Publications by authors named "Kentaro Tsuji-Naito"

15 Publications

  • Page 1 of 1

Low-molecular-weight whey proteins promote collagen production in dermal fibroblasts via the TGF-β receptor/Smad pathway.

Biosci Biotechnol Biochem 2021 Sep 8. Epub 2021 Sep 8.

DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025, Japan.

Whey proteins (WPs) reportedly enhance cutaneous tissue regeneration in in vivo studies. However, the underlying mechanisms of such regenerative processes are poorly understood. In this study, we show that low-molecular-weight WPs (LMWPs; 1-30 kDa) accelerate the dermal collagen production via the transforming growth factor β receptor (TβR)/Smad pathway. We showed that LMWPs increased type I and III collagen expression in normal human dermal fibroblasts. Moreover, LMWPs rapidly induced Smad protein phosphorylation and nuclear translocation. Notably, type I TβR/Smad signaling inhibitor treatment or type II TβR siRNA knockdown blocked the LMWP-induced type I collagen expression. To identify the active components, we fractionated LMWPs and identified β-lactoglobulin and α-lactalbumin as potential TβR/Smad signaling inducers. Our findings unravel novel biological functions of WPs, involving the TβR/Smad-dependent induction of dermal collagen synthesis, highlighting the therapeutic potential of LMWPs in wound healing.
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http://dx.doi.org/10.1093/bbb/zbab155DOI Listing
September 2021

Restoring NAD by NAMPT is essential for the SIRT1/p53-mediated survival of UVA- and UVB-irradiated epidermal keratinocytes.

J Photochem Photobiol B 2021 Aug 12;221:112238. Epub 2021 Jun 12.

DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025, Japan.

Nicotinamide adenine dinucleotide (NAD) is a crucial coenzyme in energy production. The imbalance of NAD synthesis has been found to trigger age-related diseases, such as metabolic disorders, cancer, and neurodegenerative diseases. Also, UV irradiation induces NAD depletion in the skin. In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD salvage pathway and essential for NAD homeostasis. However, but few studies have focused on the role of NAMPT in response to UV irradiation. Here, we show that NAMPT prevents NAD depletion in epidermal keratinocytes to protect against the mild-dose UVA and UVB (UVA/B)-induced proliferation defects. We showed that poly(ADP-ribose) polymerase (PARP) inhibitor rescued the NAD depletion in UVA/B-irradiated human keratinocytes, confirming that PAPR transiently exhausts cellular NAD to repair DNA damage. Notably, the treatment with a NAMPT inhibitor exacerbated the UVA/B-induced loss of energy production and cell viability. Moreover, the NAMPT inhibitor abrogated the sirtuin-1 (SIRT1)-mediated deacetylation of p53 and significantly inhibited the proliferation of UVA/B-irradiated cells, suggesting that the NAMPT-NAD-SIRT1 axis regulates p53 functions upon UVA/B stress. The supplementation with NAD intermediates, nicotinamide mononucleotide and nicotinamide riboside, rescued the UVA/B-induced phenotypes in the absence of NAMPT activity. Therefore, NAD homeostasis is likely essential for the protection of keratinocytes from UV stress in mild doses. Since the skin is continuously exposed to UVA/B irradiation, understanding the protective role of NAMPT in UV stress will help prevent and treat skin photoaging.
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http://dx.doi.org/10.1016/j.jphotobiol.2021.112238DOI Listing
August 2021

Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide.

Biochem Biophys Res Commun 2020 01 19;522(1):151-156. Epub 2019 Nov 19.

DHC Corporation, Fundamental Research Laboratory, Division 2, 2-42 Hamada, Mihama-ku, Chiba, 261-0025, Japan. Electronic address:

Peptide transporters 1 and 2 (PEPT1 and PEPT2) are proton-coupled oligopeptide transporter members of the solute carrier 15 family and play a role in the cellular uptake of di/tri-peptides and peptidomimetics. Our previous work showed that PEPT2 is predominantly expressed within undifferentiated keratinocytes. Here we show that PEPT2 expression decreases as keratinocyte differentiation progresses and that PEPT1 alternately is expressed at later stages. Absolute quantification using quantitative polymerase chain reaction revealed that the expression level of PEPT1 is about 17 times greater than that of PEPT2. Immunohistochemical study of human skin provided evidence of PEPT1 in the epidermis. The uptake of glycylsarcosine into keratinocytes was significantly blocked by PEPT inhibitors, including nateglinide and glibenclamide. Moreover, we found that PEPT1 knockdown in differentiated keratinocytes significantly suppressed the influence of a bacterial-derived peptide, muramyl dipeptide (MDP), on the production of proinflammatory cytokine interleukin-8, implying that bacteria-derived oligopeptides can be transported by PEPT1 in advanced differentiated keratinocytes. Taken together, PEPT1 and PEPT2 may concertedly play an important role in MDP-NOD2 signaling in the epidermis, which provides new insight into the mechanisms of skin homeostasis against microbial pathogens.
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http://dx.doi.org/10.1016/j.bbrc.2019.11.044DOI Listing
January 2020

Tomatidine Reduces Palmitate-Induced Lipid Accumulation by Activating AMPK via Vitamin D Receptor-Mediated Signaling in Human HepG2 Hepatocytes.

Mol Nutr Food Res 2019 11 10;63(22):e1801377. Epub 2019 Sep 10.

Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, 599-8531, Japan.

Scope: Nonalcoholic fatty liver disease (NAFLD) has emerged as the most common chronic liver disease worldwide, defined by hepatic over-accumulation of lipids without significant ethanol consumption. Pharmacological or bioactive food ingredients that suppress hepatic lipid accumulation through AMP-activated protein kinase (AMPK) signaling, which plays a critical role in the regulation of lipid metabolism, are searched.

Methods And Results: It is found that tomatidine, the aglycone of α-tomatine abundant in green tomatoes, significantly inhibits palmitate-provoked lipid accumulation and stimulates phosphorylation of AMPK and acetyl-CoA carboxylase 1 (ACC1) in human HepG2 hepatocytes. The results also indicate that tomatidine can enhance triglyceride turnover and decline in lipogenesis by upregulating adipose triglyceride lipase (ATGL) and downregulating fatty acid synthase (FAS) via the AMPK signaling-dependent regulation of transcription factors, element-binding protein-1c (SREBP-1c) and forkhead box protein O1 (FoxO1). Furthermore, mechanistic studies demonstrate that tomatidine-stimulated AMPK phosphorylation is due to CaMKKβ activation in response to an increase in intracellular Ca concentration. Finally, it is discovered that tomatidine functions as an agonist for vitamin D receptor to elicit AMPK-dependent suppression of lipid accumulation.

Conclusion: The in vitro study suggests the potential efficacy of tomatidine as a preventive and therapeutic treatment in obesity-related fatty liver diseases.
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http://dx.doi.org/10.1002/mnfr.201801377DOI Listing
November 2019

Bifunctional effects of O-methylated flavones from Scutellaria baicalensis Georgi on melanocytes: Inhibition of melanin production and intracellular melanosome transport.

PLoS One 2017 9;12(2):e0171513. Epub 2017 Feb 9.

Fundamental Research Laboratory, Fundamental Research Department, DHC Corporation, Chiba, Japan.

The growing interest in skin lightening has recently renewed attention on the esthetic applications of Chinese herbal medicine. Although Scutellaria baicalensis Georgi is used for antipyretic and antiinflammatory purposes, its whitening effect remains unclear. This study reports three major findings: (1) S. baicalensis has a potent inhibitory effect on melanogenesis; (2) wogonin and its glycoside are the active components of S. baicalensis; and (3) O-methylated flavones from S. baicalensis, such as wogonin, inhibit intracellular melanosome transport. Using a melanin quantification assay, we showed that S. baicalensis potently inhibits melanogenesis in B16F10 cells. Componential analyses revealed that the main components of S. baicalensis are baicalin, wogonoside, baicalein, wogonin, and oroxylin A. Among these five flavones, wogonin and wogonoside consistently inhibited melanogenesis in both B16F10 melanoma cells and primary melanocytes. Wogonin exhibited the strongest inhibition of melanin production and markedly lightened the color of skin equivalents. We identified microphthalmia-associated transcription factor and tyrosinase-related proteins as potential targets of wogonin- and wogonoside-induced melanogenesis suppression. In culture, we found that the melanosomes in wogonin-treated B16F10 cells were localized to the perinuclear region. Immunoblotting analyses revealed that wogonin significantly reduced in melanophilin protein, which is required for actin-based melanosome transport. Other actin-based melanosome transport-related molecules, i.e., Rab27A and myosin Va, were not affected by wogonin. Cotreatment with MG132 blocked the wogonin-induced decrease in melanophilin, suggesting that wogonin promotes the proteolytic degradation of melanophilin via the calpain/proteasomal pathway. We determined that the structural specificities of the mono-O-methyl group in the flavone A-ring and the aglycone form were responsible for reducing melanosome transport. Furthermore, wogonin and two wogonin analogs, mono-O-methyl flavones, strongly suppressed melanosome transport. Our findings suggest the applicability of S. baicalensis in the esthetic field. Thus, we propose a novel pharmacologic approach for the treatment of hyperpigmentation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0171513PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5300169PMC
August 2017

