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Inhibitory Effects of Hoelen Extract on Melanogenesis in B16/F1 Melanoma Cells
Abstract
Melanin synthesis is regulated by melanogenic proteins, such as tyrosinase, tyrosinase-related protein 1 (TRP-1) and TRP-2. The effects of Hoelen extract on melanogenesis were investigated in B16Fl murine melanoma cells. Specifically, tyrosinase activity, cell viability and melanin content were assayed, and western blotting and RT-PCR for tyrosinase, TRP-1 and TRP-2 conducted. The results show that Hoelen significantly inhibited melanin synthesis through inhibition of TRP-2 expression, while it did not affect tyrosinase activity or its expression. Taken together, RT-PCR results showed that the depigmentation effect of Hoelen may be due to inhibition of TRP-2 gene transcription. These results suggest that Hoelen may be a useful inhibitor for the attenuation of melanogenesis and hyperpigmentation in skin cells.
... Those with overly sensitive skin may suffer from redness and irritation. Kojic acid was reported to be able to cause irritation to the skin and cause serious skin problems, such as dermatitis and skin cancer (Chang et al., 2010). Exogenous ochronosis and perdurable depigmentation were reported after long-term use of arbutin (Bhattar et al., 2015;Sunkara et al., 2020). ...
Andrographis paniculata is a well-known and highly used potential herbal plant for worldwide medicinal use. Plant studies reported that the leaf extract of A. paniculata, which has high antioxidant capacity, has antifungal, antimicrobial, antiprotozoal, antidiabetic, liver enzyme modulation, insecticidal, and toxicity properties. Nevertheless, no report regarding its antimelanogenic activity is available. Hence, this study was carried out to investigate A. paniculata leaf extract potential of the antimelanogenic property. The data shown from this study proved that the A. paniculata leaf extract is responsible for melanin content reduction and decreases the protein expression of tyrosinase (TYR) and microphthalmia-associated transcription factor in B16F1 melanoma cells. This study found that the A. paniculata leaf extract acted powerfully in the synthesis of tyrosinase-related protein-2 inhibition. TYR is a well-known essential enzyme in melanogenesis activity. These results represent that the A. paniculata leaf extract strongly decreases the expression level of the enzyme important for melanin synthesis. Therefore, the A. paniculata extract has a high capability of becoming a lightening agent and hyperpigmentation treatment in the pharmaceutical and cosmetic market.
... The hot water extract of Pleurochrysis carterae inhibited melanin synthesis by downregulating the tyrosinase and MITF levels in B16F1 melanoma cells [58]. A Hoelen extract repressed melanin synthesis by inhibiting TYRP2 gene transcription while the tyrosinase expression remained constant [59]. The white rose petal extracts (WRPE) effectively inhibited the activity of tyrosinase, while Eriobotrya japonica leaf ethanol extract (EJEE) suppressed melanin contents with their strong antioxidative activity [60,61]. ...
Antioxidants are an important strategy for treating photoaging because excessive reactive oxygen species (ROS) are produced during UV irradiation. The therapeutic effects of methanol extracts of Hygrophila erecta (Brum. F.) Hochr. (MEH) against UV-induced photoaging were examined by monitoring the changes in the antioxidant defense system, apoptosis, extracellular matrix (ECM) modulation, inflammatory response, and melanin synthesis in normal human dermal fibroblast (NHDF) cells and melanoma B16F1 cells. Four bioactive compounds, including 4-methoxycinnamic acid, 4-methoxybenzoic acid, methyl linoleate, and asterriquinone C-1, were detected in MEH, while the DPPH free radical scavenging activity was IC50 = 7.6769 µg/mL. UV-induced an increase in the intracellular ROS generation, NO concentration, SOD activity and expression, and Nrf2 expression were prevented with the MEH treatment. Significant decreases in the number of apoptotic cells, the ratio of Bax/Bcl-2, and cleaved Cas-3/Cas-3 were observed in MEH-treated NHDF cells. The MEH treatment induced the significant prevention of ECM disruption and suppressed the COX-2-induced iNOS mediated pathway, expression of inflammatory cytokines, and inflammasome activation. Finally, the expression of the melanin synthesis-involved genes and tyrosinase activity decreased significantly in the α-melanocyte-stimulating hormone (MSH)-stimulated B16F1 cells after the MEH treatment. MEH may have an antioxidative role against UV-induced photoaging by suppressing ROS-induced cellular damage.
... Mice were killed after 4-week treatment, and the lungs were separated to examine the number of lung metastasis nodules. Then the lungs were homogenized and incubated in 1 µmol/l NaOH containing 10% DMSO at 80°C for 2 h to measure the melanin content [15]. Then the homogenate was centrifuged, and the absorbance of supernatant was read at 490 nm. ...
Inhibition of BRAF improves therapeutic efficacy of BRAF-mutant melanoma. However, drug resistance to BRAF inhibitor is inevitable, and the drug resistance mechanisms still remain to be elucidated. Here, BRAF mutant cells A375 and SK-MEL-28 were chosen and treated with BRAF inhibitor vemurafenib, and the results showed that the ERK signaling pathway was blocked in these cells. Then, vemurafenib-resistant cells were constructed, and we found that drug resistance-related gene P-gp was overexpressed in the two cell lines. In addition, the histone acetylation was significantly increased on the P-gp promoter region, which suggested that the epigenetic modification participated in the P-gp overexpression. Furthermore, JQ1, a bromodomain inhibitor, was added to the vemurafenib-resistant cells and sensitizes the vemurafenib-induced melanoma cell apoptosis. In C57BL/6 mice intravenously injected with vemurafenib-resistant melanoma cells, cotreatment of vemurafenib and JQ1 also severely suppressed melanoma lung metastasis. Taken together, our findings may have important implications for the combined use of vemurafenib and JQ1 in the therapy for melanoma treatment.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0/.
... **P < 0.01 indicates a significant difference versus OSKM treatment alone. b RT-PCR demonstrating that three randomly selected 4F HFF-derived iPSCs express the pluripotent marker genes Nanog, c-Myc, Sox-2, Oct4, and KIf4 exhibit a reproductive effect in mice [17], a stimulatory effects on splenocytes [18], anti-melanogenesis activity against melanoma cells [19], and the ability to extend the lifespan of C. elegans [20]. In the present study, we analyzed 7 marker compounds found in an SGT-4 ginsenoside Rg1, ginsenoside Rb1, atractylenolide III, liquiritigenin, liquiritin apioside, liquiritin, and glycyrrhizin using HPLC analysis (Fig. 1b). ...
