Arch Dermatol Res (2007) 299:245–257 DOI 10.1007/s00403-007-0740-2
O RI G I NAL PAPE R
Inhibitory eVect of the water-soluble polymer-wrapped derivative of fullerene on UVA-induced melanogenesis via downregulation of tyrosinase expression in human melanocytes and skin tissues Li Xiao · Kenji Matsubayashi · Nobuhiko Miwa
Received: 8 January 2007 / Accepted: 24 January 2007 / Published online: 28 February 2007 © Springer-Verlag 2007
Abstract The C60-fullerene derivatives are expected, as novel and potent anti-oxidants, to more eVectively protect skin cells against oxidative stress. UVAinduced oxidative stress is considered to promote melanogenesis and serious skin damage. The eVect of any fullerene derivatives on UVA-induced melanogenesis is still unknown. Here, we evaluated eVects of a water-soluble polyvinylpyrrolidone (PVP)-wrapped fullerene derivative (named “Radical Sponge®” because of its anti-oxidant ability) on melanogenesis, which was promoted by UVA-irradiation to human melanocytes and skin tissues. Radical Sponge® markedly scavenged UVA-induced reactive oxygen species (ROS) inside human melanocytes as shown by Xuorometry using the redox indicator CDCFH-DA. After treatment with Radical Sponge® or other agents, human melanocytes and skin tissues were irradiated by UVA. Then, cellular melanin content, tyrosinase activity and the ultrastructural change of skin melanosomes were examined. Radical Sponge® showed to signiWcantly inhibit UVA-promoted melanogenesis in normal human epidermis melanocytes (NHEM) and human melanoma HMV-II cells within a non-cytotoxicity dose range. As compared with two whitening
L. Xiao · N. Miwa (&) Laboratory of Cell-Death Control BioTechnology, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Nanatsuka 562, Shobara, Hiroshima 727-0023, Japan e-mail:
[email protected] K. Matsubayashi Vitamin C60 BioResearch Corporation, c/o Mitsubishi Corporation, Chiyoda-ku, Tokyo 100-8086, Japan
agents, arbutin and L-ascorbic acid, Radical Sponge® demonstrated the stronger anti-melanogenic potential according to spectrophotometric quantiWcation for extracted melanin. In human skin cultures also, UVApromoted melanin contents were repressed by Radical Sponge® according to Fontana–Masson stain, suggesting its ability to repress UVA-induced tanning. Transmission electron microscopic ultrastructural images also proved that UVA-increased melanosomes in human skin tissue were obviously reduced by Radical Sponge®. The UVA-enhanced tyrosinase enzymatic activity in NHEM melanocytes was inhibited by Radical Sponge® more markedly than by arbutin and L-ascorbic acid. The UVA-enhanced tyrosinase protein expression, together with cell-size fatness and dendrite-formation, was also inhibited more markedly by Radical Sponge® according to immunostain and Xow cytometry using anti-tyrosinase antibody. Thus the depigmentating action of Radical Sponge® might be due to its down-regulating eVect on the tyrosinase expression, which is initiated by UVA-caused ROS generation. Keywords Fullerene · Whitening eVect · UVA · Melanogenesis · Tyrosinase
Introduction Visible pigmentation in human skin results from the synthesis and distribution of melanin. Melanogenesis is catalyzed by the enzymatic factors such as tyrosinase (EC1.14.18.1), alpha-melanocyte-stimulating hormone (alpha-MSH), ACTH, and PKC-beta in melanocytes, the key components of the skin’s pigmentary system.