β-Caryophyllene attenuates palmitate-induced lipid accumulation through AMPK signaling by activating CB2 receptor in human HepG2 hepatocytes.

Mol Nutr Food Res 2016 10 16;60(10):2228-2242. Epub 2016 Jun 16.

Department of Biological Chemistry, Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan.

Scope: Nonalcoholic fatty liver disease is currently the most common chronic liver disease worldwide, characterized by excessive hepatic lipid accumulation without significant ethanol consumption. We have performed a screening for medicinal foods that inhibit hepatocytic lipid accumulation through activation of AMP-activated protein kinase (AMPK), which is a critical regulator of the hepatic lipid metabolism.

Methods And Results: We found that clove (Syzygium aromaticum), which is commonly used as a spice, markedly inhibits palmitate-inducible lipid accumulation in human HepG2 hepatocytes. Analyses of the clove extracts found that β-caryophyllene, an orally-active cannabinoid, is the principal suppressor of the lipid accumulation, and stimulates the phosphorylation of AMPK and acetyl-CoA carboxylase 1 (ACC1). Our data also showed that β-caryophyllene prevents the translocation of sterol regulatory element-binding protein-1c (SREBP-1c) into the nucleus and forkhead box protein O1 (FoxO1) into the cytoplasm through AMPK signaling, and consequently, induces a significant downregulation of fatty acid synthase (FAS) and upregulation of adipose triglyceride lipase, respectively. Moreover, we demonstrated that the β-caryophyllene-induced activation of AMPK could be mediated by the cannabinoid type 2 receptor-dependent Ca signaling pathway.

Conclusion: Our results suggest that β-caryophyllene has the potential efficacy in preventing and ameliorating nonalcoholic fatty liver disease and its associated metabolic disorders.
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http://dx.doi.org/10.1002/mnfr.201600197DOI Listing
October 2016

H(+)/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport.

Biochem Biophys Res Commun 2016 07 20;475(4):335-41. Epub 2016 May 20.

DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025, Japan. Electronic address:

Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H(+) gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake.
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http://dx.doi.org/10.1016/j.bbrc.2016.05.093DOI Listing
July 2016

Pyrroloquinoline quinone stimulates epithelial cell proliferation by activating epidermal growth factor receptor through redox cycling.

Free Radic Biol Med 2012 Sep 21;53(6):1239-51. Epub 2012 Jul 21.

Department of Biological Chemistry, Division of Applied Life Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai 599-8531, Japan.

Pyrroloquinoline quinone (PQQ), a redox cofactor for bacterial dehydrogenases, has been implicated to be an important nutrient in mammals functioning as a potent growth factor. However, the underlying molecular mechanisms have not been elucidated. The present study revealed that PQQ induces the activation (tyrosine autophosphorylation) of epidermal growth factor receptor (EGFR) and its downstream signaling in a ligand-independent manner, leading to increased cellular proliferation in an epithelial cell line A431. PQQ inhibited protein tyrosine phosphatase 1B (PTP1B), which negatively regulates the EGFR signaling by tyrosine dephosphorylation, to oxidatively modify the catalytic cysteine through its redox cycling activity to generate H(2)O(2). PQQ-inducible intracellular ROS production and EGFR activation were significantly suppressed by the pre-treatment with antioxidants. The intracellular redox state regulates the EGFR signaling through the redox-sensitive catalytic cysteine of PTP1B and modulates cell proliferation. Our data suggest that PQQ may stimulate epithelial cell proliferation by activating EGFR by oxidation and subsequent inactivation of PTP1B via its redox cycling. Our results provide novel insight into the mechanisms by which PQQ may function as a growth factor to contribute to mammalian growth.
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http://dx.doi.org/10.1016/j.freeradbiomed.2012.07.015DOI Listing
September 2012

Concentrated bovine milk whey active proteins facilitate osteogenesis through activation of the JNK-ATF4 pathway.