Background
Sagunja-Tang (SGT-4) is a traditional herbal formula in Korean medicine that is used to treat anti-metabolic syndrome, and has antioxidant activity. In this study, we evaluated the effects of SGT-4 on the formation efficiency of induced pluripotent stem cells (iPSCs) from human foreskin fibroblasts (HFFs) by four reprogramming transcription factors: Oct4, Sox2, KIf4, and c-Myc (OSKM). MethodsSGT-4 contained four different herbal medicines that are composed of Ginseng Radix, Glycyrrhizae Radix et Rhizoma, Atractylodis Rhizoma Alba, and Poria Sclerotium. The composition of SGT-4 was analyzed by high-performance liquid chromatography (HPLC). HFFs were transfected with episomal vectors contained by four OSKM. Western blotting, RT-PCR, immunofluroescence, and in vitro differentiation were used to assess the pluripotency of the iPSC cells. ResultsSGT-4 exhibited antioxidant activity against the generation of intracellular reactive oxygen species (ROS) as well as promoted the activation of superoxide dismutase 1 (SOD1), catalase, gluthathione peroxidase 1 (GPX1), and glutathione (GSH). Moreover, the ATP level was not significantly fluctuated depending on the concentration of SGT-4 in the hiPSCs. Conclusion
Our results indicate that the SGT-4, herbal formula significantly increases the efficiency of human iPSC generation via the transcription factors (Oct4, Sox2, KIf4, and c-Myc).
... Mice were sacrificed after 3-week treatment, and the lungs were separated to examine the number of lung metastasis nodules. Then the lungs were homogenized and incubated in 1 M NaOH containing 10% DMSO at 80°C for 2 h to measure the melanin content [38]. Then the homogenate were centrifuged and the absorbance of supernate was read at 490 nm. ...
Background
Metastatic malignant melanoma is one of the most aggressive malignancies and its treatment remains challenging. Recent studies demonstrate that the melanoma metastasis has correlations with the heightened activations of protein kinase C ζ (PKCζ) and cyclooxygenase-2 (COX-2) signaling pathways. Targeted inhibitions for PKCζ and COX-2 have been considered as the promising strategies for the treatment of melanoma metastasis. Thus, the PKCζ inhibitor J-4 and COX-2 inhibitor Celecoxib were combined to treat melanoma metastasis in this study.
Methods
The Transwell assay, Wound-healing assay and Adhesion assay were used to evaluate the inhibition of combined therapy of J-4 and Celecoxib on melanoma cells invasion, migration and adhesion in vitro, respectively. The impaired actin polymerization was observed by confocal microscope and inactivated signal pathways about PKCζ and COX-2 were confirmed by the Western blotting assay. The B16-F10/C57BL mouse melanoma model was used to test the inhibition of combined therapy of J-4 and Celecoxib on melanoma metastasis in vivo.
Results
The in vitro results showed that the combination of J-4 and Celecoxib exerted synergistic inhibitory effects on the migration, invasion and adhesion of melanoma B16-F10 and A375 cells with combination index less than 1. The actin polymerization and phosphorylation of Cofilin required in cell migration were severely impaired, which is due to the inactivation of PKCζ related signal pathways and the decrease of COX-2. The combined inhibition of PKCζ and COX-2 induced Mesenchymal-Epithelial Transition (MET) in melanoma cells with the expression of E-Cadherin increasing and Vimentin decreasing. The secretion of MMP-2/MMP-9 also significantly decreased after the combination treatment. In C57BL/6 mice intravenously injected with B16-F10 cells (5 × 10⁴ cells/mouse), co-treatment of J-4 and Celecoxib also severely suppressed melanoma lung metastasis. The body weight monitoring and HE staining results indicated the low toxicity of the combination therapy.
Conclusions
This study demonstrates that the combination therapy of PKCζ and COX-2 inhibitors can significantly inhibit melanoma metastasis in vitro and in vivo, which will be an efficient strategy for treatment of melanoma metastasis in clinics.
... After incubation with emu oil or emu oil and α-MSH, the cells were harvested and washed with phosphate-buffered saline thrice by centrifugation (1500 rpm, 5 min). The melanin contents in the resultant cell pellets were determined according to previously described methods with a slight modification [4][5][6]. The cell pellets were dissolved in 500 μl of 0.5 N NaOH and incubated at 60°C for 1 h. ...
Here, we present data on the effects of emu oil, obtained from emu (Dromaius novaehollandiae) fat deposits, on melanogenesis in B16F1 murine melanoma cells. The cells were cultured in media containing different concentrations of emu oil, and the melanin content of these cells was measured using a microplate reader. Next, melanin content was measured for cells cultured with α-melanocyte-stimulating hormone. This article reports the different melanin contents as μg melanin/mg cellular protein, by using bar graphs with error bars. The present data imply that emu oil reduces the cellular melanin production.
... TRP-1 and TRP-2 have functional roles in this biosynthesis pathway. TRP-1 catalyzes the oxidation of DHICA, and TRP-2 (dopachrome tautomerase) catalyzes the conversion of dopachrome to DHICA [23]. Our results revealed all of the crude herbs and mixed extracts increased TRP-1 in G-361 cells. ...
Vitiligo is considered a preimmune stage of a disease that is not well clarified. This condition is difficult to treat because there is no definite cure. Uyghur medicine is an important part of traditional Chinese medicine. There are many types of prescriptions that are used for the treatment of vitiligo. Bairesi complex prescription is one of the active prescriptions for vitiligo that is used in the clinic. However, the intensities of melanogenesis due to uses of Bairesi complex prescription and its five constituent crude herbs have not been reported yet. In the present study, we found that the hot water extracts of Bairesi complex prescription and the crude herbs were more effective in eliciting melanin production in G-361 cells than the EtOH extracts. Furthermore, the Bairesi complex prescription exhibited less cytotoxicity and was more effective in melanin formation than the five crude herbal extracts. In the present study, we also discuss the mechanisms of melanogenesis due to the use of the Bairesi complex prescription and its single crude herbal extracts.
... However, excessive progress of melanin synthesis is often the cause of hyper-pigmentary disorders (Oh et al., 2014). For several years, natural sources that regulate hyperpigmentation have been used in commercial products (Zhong et al., 2006;Luo et al., 2009;Chang et al., 2010;Goh et al., 2013). ...