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These factors induce melanocyte mitosis, and direct the formation, transport, and distribution of the mature melanin granule, melanosome [5, 11, 13, 26, 27, 36, 40]. UV photon directly or indirectly results in up-regulation of the genes for these melanogenic factors. Several lines of evidence suggest that UV produced reactive oxygen species (ROS) and nitrogen oxide (NO) are important signals in the melanogenesis [4, 30–35, 41]. UVA makes a more intense impact on oxidative stress in the skin than UVB through generation of ROS and NO [10, 25, 37], both of which exert the pigmentation eVects via (1) activating keratinocytes and melanocytes to produce above-mentioned melanogenic factors, (2) damaging DNA, and/or repairing this damage to transmit the signals of melanogenesis transduction pathway [1, 8, 12, 16]. Tyrosinase, a rate-limiting enzyme on melanin synthesis, is the only enzyme absolutely required for melanin production, and almost all of melanogenesis stimulators exert the eVect via direct or indirect activation of tyrosinase [3]. Tyrosinase catalyzes three steps of melanin synthesis: the hydroxylation of L-tyrosine to L-dopa, the oxidation of L-dopa to dopaquinone, and the additional oxidation of 5,6-dihydroxyindole (DHI) to indole-quinone [13]. It has been reported that UVA irradiation could increase tyrosinase mRNA expression and cellular tyrosinase activity, and this eVect was considered to be due to UVA-produced NO. UVA exposure elevates PKC activity in human keratinocytes and melanocytes, and PKC further activates tyrosinase by phosphorylation of speciWc serine residues on its cytoplasmic domain moiety [19, 27]. Ultraviolet radiation (UVR) enhances not only the levels of MSH but also the responsiveness of the melanocytes to this peptide hormone, which regulates skin pigmentation via increasing intracellular cAMP to activate tyrosinase [40]. Some anti-oxidants or NO-scavengers, such as L-ascorbic acid showed the reversed eVect on UVAinduced pigmentation and tyrosinase activation [6, 28, 34, 44]. It has been reported that topical pre-applications of vitamins C and E to hairless mice showed inhibition of UVR-induced tanning [28] Simultaneous administration of L-ascorbic acid, L-cysteine, and vitamin E to brownish guinea pigs displayed the inhibitory eVects on UVA-induced pigmentation, and decreased the melanin content and tyrosinase activity in cultured B16 melanoma cells [9]. The potent tyrosinase inhibitor, arbutin, is repressive to the hyperpigmentation characterized by hyperactive melanocytes [17]. Arbutin could counteract the oxidative stress generation in mouse skin [24]. These evidences showed that
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UVA-induced ROS generation is the strongest stimulus for the induction of pigmentation in human skin [2, 18, 22]. Fullerene and its derivatives, especially water-soluble fullerene derivatives, have been characterized as novel powerful anti-oxidants, and are believed to reduce intracellular ROS and prevent oxidative cell injury with lower cytotoxicity [7, 15, 20, 21]. In our previous study [42, 43], we had successfully prepared a hydrophilic polymer-wrapped fullerene, “Radical Sponge®“ (Formula 1, average MW 6 £ 104). The absorption spectrum of Radical Sponge® is shown in Fig. 1 (maximum wavelength = 259 nm). In UVA spectra (320–400 nm) Radical Sponge® shows only slight absorbance. So the screening or sheltering eVect of Radical Sponge® on UVA light can be almost ignored. In contrast, in UVB spectra (290–320 nm), Radical Sponge® shows an appreciable absorbance. With no overlapped absorbance as one of reasons, we used UVA that intrinsically contributes to oxidative eVects in more major part than UVB does, to test the antimelanogenic eVect of Radical Sponge®. Radical Sponge® was shown to diminish the ROS amounts in terms of the molecular and cellular levels, and protect skin cells from UVB, UVA or the hydroperoxide t-BuOOH injuries. We hypothesized that Radical Sponge® will decrease UVA- and ROS-caused pigmentation. To approve this hypothesis, the following experiments were performed.
