenic effect of ZER.
2.5. Anti-Melanogenic Effect of Zingiber Offificinale (ZO) Extracts
ZER is a phytochemical derived from several plant species of the Zingiberaceae family, such as Zingiber zerumbet and Zingiber officinal [12]. Thus, we initially measured the cell viability in the absence or presence of ZO extract in B16F10 and HaCaT cells. Figure 5A shows that cell viability was not altered by ZO extract treatment. We further analyzed the anti-melanogenic effects of Zingiber officinal (ZO) extract. Because the expression of MITF and its target melanogenic genes were decreased by ZER through the activated ERK1/2 signaling pathway (Figures 2 and 3), we checked whether ZO extract suppresses α-MSH-induced MITF and its target melanogenic proteins expression in B16F10 cells. Similar to the anti-melanogenic effect of ZER, α-MSH-induced MITF, tyrosinase, and TYRP2 were significantly decreased by the ZO extract in a dose-dependent manner (Figure 5B). Interestingly, ERK1/2 phosphorylation was also increased in ZO extract-treated cells (Figure 5B). Expression of MITF and its target genes, such as tyrosinase, TYRP1, and TYRP2, was significantly decreased upon 5 µg/µL ZO extract treatment (Figure 5C). Moreover, both extracellular and intracellular melanin content was significantly reduced to approximately 40% upon 5 µg/µL ZO extract treatment (Figure 5D). In addition, decreased tyrosinase activity was observed in cells that were treated with ZO extract (Figure 5E). These results suggest that Zingiber officinal (ZO) extract suppresses melanogenesis by attenuating MITF-mediated melanogenic gene expression.
For more info: david.deng@wecistanche.com WhatApp:86 13632399501