We already reported that genetically engineered resveratrol-enriched rice (RR) showed to down-regulate skin melanogenesis. To be developed to increase the bioactivity of RR using calli from plants, RR was adopted for mass production using plant tissue culture technologies. In addition, high-pressure homogenization (HPH) was used to increase the biocompatibility and penetration of the calli from RR into the skin. We aimed to develop anti-melanogenic agents incorporating calli of RR (cRR) and nanoparticles by high-pressure homogenization, examining the synergistic effects on the inhibition of UVB-induced hyperpigmentation. Depigmentation was observed following topical application of micro-cRR, nano-calli of normal rice (cNR), and nano-cRR to ultraviolet B (UVB)-stimulated hyperpigmented guinea pig dorsal skin. Colorimetric analysis, tyrosinase immunostaining, and Fontana-Masson staining for UVB-promoted melanin were performed. Nano-cRR inhibited changes in the melanin color index caused by UVB-promoted hyperpigmentation, and demonstrated stronger anti-melanogenic potential than micro-cRR. In epidermal skin, nano-cRR repressed UVB-promoted melanin granules, thereby suppressing hyperpigmentation. The UVB-enhanced, highly expressed tyrosinase in the basal layer of the epidermis was inhibited by nano-cRR more prominently than by micro-cRR and nano-cNR. The anti-melanogenic potency of nano-cRR also depended on pH and particle size. Nano-cRR shows promising potential to regulate skin pigmentation following UVB exposure.
... Numerous studies have sought to identify the factors involved in controlling melanin synthesis . A number of natural products have been reported to inhibit melanogenesis by regulating melanogenic enzymes, including Hoelen extracts [8], sesamol (3,4-methylenedioxyphenol) [9]. In addition, Arthrophytum scoparium extract [10], Caffeoylserotonin [11] and the aqueous fraction from Cuscuta japonica [12] have been shown to inhibit melanogenesis by regulating MITF. ...
We investigated the potential melanogenic effect of compounds from Zingiber cassumunar Roxb. Our data revealed that chloroform-soluble extract of Z. cassumunar enhanced melanin synthesis in B16F10 melanoma cells. Among the components of the chloroform extract, (E)-4-(3,4-dimethoxyphenyl)but-3-en-1-ol (DMPB) increased melanogenesis in both B16F10 cells and human primary melanocytes. In B16F10 cells, DMPB enhanced the activation of ERK and p38, and the level of tyrosinase. Although the level of microphthalmia-associated transcription factor was unchanged in DMPB-treated B16F10 cells, DMPB increased levels and nuclear localization of upstream stimulating factor-1 (USF1). Consistently, DMPB-mediated melanin synthesis was diminished in USF1-knockdown cells. Furthermore, DMPB induced hyperpigmentation in brown guinea pigs in vivo. Together, these data suggest that DMPB may promote melanin synthesis via USF1 dependent fashion and could be used as a clinical therapeutic agent against hypopigmentation-associated diseases.
... Total cellular RNA was prepared using the TRIzol reagent according to the manufacturer's protocol. RT-PCR was performed using the Veriti Thermal Cycler (Applied Biosystems, Foster City, CA, USA) using primers for tyrosinase and Tyrp1 (Chang et al., 2010), Dct (Liang, 2011), and MITF (Hasegawa et al., 2010). PCR products were separated by gel electrophoresis on 2.0% agarose and visualized with ethidium bromide staining under UV illumination. ...
Significance:
In our efforts to find new skin lightening agents, NED-180, a Piper amide derivative, was investigated for its ability to inhibit melanogenesis. It significantly decreased melanogenesis by regulating melanogenic enzymes through activation of PI3K and ERK pathways without cytotoxicity. Interestingly, NED-180 reduced TPA-induced activation of TRPM1 that can be a new target for anti-melanogenesis. Therefore, NED-180 may be a promising potential therapeutic agent for hyperpigmentation-related skin diseases. This article is protected by copyright. All rights reserved.
... In the ancient pharmacopoeia, this mixture had been used for a relatively long period (used as a mask at night and rinsed off the next morning). The herbal constituents of this topical Chinese medicinal formula are known to cause appreciable antityrosinase activity and suppress tyrosinase synthesis [7][8][9][10]; thus, many BZK-based pharmaceutical and cosmetic products such as BZK medicinal powder and extracts mixed with aqueous, petrolatum or olive-oil vehicles are prescribed by traditional Chinese medicine practitioners in the Chinese community. ...
Topical traditional Chinese medicine- (TTCM-) related contact dermatitis is not uncommon but ignored. Patch and photopatch tests using 6 individual herbal ingredients and Bai-Zhi-Kao (BZK; 白
芷
膏), a skin-lightening TTCM preparation, were conducted on 30 participants. Twenty-five subjects showed at least 1 positive reaction, including 6 (20.0%) participants who reacted to BZK. The majority reacted to Radix Ampelopsis japonica (Bai-Lian; 白
蘞) (60.0%), whereas few reacted to Rhizoma Bletilla striata (Bai-Ji; 白
芨) (16.7%), Rhizoma Atractylodis macrocephalae (Bai-Zhu; 白
朮) (10.0%), Radix Angelicae dahuricae (Bai-Zhi; 白
芷) (3.3%), and Herba asari (Xi-Xin; 細
辛) (3.3%). In the photopatch test, 3 participants (10.0%) reacted positively to BZK and 10 to ≥1 constituent; however, all reacted to Radix Angelicae dahuricae (26.7%), Radix Ampelopsis japonica (13.3%), and Rhizoma Bletilla striata (3.3%). In contrast, no subjects showed positive reactions to Sclerotium Poria cocos (Bai-Fu-Ling; 白
茯
苓). Thus, BZK and its constituents might present potential latent risk of contact dermatitis owing to the possible presence of Radix Ampelopsis japonica and Radix Angelicae dahuricae. Furthermore, TTCMs, particularly cosmetic products, must be used carefully, with ample warning of potential contact dermatitis risk.
... Many extracts from plants and fungi extraction inhibit melanogenesis and have been incorporated into cosmetic preparations. For example, the Hoelen extract inhibits melanogensis and hyperpigmentation in skin cells (17). Other anti-melanogenesis compounds include lucidone extract from Lindera erythrocapa (18), palmitoleic acid (19), and the hirseins extract from Thymelaea hirsute (20). ...