Biosci Biotechnol Biochem 2012 7;76(6):1150-4. Epub 2012 Jun 7.

DHC Corporation Laboratories, Chiba, Japan.

Concentrated fractions of low molecular weight whey proteins (1-30 kDa), that is concentrated bovine milk whey active proteins (CBP), have been found to enhance bone formation in both in vivo and clinical studies, but the underlying mechanisms are poorly understood. In this study, we found that CBP promoted osteoblastic differentiation in normal human osteoblasts, and determined the involvement of the c-jun NH2-terminal kinase (JNK)-activating transcription factor 4 (ATF4) pathway. We observed that alkaline phosphatase activity and mineralization were significantly induced by CBP treatment. In addition, mRNA expression of ATF4 was intensely elevated in CBP-treated osteoblasts, indicating that the late-phase events of differentiation were promoted. We found that CBP activated the phosphorylation of JNK and extracellular signal-regulated kinase (ERK). Furthermore, pathway analyses using the various signaling pathway-specific inhibitors revealed that JNK activation, but not ERK activation, is essential for CBP-induced mineralization and ATF4 expression. Our results indicate that the JNK-mediated ATF4 pathway is required for CBP-promotive osteogenesis.
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http://dx.doi.org/10.1271/bbb.110990DOI Listing
November 2012

BBF2H7-mediated Sec23A pathway is required for endoplasmic reticulum-to-Golgi trafficking in dermal fibroblasts to promote collagen synthesis.

J Invest Dermatol 2012 Aug 12;132(8):2010-8. Epub 2012 Apr 12.

DHC Corporation Laboratories, Division 2, Chiba, Japan.

Collagen fibers, structural elements responsible for mechanical strength in skin, are synthesized constitutively in response to cytokines such as IGF-I. However, little is known about their intracellular trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus during synthesis. We demonstrate herein that the BBF2 human homolog on chromosome 7 (BBF2H7)-mediated Sec23A pathway is involved in regulation of intracellular procollagen trafficking. The mRNA and protein expression of BBF2H7, Sec23A, and type I and III collagen (COL1 and COL3) was induced by IGF-I stimulation. In addition, the cleaved form of BBF2H7 was detected in IGF-I-treated cultures, indicating that activation occurs concurrently with its expression. Knockdown with small interfering RNAs targeting BBF2H7 caused a significant reduction in the expression of COL1 and COL3, regardless of IGF-I treatment. Both mitogen-activated protein kinase and phosphatidylinositol-3 kinase pathways via IGF-I receptor activation were required for BBF2H7 induction. Using immunofluorescence microscopy, we showed that Golgi apparatus dysmorphology is due to coat protein complex II vehicle hypoplasia caused by the absence of BBF2H7 and Sec23A. The BBF2H7-mediated Sec23A pathway was required for ER-to-Golgi procollagen trafficking to promote collagen synthesis. This role of growth factors such as IGF-I, which to our knowledge is previously unreported, suggests antiaging strategies.
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http://dx.doi.org/10.1038/jid.2012.103DOI Listing
August 2012

Cinnamon extract promotes type I collagen biosynthesis via activation of IGF-I signaling in human dermal fibroblasts.

J Agric Food Chem 2012 Feb 27;60(5):1193-200. Epub 2012 Jan 27.

Department of Biological Chemistry, Division of Applied Life Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Naka-ku, Sakai, Japan.