Materials and methods Cell culture Human normal epidermis melanocytes (NHEM) were provided from Kurabo Bio. Int., Osaka, Japan and cultured in Medium154s (M-154-500S, Kurabo) containing 0.2% BPE, 0.5% heat-inactivated (56°C, 30 min) fatty acid-free fetal bovine serum (FBS), 3 ng/ml rFGF-B, 5 £ 10¡7 M hydrocortisone, 5 g/ml insulin, 5 g/ml transferring, 10 ng/ml PMA, and 3 g/ml heparin at 37°C in a humidiWed atmosphere containing 5% CO2. Human melanoma cells, HMV-II were kindly gifted by Dr. T. Kasuga of Tokyo Med. Dent. Univ. [23] and maintained in Ham F12 medium (Nissui Seiyaku, Tokyo, Japan) containing 15% FBS (manufactured by Trace ScientiWc Ltd., Melbourne, VIC, Australia), 100 units/ml of penicillin (Sigma, St. Louis, MO, USA), and 100 g/ml of streptomycin (Nissui Seiyaku). Other cell culture reagents were provided from Sigma, St. Louis, MO, USA. For each study, cells were plated onto 10-cm tissue culture dishes and grew for
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Formula 1 Chemical structure of polyvinylpyrrolidone (PVP)-wrapped fullerene derivative, named “Radical Sponge®”
Fig. 1 Absorption spectra in the UV-visible regions of Radical Sponge® aqueous solution (0.06 mg/ml)
4–7 days up to near 80% conXuence. The culture medium was replaced by fresh medium for 48 h before the experiment. Skin culture Human fresh skin tissues from Wve volunteers were kindly provided as samples for Fontana–Masson stain, and transmission electron microscopy by surgeon practitioners including Dr. Yuko Ito of Institute of Ito Beauty Plastic Clinic, Tokyo, Japan who obtained the sign of the informed-consents forms for the dermatological surgery and biopsy proposal. The experiment was conducted in accord with the Declaration of Helsinki. The skin biopsy was vertically separated into several pieces, each of which was cultured in a diVusion chamber (Fig. 2). The culture conditions were close to the nature milieu of the in vivo skin, because the lowest edge of the dermal layer could absorb the nourishment from the medium to avoid immersing of the whole skin tissue in the medium. The skin tissue (0.5 £ 0.5 cm2) was subjected to removal of the subcutaneous fat and
Fig. 2 A diVusion chamber for skin culture a vertically separated piece of skin biopsy was held with biocompatible TG polymer and cyanoacrylate bond, and cultivated with culture medium that was poured in the lower compartment. Diverse anti-oxidant agents were added on the surface of the skin piece, which was irradiated with UVA ray after rinsing the agent
mounted on the diVusion chamber in a 12-well plate (one tissue piece per 1 ml of a culture medium, DMEM containing 0.5% FBS) for 1 h before application in Radical Sponge® aqueous solution at 37°C in a humidiWed atmosphere containing 5% CO2. Histological melanin stain At the end of the incubation period, the tissues were embedded in OCT compound, frozen and vertically sliced into 4-m thick sections with a cryostat. The sections were mounted on polylysine-coated glass slides, dried and further Wxed in 4% buVered formaldehyde. Melanin pigments were visualized with the Fontana– Masson stain. The specimens were observed with a light microscope (ECLIPSE E 600, Nikon Co., Tokyo, Japan). The scanning of skin melanocytes layer (in the upper vicinity of the basement membrane) was performed and quantitatively analyzed by an Image-pro Express 4.0 software.