Cordycepin has been a traditional medicine in China and Korea for centuries. This study explored the inhibitory effect of cordycepin on melanogenesis and the relative molecular mechanisms. Cordycepin inhibited melanin synthesis-related enzymes, such as tyrosinase, tyrosinase-related protein-1 (TRP1) and tyrosinase-related protein-2 (TRP2). α-MSH and IBMX were reported as melanin synthesis enhancers. Both of them could increase intracellular melanin synthesis by activation of the microphthalmia-associated transcription factor (MITF) signaling pathway. In the MITF pathway, the phosphorylation of cAMP related binding protein (CREB) activated the transcription of MITF, resulting in increasing melanin synthesis. Cordycepin also decreased the phosphorylation of CREB induced by α-MSH and IBMX in B16F10 melanoma cells. Accordingly, cordycepin inhibited melanogenesis signaling pathways by activating ERK and AKT signaling pathways to regulate the suppression of MITF and its downstream pathways including tyrosinase, TRP1 and TRP2. These results indicate the role of cordycepin as a potent depigmenting agent for cosmetics.
Background
Korean cosmetics are widely exported throughout Asia. Cosmetics exploiting traditional Korean medicine lead this trend; thus, the traditional medicinal literature has been invaluable in terms of cosmetic development. We sought candidate medicinal herbs for skincare.
Methods
We used data mining to investigate associations between medicinal herbs and skin-related keywords (SRKs) in a classical text. We selected 26 SRKs used in the Donguibogam text; these referred to 626 medicinal herbs. Using a term frequency-inverse document frequency approach, we extracted data on herbal characteristics by assessing the co-occurrence frequencies of 52 medicinal herbs and the 26 SRKs.
Results
We extracted the characteristics of the 52 herbs, each of which exhibited a distinct skin-related action profile. For example Ginseng Radix was associated at a high-level with tonification and anti-aging, but Rehmanniae Radix exhibited a stronger association with anti-aging. Of the 52 herbs, 46 had been subjected to at least one modern study on skincare-related efficacy.
Conclusions
We made a comprehensive list of candidate medicinal herbs for skincare via data mining a classical medical text. This enhances our understanding of such herbs and will help with discovering new candidate herbs.
The effect of four different retinoids [retinol, 13-cis-retinoic acid (Ro4-3780), beta-all-trans-retinoic acid, and aromatic retinoic acid ethyl ester (Ro10-9359)] on the cellular proliferation (cell number) and biochemical differentiation (tyrosinase activity) of a human melanoma cell line (MIRW) and three subclones was assessed. All four retinoids (10(-6) M) inhibited the cellular proliferation (36 to 42%) and stimulated tyrosinase activity (58 to 72%) in the parent cell line to a similar extent. In contrast, the effects of the different retinoids on three derived melanoma clones was dissimilar. For example, in clone A6, beta-all-transretinoic acid stimulated tyrosinase activity by 48% but caused only a 7% inhibition of cellular proliferation. This retinoid caused a more pronounced effect in the other two subclones, stimulating tyrosinase from 135 to 195% and inhibiting growth 19 to 33%. Al three melanoma clones demonstrated increased tyrosinase activity (110 to 225%) and reduced proliferation (37 to 52%) following exposure to 13-cis-retinoic acid. This retinoid was found overall to be the most effective stimulator of tyrosinase, while retinol was observed to be the least active, stimulating enzyme activity slightly (25%) in only one of three clones. Retinol inhibited proliferation 27 to 33% in two of three melanoma subclones. The aromatic retinoic acid ethyl ester elevated tyrosinase levels in two clones but inhibited the enzyme in one melanoma line. Cellular proliferation, however, was reduced in all three clones. These results suggest that retinoid-induced changes in human melanoma cell growth and differentiation reflect underlying cellular differences and diverse biochemical interactions.
We have cloned and sequenced the human genomic DNA segments encoding the 5'-flanking region and the first two exons of the DOPAchrome tautomerase (DT)/tyrosinase-related protein 2 (TRP-2) gene. The DT gene is a member of the tyrosinase gene family and specifically expressed in melanin-producing cells. A transcriptional initiation site of the DT gene was identified by S1 nuclease-mapping and primer-extension analyses using RNA prepared from human pigmented melanoma cells. To study the mechanism for pigment cell-specific expression of the human DT gene, we analyzed the promoter function of its 5'-flanking region by transient expression assays. The fusion genes, containing the DT gene promoter upstream from a firefly luciferase reporter gene, were introduced into human pigmented melanoma cells and HeLa cells, and the pigment cell-specific promoter activity was evaluated by comparing the luciferase activity expressed in both cell lines. A series of 5' deletion studies of the human DT gene promoter revealed that the 32-bp element, located between -447 and -415, is sufficient to confer pigment cell-specific expression of a reporter gene on a homologous promoter, but not on a heterologous simian virus 40 promoter. Internal deletion studies using a homologous or a heterologous promoter revealed that the pigment cell-specific expression of a reporter gene mediated by the 32-bp element is dependent on the presence of another region of the DT gene spanning from -268 to -56, which was termed the proximal region. However, the proximal region by itself is not sufficient to confer cell type-specific expression. These results indicate that the presence of two regulatory regions, the 32-bp element and the proximal region, is required for pigment cell-specific expression of the DT gene. Both regulatory regions contain a CANNTG motif, a well known binding site for a large family of transcription factors possessing a basic helix-loop-helix structure.
Hoelen is used to treat gastric disease in Eastern traditional medicine. The efferent activity on the gastric vagus nerve was studied in the rat, and the activity was found to increase after administration of Hoelen into the duodenum. In addition, we examined the effect of Hoelen fractions. Both the fraction containing polysaccharide and the triterpenoid-rich fraction increased activity significantly. Hoelen would thus appear useful for treating gastric disease by activating efferent activity of the gastric vagus nerve through the action of triterpenoid and polysaccharide.
Tyrosine-based sorting signals conforming to the motif YXXØ (Y is tyrosine, X is any amino acid, and Ø is an amino acid with a bulky hydrophobic side chain (leucine, isoleucine, phenylalanine, methionine,
valine)) interact with the medium (μ) subunits of clathrin adaptor (AP) complexes. We have analyzed the selectivity of interaction
between YXXØ signals and the μ1, μ2, and μ3 (A or B) subunits of the AP-1, AP-2, and AP-3 complexes, respectively, by screening a combinatorial
XXXYXXØ library using the yeast two-hybrid system. All the medium subunits were found to prefer proline at position Y+2, suggesting
that YXXØ signals are stabilized by a bend in the polypeptide backbone. Other than for this common preference, each medium subunit
favored specific sets of residues at the X and Ø positions; these preferences were consistent with the proposed roles of the different adaptor complexes in rapid endocytosis
and lysosomal targeting. A considerable specificity overlap was also revealed by these analyses, suggesting that additional
factors, such as the context of the signals, must be important determinants of recognition.