The breakdown of collagenous networks with aging results in hypoactive changes in the skin. Accordingly, reviving stagnant collagen synthesis can help protect dermal homeostasis against aging. We searched for type I collagen biosynthesis-inducing substances in various foods using human dermal fibroblasts and found that cinnamon extract facilitates collagen biosynthesis. Cinnamon extract potently up-regulated both mRNA and protein expression levels of type I collagen without cytotoxicity. We identified cinnamaldehyde as a major active component promoting the expression of collagen by HPLC and NMR analysis. Since insulin-like growth factor-I (IGF-I) is the most potent stimulator of collagen biosynthesis in fibroblasts, we examined the effect of cinnamaldehyde on IGF-I signaling. Treatment with cinnamaldehyde significantly increased the phosphorylation levels of the IGF-I receptor and its downstream signaling molecules such as insulin receptor substrate-1 and Erk1/2 in an IGF-I-independent manner. These results suggested that cinnamon extract is useful in antiaging treatment of skin.
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http://dx.doi.org/10.1021/jf2043357DOI Listing
February 2012

α-Lipoic acid induces collagen biosynthesis involving prolyl hydroxylase expression via activation of TGF-β-Smad signaling in human dermal fibroblasts.

Connect Tissue Res 2010 Oct;51(5):378-87

DHC Corporation, Laboratories Division 2, Mihama-ku, Chiba, Japan.

The collapse of collagenous networks with aging results in comprehensive changes in the functional properties of skin. α-Lipoic acid (LA) is known to possess beneficial effects against skin aging, effects often presumed to be its antioxidant potential. However, the effects of LA on fibrillogenesis in dermal fibroblasts have not been adequately assessed. In this study, we demonstrated for the first time that LA enhances the biosynthesis of new collagen in normal human dermal fibroblasts (NHDFs). By using a quantitative dye-binding method and immunochemical approaches, we showed that LA effectively increased the expression and subsequently the deposition of type I collagen in NHDFs. LA also facilitated the expression of a collagen-processing enzyme, prolyl-4-hydroxylase, pointing to the existence of a posttranslational mechanism among the LA-mediated effects on collagen synthesis. In addition, we determined that both Smad 2/3 were rapidly phosphorylated by treatment with LA within 30 min, indicating that LA enhances type I collagen synthesis through the activation of Smad signaling. Pretreatment of SB431542, a specific transforming growth factor-β (TGF-β) receptor type I (TβRI) kinase inhibitor, blocked LA-mediated Smad 2/3 phosphorylations and both type I collagen and prolyl-4-hydroxylase expression, suggesting that LA-mediated cell responses are regulated by TβRI kinase-dependent pathway. Levels of TGF-β secretion after 4 hr of treatment with LA were not remarkably elevated, indicating that LA may be able to mimic TGF-β-mediated cell response. The study results produced new insights into the molecular pharmacology of LA in NHDFs, with potential applications in the treatment of aging skin.
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http://dx.doi.org/10.3109/03008200903486188DOI Listing
October 2010

Aldehydic components of cinnamon bark extract suppresses RANKL-induced osteoclastogenesis through NFATc1 downregulation.

Bioorg Med Chem 2008 Oct 14;16(20):9176-83. Epub 2008 Sep 14.

DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025, Japan.

Several major bone diseases are directly attributable to bone loss, including osteoporosis, bone metastasis, and rheumatoid arthritis. The nuclear factor of activated T cell 1 (NFATc1), a transcription factor, has recently been shown to play an essential role in osteoclastogenesis. In this study, we found that of several herbs, Cinnamomum zeylanicum (C. zeylanicum) exhibited the strong inhibitory effects on osteoclastogenesis and that its mechanism of action involves the suppression of NFATc1-mediated signal transduction. C. zeylanicum dose-dependently inhibited osteoclast-like cell formation at concentrations of 12.5-50 microg/ml without affecting cell viability. Resorption pit assays have shown that C. zeylanicum also inhibits the bone-resorbing activity of mature osteoclasts. Treatment with C. zeylanicum inhibited the receptor activator of nuclear factor-kappaB ligand (RANKL)-induced NFATc1 and c-fos expression. Additionally, C. zeylanicum moderately inhibited phosphorylation of IkappaB-alpha, suggesting that the c-fos/NFATc1 pathway, rather than the nuclear factor-kappaB (NF-kappaB) pathway, is the primary target of C. zeylanicum during RANKL-induced osteoclastogenesis. Using an HPLC-DAD system, we identified three major peaks for four characteristic components in the C. zeylanicum extract and identified an unknown peak as 2-methoxycinnamaldehyde via HPLC and a 2D-COSY (1)H NMR study. We identified cinnamaldehyde and 2-methoxycinnamaldehyde as active components reducing osteoclast-like cell formation and inhibiting NFATc1 expression. Notably, in a resorption pit assay, 2-methoxycinnamaldehyde exhibited remarkable inhibition rates of 95% at 2 microM on bone resorption. In summary, this study points to the conclusion that C. zeylanicum inhibits RANKL-induced osteoclastogenesis. This finding raises prospects for the development of a novel approach in the treatment of osteopenic disease.
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http://dx.doi.org/10.1016/j.bmc.2008.09.036DOI Listing
October 2008