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Transmission electron microscopic specimen preparation Skin tissues after tissue culture and subsequent treatments were Wxed with a fresh solution of 4.0% glutaraldehyde and 1.0% osmium tetroxide in 0.1 M sodium cacodylate buVer at pH 7.2, 4°C for 1 h. Prestaining was done with 0.5% uranyl acetate in 0.04 M veronal acetate buVer at pH 6.1 for 30 min. Dehydration was done at 4°C and 2-min periods of, respectively, 50, 70, 80, 90, 95, and 100% ethanol. After inWltration (vol/vol absolute ethanol to Epon 812 mixture), the samples were embedded in Epon 812 mixture and polymerized for 48 h at 60°C. After hardening at 60°C overnight, thin sections were cut with a Reichert Ultracut E ultramicrotome (Reichert-Jung, Vienna, Austria). All sections were stained with uranyl acetate and Reynold’s lead citrate. Sections were observed with a Hitachi transmission electron microscope JEM-1200 EX II. UVA irradiation After the culture medium was removed, melanocytes and skin tissues were irradiated, in 200 l of PR-free DMEM containing no fullerene, with Xuorescent lamps. The samples were previously administered with Radical Sponge® that were rinsed three times by PRfree DMEM before UVA irradiation. The emission maximum of the lamp was centered at 365 nm with sheltering of UVB ray by a silica glass Wlter. The control samples were kept in the dark under the same conditions. Fresh medium containing 10% FBS was added after exposure to UVA ray, and the samples were further incubated at 37°C for 48 h. Measurement of intracellular ROS level In order to investigate the inXuence of Radical Sponge® on the ROS level in melanocytes, we used 6-caboxy-2⬘, 7⬘-dichlorodihydroXuorescein diacetate (CDCFH) (Molecular Probes, Eugene, OR, USA) as a redox indicator and used Xuorometry as a detection system. The CDCFH-DA dye was taken up by cells, esterolyzed to be membrane-impermeable CDCFH, and oxidized to be highly Xuorescent CDCF by reaction with ROS such as hydroperoxide and hydrogen peroxide [38]. The cultures were rinsed with PBS(¡) and incubated in phenol red-free DMEM containing 7 M CDCFH-DA for 30 min. The Xuorescence intensity of the oxidative form of CDCFH was measured at 534 nm of an emission wavelength after excitation at 510 nm with a Xuorescence plate reader CytoFluor 2350
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(Millipore, Bedford, MA, USA). The methanol-killed cells were treated as the blank ones. Then cells were trypsinized and daubed on the slide glass and observed using a Xuorescence microscope (Nikon ECLIPSE E 600, Nikon Co.). Determination of melanin content The melanin content of melanocytes was performed using the modiWed method [45]. The cells were harvested by trypsin and washed twice with PBS. Then, samples were resuspended in 200 l of Milli Q water and 1 ml of ethanol–ether 1:1 (vol/vol) to remove opaque substances other than melanin. About 15 min later, samples were centrifuged at 3,000 rpm for 5 min. The precipitates were air-dried and dissolved in 500 l of 1 N NaOH. Samples were then heated at 80°C for 1 h and cooled. The amount of melanin was determined spectrophotometrically based on absorbance at 475 nm. Cellular melanin pigments were visualized with the Fontana–Masson stain. The results were observed with a light microscope and quantitatively analyzed by an Image-pro Express 4.0 software. Assay of cellular tyrosinase activity Cellular tyrosinase activity was measured using a modiWed version of a previously reported method [45]. NHEM cells were solubilized in 1 ml PBS(¡) containing 0.5% Triton X-100, 1 l protease inhibitor cocktail (Sigma, P 8340). After sonication for 20 s on ice, the extracts were clariWed by centrifugation at 15,000 rpm for 15 min at 4°C. The reaction mixture consisted of 0.05% L-DOPA solution and 30 g of protein cell extract in a total volume of 0.2 ml sodium phosphate buVer (0.1 M, pH 7.0). Protein content was measured using the Bio-Rad Protein Assay Kit (Bio-Rad, Richmond, CA, USA) with BSA as the standard. The absorbance of dopachrome formation was monitored using a spectrophotometer (HITACHI U-2800) and a microplate reader (Bio-Rad, Model 3550) at 490 nm after 1 h incubation at 37°C. ImmunoXuorescence Indirect stain Cells were grown overnight on chamber slide, after reagent administered and UVA irradiation, washed three times in PBS(¡) then Wxed in 4% PFA/PBS(¡) for 20 min at room temperature. After three additional washes with PBS(¡), cells were incubated with 1% skim milk in PBS for 20 min to suppress non-speciWc
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binding of IgG. Cells were incubated with 0.5%Triton X 100/PBS(¡) for 3 min and washed with PBS one time then incubated with primary antibody tyrosinase M19 (Santa Cruz Bio Inc., sc-7834, CA, USA) diluted in PBS(¡) for 1 h at room temperature. Cells were washed three times with 1% skim milk then incubated for 1 h at room temperature with FITC-labeled second antibody diluted in PBS(¡) After three further washes with 1% skim milk, and one wash in PBS(¡), cells were mounted in glycerol mounting medium (Sigma) and examined using a Xuorescence microscope (Nikon ECLIPSE E 600, Nikon Co.). Direct stain Cells were harvested by trypsinization and washed once by ice-cold PBS(¡) at 4°C, followed by centrifugation for 5 min at 2,000 rpm and removal of PBS. Cells received 1 ml of FCM Wxation buVer at 4°C (Santa Cruz Bio Inc., sc-3622) per 106 cells and were incubated on ice for 15–30 min. After washed twice in 4°C PBS(¡), cells were immersed in 1 ml of 70% methanol on ice for 15 min to promote the membrane permeability. Cells were washed twice with 4°C FCM wash buVer (Santa Cruz Bio Inc., sc-3624) and stained for the intracellular space with phycoerythrin (PE)conjugated anti-tyrosinase antibody (M19) (Santa Cruz Bio Inc., sc-7834) for 1 h. Cells were washed twice with 1 ml of FCM wash buVer then resuspended in 500 l of fresh FCM wash buVer. Samples were analyzed with a COULTEREPICSXL™ Xow cytometer using an EXPO32™ software (Beckmen Couler Co., Miami, FL, USA) within 24 h. Cell viability assay Cell viability was evaluated by photometric assay using the formazan-forming redox indicator dye WST-1 (2(4-iodophenyl)-3-(4-nitrophenyl)-5-(2-disulfophenyl)2H-tetrazolium, monosodium salt, Wako Pure Chem., Osaka, Japan). At diVerent time points, the cells were rinsed twice with PR-free DMEM medium containing 10% WST-1 at 37°C. Cell viability was determined based on mitochondrial conversion of WST-1 to yellowish formazan, being indicative of the number of viable cells [14]. The absorbance was read at 450 nm with an absorbance multiplate reader. Statistical analysis All data were processed statistically by the software of SPSS 11.5 for Windows, and are expressed as mean § standard deviation of 3–5 independent experi-
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ments. SigniWcant diVerences were determined by LSD or Tamhane’s T2 test.