Eight compounds were isolated from the roots of Glycyrrhiza glabra by the tyrosinase inhibitory activity guided fractionation, and their structures were identified as liquiritigenin (1), isoliquiritigenin (2), isoliquiritigenin-2-O-methyl ether (3), liquiritin (4), isoliquiritin (5), ononin (6), glycycoumarin (7), glycyrol (8) by analysis of spectral data. Compound 3 exhibited the most potent inhibitory effect on mushroom tyrosinase activity (IC50, 47 M).
Pachymic acid, 3-O-acetyl-16 α-hydroxytrametenolic acid, and poricoic acid B had been isolated from the sclerotium of Poria cocos Wolf. These compounds showed a strong inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate-induced inflammation in mice. At 0.2 μmol/mouse, these compounds markedly inhibited the promoting effect of 12-O-tetradecanoylphorbol-13-acetate (1 μg/mouse) on skin tumor formation following initiation with 7,12-dimethylbenz[a]anthracene (50 μg/mouse).Copyright © 1996 S. Karger AG, Basel
Abstract This study was conducted to evaluate the effects of unsaturated fatty acids on ultraviolet-induced hyperpigmentation of the
skin. An efficient lightening effect was observed following topical application of linoleic acid or α-linolenic acid to UV-stimulated
hyperpigmented dorsal skin of brownish guinea pigs. The number of melanocytes in the treated skin was similar to the number
in the skin of the pigmented control, indicating that the pigment-lightening effect was not due to depletion of melanocytes.
In vitro experiments using cultured murine melanoma cells showed that melanin production was inhibited most effectively by
α-linolenic acid, followed by linoleic acid and then by oleic acid. Furthermore, the turnover of the stratum corneum, which
plays an important role in the removal of melanin pigment from the epidermis, was accelerated by linoleic acid and by α-linolenic
acid. Taken together, the results suggest that the pigment-lightening effects of linoleic acid and α-linolenic acid are, at
least in part, due to suppression of melanin production by active melanocytes, and to enhanced desquamation of melanin pigment
from the epidermis.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
The regulation of the final steps of the melanogenesis pathway, after L-2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome) formation, is studied. It is shown that both tyrosinase and dopachrome tautomerase are involved in the process. In vivo, it seems that tyrosinase is involved in the regulation of the amount of melanin formed, whereas dopachrome tautomerase is mainly involved in the size, structure and composition of melanin, by regulating to the incorporation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into the polymer. Moreover, using L-3,4-dihydroxyphenylalanine (dopa) and related compounds, it was shown that the presence of dopachrome tautomerase mediates an initial acceleration of melanogenesis since L-dopachrome is rapidly transformed to DHICA, but that melanin formation is inhibited because of the stability of this carboxylated indole compared to 5,6-dihydroxyindole (DHI), its decarboxylated counterpart obtained by spontaneous decarboxylation of L-dopachrome. Using L-dopa methyl ester as a precursor of melanogenesis, it is shown that this carboxylated indole does not polymerize in the absence of DHI, even in the presence of tyrosinase. However, it is incorporated into the polymer in the presence of both tyrosinase and DHI. Thus, this study suggests that DHI is essential for melanin formation, and the rate of polymerization depends on the ratio between DHICA and DHI in the medium. In the melanosome, this ratio should be regulated by the ratio between the activities of dopachrome tautomerase and tyrosinase.
There has been great interest lately in the cloning of pigment-related genes; several laboratories have succeeded in isolating melanocyte-specific genes which have many of the characteristics expected for tyrosinase. In this paper, we review the selection criteria, the physical properties, and the functional characteristics of several of these gene products. Two of the clones map to the brown (b) and albino (c) loci, genes that are involved in the regulation of the quantity and quality of melanin production. The functional characteristics of these gene products are not easily reconciled with existing schemes of melanogenesis, and a reevaluation of our concepts of melanogenic regulation may be necessary. The altered expression of these gene products in normal and in transformed melanocytes, and the alternative mRNA processing that occurs in those cells, makes this system an appropriate and interesting one for studies of normal metabolic regulation of gene expression, as well as altered gene expression by neoplastic cells.
1. Tyrosinase is a copper-containing enzyme responsible for the production of melanin pigment throughout the phylogenetic spectrum. 2. In mammals, tyrosinase is a glycosylated enzyme found specifically in melanocytes--cells functional in the production and secretion of pigment granules. 3. Although many factors determine the type, quantity and quality of the melanin produced, tyrosinase activity is the critical factor that ultimately regulates melanogenesis.
Melanins were purified from melanoma tissues and black hair of mice by HCl treatment. This treatment hydrolyzed protein and other macromolecules leaving melanin as insoluble material. These melanin preparations were dissolved in 0.5 N dimethyl n undecyl n dodecyl ammonium hydroxide in toluene. Their extinction coefficients were about 20 cm2mg-1 at 400 nm. Melanins produced by autoxidation of dopa or oxidation of dopa with mushroom tyrosinase had a distinctly higher extinction coefficient of about 28 cm2mg-1. The molar amount of tyrosine converted to melanin by cell free extracts and whole cells of a cultured line of melanoma cells was measured. The increase in the melanin content during the incubation was assayed colorimetrically. From the results the molar extinction coefficient of monomer units of melanin produced by melanocytes was calculated to be 3000-3100 cm-1M-1. The mean molecular weight of monomer units of purified melanin was calculated as 150-160. This is slightly higher than that of indole quinone, which is thought to be the main component of melanin.
A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. We have used the assay to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.
Glycosylation inhibitors, glucosamine or tunicamycin, have been found to be specific inhibitory modulators for melanogenesis, which is accentuated generally in malignant melanoma cells. Exposure to glucosamine (1 mg/ml) or tunicamycin (0.2 to 0.4 micrograms/ml) induces a marked pigment loss within melanoma cells in vitro with a decrease in their grown curves. This melanogenic inhibition occurs without a substantial decrease in the synthesis of DNA, RNA, and protein in comparison with a specific, marked suppression of carbohydrate synthesis as revealed by suppressed mannose incorporation into these cells. Assay of tyrosinase of glucosamine- or tunicamycin-induced unpigmented melanoma cells has revealed a selective and marked decrease in the melanosome-rich large-granule fraction, but no substantial decrease in the total activity of remaining subcellular fractions. Electrophoresis of tyrosinase in the 30,000 X g supernatant fraction demonstrates an increase in the T1 form of soluble tyrosinase, while a disappearance of or marked decrease in membrane-bound tyrosinase, T3, is seen in the small- and large-granule fractions. Glycoprotein synthesis in the melanogenic subcellular compartments of pigment cells seems to play an integral role in melanogenesis which is principally enhanced in their carcinogenic status.