Modulating effects of a novel skin-lightening agent, alpha-lipoic acid derivative, on melanin production by the formation of DOPA conjugate products.

Bioorg Med Chem 2007 Mar 31;15(5):1967-75. Epub 2006 Dec 31.

DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025, Japan.

Sodium zinc dihydrolipoylhistidinate (DHLHZn) is a compound of Zn(2+)/dihydrolipoic acid derivate complex, which was developed for cosmetic/medical use. To characterize DHLHZn as a novel skin-lightening agent, inhibitory actions of DHLHZn on tyrosinase (including its reaction pathway) have been elucidated in this study. In a B16 melanoma cell system, DHLHZn was active in suppressing the synthesis of melanins as well as alpha-arbutin, well known as a depigmenting drug. Furthermore, in a tyrosinase assay, DHLHZn showed stronger inhibitory effect on DOPAchrome formation than other tyrosinase inhibitors such as kojic acid. Our previous report demonstrated that the sulfhydryl groups of lipoyl motif react with DOPAquinone to form lipoyl DOPA conjugates. We therefore postulated that conjugated products between DHLHZn and DOPAquinone might be formed. Upon reaction of DHLHZn with L-DOPA following tyrosinase-catalyzed oxidation, the formation of DHLH DOPA conjugated products was confirmed by HPLC-tandem mass spectrometry using reserpine as the internal standard. In addition, the inhibitory kinetics analyzed by a Lineweaver-Burk plot exhibited the reversibility of DHLHZn as a competitive inhibitor with a KI value of 0.35 microM. These results indicate that this covalent reaction might contribute to alternating DOPAquinone, which is a tyrosinase reaction product, and result in the competitive inhibitory effect of DHLHZn on DOPAchrome formation. DHLHZn may thus serve as a potentially effective skin-lightening agent, an effectiveness that is based on the compound's covalent scavenging of DOPAquinone resulting in depigmentation.
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http://dx.doi.org/10.1016/j.bmc.2006.12.042DOI Listing
March 2007

Evidence for covalent lipoyl adduction with dopaquinone following tyrosinase-catalyzed oxidation.

Biochem Biophys Res Commun 2006 Apr 28;343(1):15-20. Epub 2006 Feb 28.

DHC Corporation Laboratories, Division 2, 2-24 Hamada, Mihama-ku, Chiba 261-0025, Japan.

Previous studies have examined the conjugation of sulfhydryl compounds such as L-cysteine and glutathione with DOPA-quinone following the oxidation of tyrosine and DOPA by tyrosinase. These covalent reactions play a key role in the regulation and metabolism of pigment cells. We report on the first direct evidence for the formation of lipoyl adducts in reactions of thiol groups with DOPA-quinone in dihydrolipoic acid (6,8-dimercaptooctanoic acid [DHLA]). Incubating DHLA with DOPA-quinone followed by tyrosinase-catalyzed oxidation resulted in the three products predicted by HPLC-UV and LC-ESI(-)-MS analyses for DHLA DOPA conjugates. In the current study, we identified 5-S-lipoyl-DOPA among the principal products isolated by HPLC and characterized by FAB(-)-MS, ESI(-)-MS/MS, and 1H NMR, 2D-COSY studies. Collectively, these results suggest that DHLA undergoes sulfhydryl conjugation with DOPA-quinone, pointing to the involvement of thiol-reactive metabolites.
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http://dx.doi.org/10.1016/j.bbrc.2006.02.118DOI Listing
April 2006
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