Results Scavenging eVect of Radical Sponge® on UVA-enhanced intracellular oxidative stress in human melanocytes To examine whether Radical Sponge® could inXuence UVA-induced elevation of intracellular ROS level in human melanocytes HMV-II, we quantiWed the intracellular ROS by CDCFH method. HMV-II cells were previously administered with or without varying concentrations of Radical Sponge®, and then irradiated with UVA. The intracellular ROS level detection was performed at 1 h after UVR. As shown in Fig. 3a, at 1 h after irradiation with UVA (60 J/cm2), signiWcant accumulation of intracellular ROS in non-pretreated cells was observed. In the cells pretreated with Radical Sponge® at 25, 50, and 75 M, the ROS level was markedly reduced to 32, 65, and 82% versus the sham-irradiated level, respectively. After UVA irradiation, the high Xuorescence intensity and morphological abnormalities such as blebs on the cell surface in the nonpretreated HMV-II cells were observed, whereas Radical Sponge®-pretreated cells showed much lower Xuorescence intensity and intact shape. The UVA-induced intracellular ROS enhancement and cell degeneration were prevented by addition of 25 M Radical Sponge® (Fig. 3b). Inhibitory eVect of Radical Sponge® on UVA-induced melanin production in human melanocytes and melanoma cells We examined the inhibitory eVects of Radical Sponge® on melanogenesis in two-type melanocytes: NHEM and human melanoma HMV-II cells which were treated by weak and strong UVA irradiation, whereas two antimelanogenic agents, arbutin, and L-ascorbic acid were used for comparison (Fig. 4). NHEM cells were previously administered with or without varying concentrations of Radical Sponge®, arbutin or L-ascorbic acid for 3 h, and then irradiated with UVA. The melanin content was performed at 24 h after UVR. As shown in Fig. 4a, at 24 h after irradiation with UVA (0.1 J/cm2), the melanin content in non-pretreated NHEM cells was increased to 190% versus that in the control cells. Whereas, Radical Sponge® signiWcantly reduced UVAstimulated melanin production in a dose-dependent manner, and the melanin content in Radical Sponge®
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Fig. 3 Repressive eVects of Radical Sponge® on intracellular oxidative stress in UVA-irradiated HMV-II cells. a Human melanoma HMV-II cells were seeded and cultivated for 24 h, and then administered with Radical Sponge® at diVerent concentrations for 3 h. The cells were rinsed and further incubated in phenol redfree DMEM containing 7 M CDCFH-DA for 30 min. Then, the cells were irradiated with UVA (60 J/cm2). At 1 h after UVR, the cells were assayed using a Xuorescence plate reader (em 458 nm;
ex 530 nm). Data are expressed as % of the control, and each column and bar represent the mean § SD of Wve independent experiments. *P < 0.05, ***P < 0.001 as compared with the nonpretreated cells. b HMV-II cells then were trypsinized and daubed on the slide glass and observed using a Xuorescence microscope. According to Xuorescence intensity being proportional to intracellular ROS amounts, pseudo-colors are expressed from blue via yellow to red. Scale bar indicated 15 m
(50 M)-treated cells was 54.6% of that in non-pretreated cells. Arbutin and L-ascorbic acid reduced the melanin production to 69.5–73.8 and 75.2–77.3%, respectively, of that in non-pretreated cells at the concentration range of 500–100 M. Radical Sponge® (50 M) was more eVective (P < 0.05 versus arbutin at 500 M) than arbutin and L-ascorbic acid. Unlike normal human epidermal melanocytes, melanoma cells HMV-II did not show a marked promotion in melanin synthesis by UVA irradiation at low doses (data are not shown), and so we chose the dose of 33 J/ cm2, which could increase to the melanin content of 150%, but does not aVect the cell viability (data are not shown). In contrast, NHEM cells markedly promoted their melanin synthesis even at a UVA dose as low as 0.1 J/cm2 [44]. As one example of the similar phenomena, Ramirez-Bosca et al. previously reported that more marked UV-promotion of melanin contents is
observed in normal melanocytes than in melanoma cells [29]. Figure 4b showed that, UVA at a dose of 33 J/cm2 caused a marked increase in cellular melanin granules in non-administered HMV-II cells, in comparison to those that were pre-administered by Radical Sponge® at 25 M. The quantitative results showed that Radical Sponge® decreased the melanin formation to 65% of the non-pretreated cells. The color of about 1 £ 106 cell pellets showed that, after UVA irradiation (33 J/cm2), HMV-II cells showed densely brown in the absence of Radical Sponge®, whereas, Radical Sponge®-pretreated cells showed light-brown, being the most marked at a dose of 25 M. Thus, Radical Sponge® showed excellent inhibitory eVects on UVAinduced melanogenesis, suggesting that, irrespective of UVA irradiances or irradiational conditions, melanin formation was consistently inhibited by Radical Sponge® in both melanocytes and melanoma cells.