Several genes critical to the enzymatic regulation of melanin production in mammals have recently been cloned and mapped to the albino, brown and slaty loci in mice. All three genes encode proteins with similar structures and features, but with distinct catalytic capacities; the functions of two of those gene products have previously been identified. The albino locus encodes tyrosinase, an enzyme with three distinct melanogenic functions, while the slaty locus encodes tyrosinase-related protein 2 (TRP2), an enzyme with a single specific, but distinct, function as DOPAchrome tautomerase. Although the brown locus, encoding TRP1, was actually the first member of the tyrosinase gene family to be cloned, its catalytic function (which results in the production of black rather than brown melanin) has been in general dispute. In this study we have used two different techniques (expression of TRP1 in transfected fibroblasts and immunoaffinity purification of TRP1 from melanocytes) to examine the enzymatic function(s) of TRP1. The data demonstrate that the specific melanogenic function of TRP1 is the oxidation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) to a carboxylated indole-quinone at a down-stream point in the melanin biosynthetic pathway. This enzyme activity appears to be essential to the further metabolism of DHICA to a high molecular weight pigmented biopolymer.
An in vitro cell culture assay was developed to identify inhibitors of melanogenesis and agents which produce cytostatic or cytotoxic effects specifically in melanocytes. A total of 50 compounds related to tyrosine, dihydroxyphenylalanine, and hydroquinone (HQ) were tested in vitro in order to determine their effects upon a murine melanocyte cell line, Mel-Ab, that produces copious amounts of melanin in culture. The agents that demonstrated an inhibition of growth or pigment production by 50% (IC50) at < 100 micrograms/ml were considered active. The cytotoxicity of melanocyte-active compounds were also tested in vitro on a control nonmelanocyte cell line (HT 1080), using a simple crystal violet staining method to quantitate adherent cell number after treatment. The cell culture assay was validated with known potent melanocyte cytotoxic agents, including HQ and 4-S-cysteaminylphenol (4-S-CAP). Although most cytotoxic chemicals were nonspecific in this primary screen (i.e. killing both Mel-Ab and HT-1080 cells), several of the compounds tested exhibited high melanocyte-specific cytotoxicity, similar to HQ and 4-S-CAP. Potentially these compounds may be useful as either antimelanoma or skin depigmentation agents. All of the compounds identified as active in this primary screen were cytotoxic or cytostatic to melanocytes, except for the methyl ester of gentisic acid, which uniquely inhibited the de novo synthesis of melanin without cytotoxicity.
The levels of tyrosinase mRNA and tyrosinase activity were analyzed in two amelanotic melanoma cell lines, D1(178) (hamster) and G-361 (human). Neither tyrosinase mRNA nor tyrosinase activity were detected in D1(178) cells. On the other hand, both tyrosinase mRNA and weak tyrosinase activity were detected in G-361 cells. Assuming that the different types of melanogenic inhibitors affected melanogenesis in these two amelanotic melanoma cells in different manners, we performed a screening of melanogenic inhibitors in these two cell lines. As an isolated tyrosinase suppressive melanogenic inhibitor, ascorbic acid and glutathione were identified from D1(178) cells and G-361 cells, respectively. Furthermore, lactic acid was identified from D1(178) cells as an isolated tyrosinase non-suppressive melanogenic inhibitor. B-16 mouse melanotic melanoma cells were depigmented by treatment with lactic acid. The melanogenesis suppression by lactic acid in B-16 cells was found to be due to inhibition of tyrosinase gene expression.
Tyrosinase is the key enzyme in pigment synthesis, initiating a cascade of reactions which convert the amino acid tyrosine to the melanin biopolymer. Two other tyrosinase-related proteins (TRP) are known, TRP-1 (probably DHICAoxidase) and TRP-2 (DOPAchrome tautomerase). These proteins show about 40% homology, and recent results have indicated that the genes might be derived from a common ancestor. We will discuss recent findings on genomic organization, and on the proteins and their presumed function, which is important for eumelanin synthesis in mouse and man.
Fu-Ling, the sclederma of Poria cocos (Schw.) Wolf, has long been used as a sedative and diuretic in traditional Chinese herbal medicine. Our study demonstrated that the substances extracted from Fu-Ling by 50% hot ethanol significantly augmented the secretion of interleukins IL-1 beta and IL-6 6 h after in vitro cultivation of human peripheral blood monocytes. The augmented effect was dose dependent. Tumour necrosis factor-alpha (TNF-alpha) secretion was also increased as the cells were treated with 0.4 mg/ml or higher doses of Fu-Ling extract. By contrast, Fu-Ling extract significantly suppressed the secretion of transforming growth factor-beta (TGF-beta) 3 h after the in vitro drug treatment. The suppressive effect was shown at doses as low as 0.2 mg/ml of Fu-Ling extract. Since Fu-Ling extract enhanced the secretion of immune stimulators (IL-1 beta, IL-6 and TNF-alpha) but suppressed the secretion of an immune suppressor (TGF-beta), the substance in 50% hot ethanol extract of Fu-Ling might have potentiated the immune response. Fu-Ling extract was further fractionated by reverse-phase column chromatography and high-performance liquid chromatography (HPLC). The components showing activity in modulating the cytokine secretion were relatively high in hydrophobicity.
Pachymic acid, 3-O-acetyl-16 alpha-hydroxytrametenolic acid, and poricoic acid B had been isolated from the sclerotium of Poria cocos Wolf. These compounds showed a strong inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate-induced inflammation in mice. At 0.2 mumol/mouse, these compounds markedly inhibited the promoting effect of 12-O-tetradecanoylphorbol-13-acetate (1 microgram/mouse) on skin tumor formation following initiation with 7,12-dimethylbenz[a]anthracene (50 micrograms/mouse).
The triterpenes isolated from P. cocos and their derivatives were examined for anti-emetic activity. Some of these triterpenes inhibited emetic action induced by oral administration of copper sulfate pentahydrate to leopard frog. The triterpenes having an exomethylene group at C-24 showed anti-emetic activity to frogs.