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Fig. 4 Inhibitory eVect of Radical Sponge® on UVA-induced melanogenesis in human melanocytes. a Normal human epidermal melanocytes NHEM, were incubated with Radical Sponge®, arbutin or L-ascorbic acid at diVerent concentrations for 3 h. At 24 h after UVA-irradiation (0.1 J/cm2), the melanin content per 5 £ 105 cells was determined with a spectrophotometer. Data are expressed as % of control, and each column and bar represent the mean § SD of Wve independent experiments. *P < 0.05 as compared with “arbutin (500 M)-treated cells.” RS Radical Sponge®, L-AA L-ascorbic acid. b HMV-II cells were seeded on a chamber slide and cultivated for 24 h, and then incubated with Radical Sponge® for 3 h. After UVA-irradiation at a dose as high as 33 J/ cm2 and further cultivation for 24 h, the cellular melanin granules were visualized with Fontana–Masson stain. The intracellular
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scanning from one end to another end of a cell was performed and densitometrically analyzed by the Image-pro Express 4.0 software. Scale bar indicated 10 m (Middle). HMV-II cells were seeded in a 6-well plate and treated as above-described. After UVA irradiation and further 24-h cultivation, about 1 £ 106 cells were collected into a microtube, and then the pellets color were observed using a Nikon digital camera DXM 1200 system. Scale bar indicated 0.5 mm (Bottom). c Top: HMV-II cells were cultured with Radical Sponge®, arbutin or L-ascorbic acid at graded concentrations for consecutive 7 days. The melanin content per 2 £ 106 cells was determined with a spectrophotometer. Data are expressed as % of control, and each column and bar represent the mean § SD of Wve independent experiments. **P < 0.01, ***P < 0.001 as compared with “Control.” Bottom: the precipitates of 2 £ 106 HMV-II cells
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The eVect of Radical Sponge® on melanogenesis of the basal level, in HMV-II cells that did not undergo any stimulants such as UVA, was also examined (Fig. 4c). HMV-II cells were cultured in Ham F12 culture medium containing various concentrations of Radical Sponge®, arbutin and L-ascorbic acid for 7 days. The melanin amount per 2 £ 106 cells was measured. As compared with the control, the melanin contents of Radical Sponge®-, arbutin- and L-ascorbic acid-treated cells were all diminished in a dose-dependent manner. Radical Sponge® (50 M) showed a more marked anti-melanogenic eVect than arbutin and L-ascorbic acid. Inhibitory eVect of Radical Sponge® on melanin synthesis in human skin organ culture The eVect of Radical Sponge® on skin pigmentation was examined using the fresh human skin organ culture. Human skin tissues of Wve volunteers were previously applied with or without Radical Sponge® for 4 h and irradiated with UVA at 60 J/cm2. After 24 h, melanin pigments were visualized with the Fontana–Masson stain. The specimens were observed with a light microscope and quantitatively analyzed by an Image-pro Express 4.0 software. All Wve samples showed that UVA-increased melanin pigments were signiWcantly inhibited by Radical Sponge® at 100 M. The quantitative data showed that application of Radical Sponge® diminished melanin formation down to 78% of the non-applied skin (P < 0.05 vs. 0 M) (Fig. 5a). The typical samples which were provided from a 23-year-old male volunteer showed that, before Fontana–Masson stain, the cryo-section which was obtained from UVA (60 J/cm2)-irradiated skin showed a clearly black and brownish zone in the stratum basale, but did not show when Radical Sponge® at 100 M was pre-irradiationally applied to the surface of the tissue for 4 h. The Fontana–Masson stain showed that the amount of melanin pigments was markedly increased by UVA irradiation, which was prevented by Radical Sponge® at 100 M (Fig. 5b). To further investigate the ultrastructural change in UVA-irradiated skin, after above-mentioned treatments, skin tissues were Wxed with ruthenium tetroxide and observed with a Hitachi transmission electron microscope (TEM) JEM-1200 EX II. According to the TEM images, after UVA irradiation, a largely increased melanosome and the melanin migration could be observed in the stratum basale, whereas the control and Radical Sponge®-applied tissues showed sparse melanosome in keratinocytes or melanocytes (Fig. 5c). These results indicate that Radical Sponge®