There is no doubt that visual impressions of body form and color are important in the interactions within and between human communities. Remarkably, it is the levels of just one chemically inert and stable visual pigment known as melanin that is responsible for producing all shades of humankind. Major human genes involved in its formation have been identified largely using a comparative genomics approach and through the molecular analysis of the pigmentary process that occurs within the melanocyte. Three classes of genes have been examined for their contribution to normal human color variation through the production of hypopigmented phenotypes or by genetic association with skin type and hair color. The MSH cell surface receptor and the melanosomal P-protein are the two most obvious candidate genes influencing variation in pigmentation phenotype, and may do so by regulating the levels and activities of the melanogenic enzymes tyrosinase, TRP-1 and TRP-2.
The pigment melanin in human skin is a major defense mechanism against ultraviolet light of the sun, but darkened skin color, which is the result of increased and redistributed epidermal melanin, could be a serious aesthetic problem. Epidemiologically, it is well known that the consumption of green tea may help prevent cancers in humans and also reduce several free radicals including peroxynitrite. In the present study, to assess the efficacy of the inhibition of mushroom tyrosinase (monophenol monooxygenase EC 1.14.18.1), ten kinds of Korean traditional teas were screened for their tyrosinase inhibitory activity. Green tea was the strongest inhibitor, and the major active constituents in the tea are (-)-epicatechin 3-O-gallate (ECG), (-)-gallocatechin 3-O-gallate (GCG), and (-)-epigallocatechin 3-O-gallate (EGCG). All are catechins with gallic acid group as an active site. The kinetic analysis for inhibition of tyrosinase revealed a competitive nature of GCG with this enzyme for the L-tyrosine binding at the active site of tyrosinase.
Tyrosinase related protein (TRP)-1 and TRP-2 are known to regulate the quality of melanin, and recently their potential role in inhibiting apoptosis have also been reported. To study the role of tyrosinase, TRP-1 and TRP-2 in the growth, differentiation and cell death of ultraviolet B (UVB) irradiated melanocytes, the expression of these proteins in amelanotic and melanotic cells was examined. Expression of tyrosinase and TRP-1 correlated with melanin content, which was upregulated after repeated irradiation of melanotic cells by low doses of UVB. In contrast, the expression and activity of TRP-2 correlated with cell proliferation, but not with pigmentation. In one melanotic melanoma cell line, significant suppression of cell proliferation was observed after low or high doses of UVB irradiation, possibly due to apoptotic changes. TRP-2 expression was remarkably reduced in UVB-irradiated cells, and transfection with TRP-2 expression vector rescued these cells from UVB-induced apoptosis. These results indicate that TRP-2 expression is closely associated with the regulation of cell growth/survival of melanocytes exposed to UVB and that TRP-2 plays a role in protecting melanoma cells from UVB-induced apoptosis.
For the development of peptide-based immunotherapies, the identification of additional tumor antigens and T-cell epitopes is required. Because HLA-A(*)0201 is the most common allele in Caucasians, who represent the majority of patients with melanomas, 6 peptides carrying an HLA-A(*)0201 motif were synthesized from tyrosinase-related protein-2 (TRP2) melanoma antigen and tested for binding affinity to the HLA allele using processing-defective T2 cells. These peptides were then pulsed onto autologous dendritic cells and used to stimulate in vitro CD8(+)-enriched T cells isolated from peripheral blood of HLA-A(*)02(+) healthy donors or melanoma patients for the induction of specific cytotoxic T lymphocytes (CTLs). One peptide, TRP2(288-296) (SLDDYNHLV), the best HLA-A(*)0201 binder, elicited specific CTLs from 1 of 4 patients and 3 of 4 healthy donors. The induced CTLs from the patient and from 1 donor efficiently recognized HLA-A(*)02(+) TRP2(+) melanomas as well as COS-7 cells expressing HLA-A(*)0201 and TRP2 in an HLA class I-restricted manner, as assessed by cytokine production and direct cytolysis. The remaining 2 CTL lines derived from 2 donors displayed low T-cell receptor avidity, which could lyse melanoma cells in the presence of exogenous peptide. Since TRP2 is an antigen expressed in most melanomas, identification of the TRP2/HLA-A(*)0201 peptide SLDDYNHLV may facilitate the design of present peptide-based immunotherapies for the treatment of a large fraction of melanoma patients.
Banxia Houpu Decoction, having been used for the treatment of depression-related diseases since ancient times, is a traditional Chinese medicinal empirical formula consisting of Pinellia ternata, Poria cocos, Magnolia officinalis, Perilla frutescens and Zingiber officinale. The effects of the total decoction extract and five fractions therefrom, were evaluated in mice by tail suspension and forced swimming tests. The total 90% ethanol extract of the decoction was shown to possess an antidepressant activity that was close to that of Prozac, an antidepressant agent being applied clinically. Furthermore, the active principles were desmonstrated to be mainly in the aqueous (Bx4) and lipophic (Bx5) parts of the decoction extract while the polyphenol fraction (Bx2) exhibited a moderate action.
The antioxidant properties of twenty medical herbs used in the traditional Mediterranean and Chinese medicine were studied. Extracts from Forsythia suspensa, Helichrysum italicum, Scrophularia auriculata, Inula viscosa, Coptis chinensis, Poria cocos and Scutellaria baicalensis had previously shown anti-inflammatory activity in different experimental models. Using free radical-generating systems H. italicum. I. viscosa and F. suspensa protected against enzymatic and non-enzymatic lipid peroxidation in model membranes and also showed scavenging property on the superoxide radical. All extracts were assayed at a concentration of 100 microg/ml. Most of the extracts were weak scavengers of the hydroxyl radical and C. chinensis and P. cocos exhibited the highest scavenging activity. Although S. baicalensis inhibited the lipid peroxidation in rat liver microsomes and red blood cells, the extract showed inhibitory actions on aminopyrine N-demethylase and xanthine oxidase activities as well as an pro-oxidant effect observed in the Fe3+-EDTA-H2O2 system. The results of the present work suggest that the anti-inflammatory activities of the same extracts could be explained, at least in part, by their antioxidant properties.