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could inhibit UVA-induced melanogensis in human skin. Inhibitory eVect of Radical Sponge® on UVA-induced cellular tyrosinase activation and the expression of tyrosinase protein in melanocytes To investigate the mechanism of inhibition of melanogenesis by Radical Sponge®, we further observed the eVect of Radical Sponge® on UVA-induced tyrosinase activation and compared with that of arbutin and Lascorbic acid in human melanocytes. NHEM cells were pre-administrated with Radical Sponge®, arbutin and L-ascorbic acid for 3 h and irradiated with UVA at 0.1 J/cm2, and then the cellular tyrosinase activity was assessed at 24 h after UVA irradiation. As shown in Fig. 6, UVA enhanced cellular tyrosinase activity to a level 136% higher than control that was inhibited by all of these agents at graded concentrations. Radical Sponge® and arbutin showed an anti-melanogenic eVect in a dose-dependent manner, whereas, L-ascorbic acid showed a reversed dose-depended manner. The tyrosinase activity was suppressed to 62.6% by L-ascorbic acid at 100 M, 53.5% by arbutin at 500 M and 50% by Radical Sponge® at 50 M compared with positive control. Radical Sponge® was more potent than arbutin and L-ascrobic acid (P < 0.05). It has been reported that UVA irradiation could not only increase cellular tyrosinase activity, but also tyrosinase protein expression [44]. So the regulation of tyrosinase protein expression by Radical Sponge®, arbutin and L-ascorbic acid was examined using two immunoXuorescence methods including indirect and direct intracellular staining. From the Xuorescence images which were observed with a Xuorescence microscope (ECLIPSE E 600, Nikon Co.) by indirect immunoXuorescence method, 24 h after UVA irradiation, most of NHEM cells including arbutin (500 M)administrated cells showed obvious cytoplasmic staining and dendritic shape, whereas the above-mentioned changes of NHEM cells which were administered by Radical Sponge® or L-ascorbic acid were slight, especially for Radical Sponge® at 50 M and L-ascorbic acid at 100 M (Fig. 7a). HMV-II cells were stained with PE-conjugated antityrosinase antibody, and analyzed with a Xow cytometer. The histograms on PE Xuorescence intensity that, as compared with the higher PE-based Xuorescence intensity of the non-treated cells, the control and Radical Sponge®- or L-ascorbic acid-treated cells were weakly Xuorescent. On the contrary, arbutin-treated cells showed a PE Xuorescence intensity similar to that of non-treated cells. These results suggested that both
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Fig. 5 Microscopic views and transmission electron micrograph of UVA-irradiated or Radical Sponge®administered human skin tissues. a Human skin tissues (n = 5) were administrated with Radical Sponge® at 100 M or PBS(¡) on the top surface of the skin piece for 4 h. After rinsed twice with PBS(¡), the skin tissues were irradiated by UVA at 60 J/cm2, and the control tissues were sham-irradiated. After UVA irradiation and the subsequent 24-h cultivation, the cryo-sections of cultured skin tissues were prepared and stained by Fontana–Masson method. The melanin content of skin melanocytes layer was quantitatively analyzed by an Imagepro Express 4.0 software. Data are expressed as % of control, and each column and bar represent the mean § SD of Wve independent experiments. *P < 0.05 as compared with “Control.” b the sections were observed before or after the stain under the microscope (Nikon ECLIPSE E600). MagniWcation £400. Scale bar = 50 m. Em epidermis, Bm basement membrane, Der dermis, Red arrow melanocytes layer. c Human skin tissues were previously administered with Radical Sponge® and treated by UVA irradiation as described in Fig. 2. The ultrastructure was observed with a transmission electron microscope. The scale indicates 1 m. Bm basement membrane KC keratinocyte MC melanocyte D dermis N nucleus
Radical Spong and L-ascorbic acid could down-regulate the expression level of tyrosinase protein which was unaVected by arbutin (Fig. 7b). In vitro cytotoxicity To examine a possibility that the above-shown inhibitory eVects of Radical Sponge®, arbutin and L-ascorbic
acid on melanogenesis and tyrosinase might be caused by the putative cytotoxicity, we evaluated the cell viability of 48-h cultures of human melanoma HMV-II cells in the presence and absence of the agents using WST-1 assay. Radical Sponge® caused no signiWcant change in cell viability at Wnal concentrations of 25–250 M, and, within the concentration range, antimelanogenic (Fig. 4a) and tyrosinase-repressing
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Fig. 6 Inhibitory eVects of Radical Sponge® on cellular tyrosinase activity in NHEM cells NHEM cells were previously administered with Radical Sponge®, arbutin or L-ascorbic acid and exposed to UVA ray at 0.1 J/cm2 as described in Fig. 1. The cellular tyrosinase activity per 30 g protein was determined with a spectrophotometer and a microplate reader. Data are expressed as % of control, and each column and bar represent the
mean § SD of Wve independent experiments. *P < 0.05 versus “arbutin (500 M)-treated cells”. Left Spectrophotometric determination of dopachrome formation. UVR UVA-irradiation 0.1 J/ cm2, RS50 Radical Sponge® at 50 M, Arbutin 500 arbutin at 500 M, L-AA 100 L-ascorbic acid at 100 M. Right the absorbance at a wavelength of 490 nm that was attributed to dopachrome formation. *P < 0.05 versus arbutin at 500 M
(Figs. 6, 7a, b) eVects were conWrmed to be appreciable. Arbutin and L-ascorbic acid did not reduce cell viability at the concentrations below 2,000 M (Fig. 8). These results indicated that Radical Sponge®, arbutin and L-ascorbic acid exert the inhibitory eVects on melanogenesis and tyrosinase activity in human melanocytes at non-cytotoxic concentrations.