The structures of two novel 3,4-seco-lanostane-type triterpenes isolated from the sclerotium of Poria cocos were established to be 16alpha-hydroxy-3,4-seco-lanosta-4(28),8,24-triene-3,21-dioic acid (1; poricoic acid G) and 16alpha-hydroxy-3,4-seco-24-methyllanosta-4(28),8,24(24(1))-triene-3,21-dioic acid (2; poricoic acid H) on the basis of spectroscopic methods. These two, and eight other known compounds isolated from the sclerotium, poricoic acid B (3), poricoic acid A (4), tumulosic acid (5), dehydrotumulosic acid (6), 3-epidehydrotumulosic acid (7), polyporenic acid C (8), 25-hydroxy-3-epidehydrotumulosic acid (9), and dehydroabietic acid methyl ester (10), showed potent inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Evaluation of the cytotoxicity of compounds 1 and 4 against human cancer cell lines revealed that 1 was significantly cytotoxic to leukemia HL-60 cells [GI(50) (concentration that yields 50% growth) value 39.3 nM], although it showed only moderate cytotoxicity to the other cells. Compound 4 exhibited moderate cytotoxicity to all of the cancer cell lines tested.
Hoelen, sclederma of Poria cocos Wolf, has long been used as a sedative and diuretic in traditional medicine. Formerly, we demonstrated that Hoelen in vitro protects red blood cells from AAPH-induced hemolysis. In this study, tests were carried out to identify the main ingredient of Hoelen that has the scavenging effect on free-radicals. Triterpene carboxylic acids isolated from the methanol extract of Hoelen, i.e. pachymic acid, polyporenic acid, 3-epidehydrotumulosic acid, 3beta-hydroxylanosta-7,9(11), 24-trien-21-oic acid and 3-o-acetyl-16 alpha -hydroxytrametenolic acid, were found to have inhibitory activities against AAPH-induced lysis of red blood cells.
In our previous studies, we showed that PCSC, a polysaccharide isolated from Poria cocos, activated macrophages to induce the translocation of NF-kappaB/Rel into nucleus and DNA binding to its cognate site in the promoter of iNOS gene [Int. Immunopharmacol. 3 (2003) 1353]. In the present study, we investigated the role of p38 kinase pathway and membrane receptors (CD14, Toll-like receptor 4 (TLR4), and CR3) in mediating nitric oxide (NO) production and NF-kappaB/Rel activation induced by PCSC. Treament of RAW 264.7 cells with PCSC resulted in significant activation of p38. The specific p38 inhibitor SB203580 abrogated the PCSC-induced NF-kappaB/Rel activation and NO generation, whereas the selective mitogen-activated protein kinase/extracellular signal-regulated kinase 1 (MEK-1) inhibitor PD98059 did not affect the NF-kappaB/Rel and NO induction. Treatment of RAW 264.7 cells with anti-CD14 Ab, anti-TLR4 Ab, and anti-CR3 Absignificantly blocked PCSC-induced NO production activation. In conclusion, we demonstrate that PCSC induces NF-kappaB/Rel activation and iNOS expression through the CD14, TLR4, and CR3 membrane receptor and p38 kinase which is critically involved in the signal transduction leading to NF-kappaB/Rel activation in murine macrophages.
Pigmentation not only provides a wide range of cosmetic coloration to the skin, hair and eyes, but also provides the underlying tissue significant protection from ultraviolet (UV) damage, which can lead to photoaging and photocarcinogenesis. The melanin pigment is synthesized and deposited within a unique, membrane-bound organelle termed the melanosome. Recent advances in molecular biology and biochemistry have allowed a greater appreciation of how melanocytes generate this organelle and how its biogenesis, structure and function is regulated by the environment. Melanosomes serve as ideal models for the study of organelle biogenesis, protein trafficking, organelle movement and cell-cell interactions that occur during the transfer of melanosomes to keratinocytes. Our understanding of the mechanisms behind a wide range of human pigmentary diseases have grown remarkably as melanosomes have been unraveled.
Tyrosinase related protein (TRP)-1 and -2 regulate the main steps in melanin synthesis and are immune targets in skin cancer or autoimmune pigmentary disorders. We found that ionophore monensin (Mon) and the quaternary amine chloroquine (CQ) discriminate between the traffic routes of TRP-2 and TRP-1. TRP-2 N-glycan processing is interrupted by Mon between ER and trans-Golgi, whereas this process continues for TRP-1. Mature TRP-2 is diverted by CQ treatment to a degradation pathway which depends on functional vacuolar ATPases. Conversely, the subcellular distribution and stability of TRP-1 were not affected by CQ. We propose that TRP-2 is sorted and trafficked in the early secretory pathway with a cargo which does not include TRP-1; post Golgi, TRP-2 intersects the endocytic pathway following a route via early endosomes, possibly by rapid recycling from the plasma membrane. These data show that highly structural homologous glycoproteins use distinct trafficking pathways in the same cell.
Kojic acid derivative 2 was synthesized by joining two pyrone rings through an ethylene linkage by Horner-Emmons reaction of phosphonate 6 with aldehyde 7. The intermediates 6 and 7 were derived from kojic acid. The tyrosinase inhibitory activity of 2 was about 8 times more potent (IC(50) = 3.63 microM) than that of kojic acid (IC(50) = 30.61 microM). Compound 2 also exhibited potent melanin synthesis inhibitory activity (19.53% inhibition at 5 mug) indicating that the connection of two pyrone rings of kojic acid through a suitable linker can be an useful strategy for identification of potent tyrosinase inhibitors.
The effect of coumarin derivatives on melanogenesis was investigated in B16 murine melanoma cells.
Melanin content and tyrosinase activity were analyzed spectrophotometrically. The expression of tyrosinase, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2) were measured either by reverse transcription-polymerase chain reaction (RT-PCR) or Western blot.
Among the coumarin derivatives studied, scoparone (6,7- dimethoxycoumarin) was the most potent; the 6- or 7-methoxy group was found to be essential for the stimulation of melanogenesis. The melanin content was greatly increased by scoparone in a dose-dependent manner; there was no cytotoxicity at the effective concentrations. Scoparone increased enzyme activity as well as protein and mRNA expression of tyrosinase. In addition, mRNA of TRP-1 and TRP-2 were also increased after treatment with scoparone. H-89, an inhibitor of protein kinase A (PKA), completely inhibited the scoparone-induced increase of melanogenesis and the tyrosinase protein.
These results suggest that scoparone-induced stimulation of melanogenesis is likely to occur at the transcriptional level of melanogenesis-related enzymes through PKA signaling.
Tyrosinase gene transcription and its control by melanogenic inhibitors
- Ando
Isolation and identification of tyrosinase inhibitor from Galla Rhois
- Kim H
Identification of a new HLA-A* 0201-restricted T-cell epitope from the tyrosinase-related protein 2 (TRP2) melanoma antigen
- Sun