non-stimulated HMV-II cells also, the signiWcantly inhibitory eVect on melanogenesis was observed for Radical Sponge® more appreciably than for arbutin or L-ascorbic acid at 50 M. These results indicated that Radical Sponge® could be expected for its wide-ranged application as a whitening cosmetic material. Second, we proved that Radical Sponge® could inhibit UVA-induced tyrosinase activation and down-regulates the expression of tyrosinase protein in two kinds of human melanocytes at the secure concentration range. As compared with two major eVective cosmetic additives, arbutin and L-ascorbic acid, Radical Sponge® showed the more marked depigmenting eVect in human melanocytes or melanoma cells. The mechanism of the anti-melanogenic eVects of Radical Sponge® could be regarded as its scavenging action toward UVA-caused ROS, the major causation of tyrosinase gene activation and melanin synthesis. The hypothetic mechanism could be considered that Radical Sponge® acts as a strong anti-oxidant could (1)
Summary and conclusions In the present study, we have proved that Radical Sponge® signiWcantly scavenged UVA-enhanced intracellular ROS in human melanocytes. The results suggested that Radical Sponge® has a potential antimelanogenic eVect on UVA-ROS cascade that caused melanogenesis. Then we aVorded more direct evidences that Radical Sponge® exerted excellent inhibitory eVects on UVA-induced melanogenesis in NHEM and human malignant melanoma cells HMV-II. In Fig. 7 ImmunoXuorescence localization and Xow cytometric (FCM) analysis of tyrosinase protein in melanocytes. a NHEM cells were seeded on a chamber slide, then administered with Radical Sponge®, arbutin or L-ascorbic acid and irradiated with UVA as described in Fig. 1a. The cells were stained using phycoerythrin (PE)-conjugated antibody M19 to detect the tyrosinase protein and observed with a Xuorescence microscope. Bottom the histogram of the Xuorescence intensity corresponding to extents of the expression of tyrosinase protein was processed by an ACTII software. The data shown are typical of three independent
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experiments, each of which contained wells in duplicate in cham- 䉴 ber slides. MagniWcation £400. Scale bar = 20 m. b HMV-II cells were incubated with Radical Sponge®, arbutin or L-ascorbic acid at diVerent concentration for 3 h. After exposed to UVA-irradiation (0.1 J/cm2) 24 h, the expression of tyrosinase protein in cytoplasm per 5 £ 105 per cells was stained with a phycoerythrin (PE)-conjugated anti-tyrosinase antibody by immunoXuorescence method. The Xuorescence intensity was analyzed with a Xow cytometer
Arch Dermatol Res (2007) 299:245–257
inhibit ROS-activated tyrosianse within the melanogenic signaling pathway (2) scavenge ROS to impair their contribution to tyrosinase-catalyzed reaction cascades.
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We used the skin culture system which bears a close resemble to the natural milieu of the in vivo skin to evaluate the eVect of Radical Sponge® on UVAinduced pigmentation. Histological and TEM studies
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Fig. 8 EVects of Radical Sponge®, arbutin, and L-ascorbic acid on cell viability in HMV-II cells. Human melanoma HMV-II cells were incubated with Radical Sponge®, arbutin or L-ascorbic acid at diVerent concentrations for 48 h. The cell viability was estimated by WST-1 assay using a microplate reader. Data are expressed as % of control, and each concentration point and bar represent the mean § SD of Wve independent experiments. Control = (0) *P < 0.05 versus the control
(based on n = 5) displayed that Radical Sponge® could prevent UVA-caused pigmentation in human skin tissue. The ultrastructural images further proved that UVA-enhanced melanosomes and melanin migration were reduced by Radical Sponge® application. According to our experiment results on permeability of Radical Sponge using anti-C60-fullerene antibody, at 3 h after addition, Radical Sponge could cross the external cellular membrane and localize to cytoplasm (data are not shown). The skin permeation of Radical Sponge was evaluated using the TESTSKINTM LSEhigh skin model (Toyobo Co., Fukui, Japan). After 3 h application, Radical Sponge permeated the epidermis and reached the basement membrane (data are not shown). Therefore, the skin tissue culture system using the human biopsy is endowed with evaluation and predictability for the preclinical responses to UV-irradiation and the application of cosmetic reagents. In our simultaneously progressing study, artiWcial UVinduced formation of melanin on human arms of 20–30 testees has been demonstrated to be repressed by preirradiational spreading of Radical Sponge, followed by preparation for another submission as a full article. From the results of cellular tyrosinase activity experiment, Radical Sponge®, arbutin and L-ascorbic acid showed to inhibit cellular tyrosinase activity in NHEM cells. Arbutin did not aVect the regulation of tyrosinase protein. This might be because arbutin could repress tyrosinase activity as a competitive inhibitor for L-tyrosine- and L-DOPA-binding site at the catalytic site of a tyrosinase molecule, but do not aVect expression of
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tyrosinase protein [17]. On the contrary, both Radical Sponge® and L-ascorbic acid act as ROS-scavengers to reduce ROS-caused tyrosinase gene transcription. Although tyrosinase is the most important key enzyme on melanogenesis, as described in the introduction section of the present manuscript, melanogenic signal pathways are related to many cellular factors and contiguous cells such as keratinocytes. Keratinocyte-derived melanogenic factors (such as endothelin-1, alpha-MSH and basic Wbroblast growth factor) interact synergistically to stimulate human melanocyte proliferation and modulate melanogenesis [39]. Therefore, it is necessary to investigate the eVects of Radical Sponge® on those factors. This will be the next experiment we will systematically and extensively perform. In conclusion, these results suggest that Radical Sponge® has strong potential as a novel skin-lightening agent in the cosmetic industry. Acknowledgments The authors thank Dr. Hiroya Takada, Ms. Akiko Tamagawa, and Mr. Koji Tani for their technical assistance.
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