Hypopigmentary effects of 4-n-butylresorcinol and resveratrol in combination.

In the present study, the effects of 4-n-butylresorcinol and/or resveratrol on melanogenesis were studied. To achieve synergistic effects and avoid potential adverse effects, combinations of the agents in low concentrations were investigated. Our results show that 1 microM of 4-n-butylresorcinol and 1 microM of resveratrol did not individually inhibit melanin synthesis. However, the combination of 4-n-butylresorcinol (1 microM) and resveratrol (1 microM) significantly reduced melanin synthesis. Furthermore, 4-n-butylresorcinol (10 microM) and resveratrol (10 microM) decreased melanogenesis much stronger. 4-n-Butylresorcinol is reported to directly inhibit tyrosinase, the rate-limiting melanogenic enzyme, without changing tyrosinase levels. Our results also showed that resveratrol did not directly inhibit tyrosinase at 0.1-10 microM. Literature has reported that resveratrol led to post-transcriptional regulation of tyrosinase. However, Western blot analysis showed that neither 4-n-butylresorcinol nor resveratrol alone decreased tyrosinase protein levels. Surprisingly, the combination of 4-n-butylresorcinol and resveratrol reduced tyrosinase levels. Therefore, these results indicate that the synergistic hypopigmentary effect of 4-n-butylresorcinol and resveratrol results from a decreased level of tyrosinase possibly resulting from synergistic action of 4-n-butylresorcinol on tyrosinase alteration by resveratrol.

Sphingosine-1-phosphate decreases melanin synthesis via microphthalmia-associated transcription factor phosphorylation through the S1P3 receptor subtype.

OBJECTIVES: Previously, we reported that sphingosine-1-phosphate (S1P) reduced melanin synthesis. In this study we have investigated S1P receptor-mediated extracellular signal-regulated protein kinase (ERK) activation and microphthalmia-associated transcription factor (MITF) phosphorylation. METHODS: To examine S1P-induced signalling pathways, electron and confocal microscopic studies, reverse transcription-polymerase chain reaction and Western blot analysis were performed. KEY FINDINGS: S1P phosphorylated MITF at Ser73, which may have resulted in a MITF mobility shift. Furthermore, 90 kDa ribosomal S6 kinase-1 (RSK-1) phosphorylation was observed after S1P treatment. In addition, PD98059 abrogated the S1P-induced MITF mobility shift and RSK-1 activation. In experiments with MITF mutants, it was shown that dual phosphorylation at Ser73 and Ser409 was indispensable for MITF degradation. We investigated further the actions of S1P on its specific receptors. The results showed that pertussis toxin completely abolished the hypopigmentary effects and ERK pathway activation by S1P, suggesting that S1P regulated melanogenesis via its receptor. The use of specific receptor antagonists indicated that the S1P(3) receptor was dominantly involved in S1P-induced ERK activation and hypopigmentation. CONCLUSIONS: The results suggested that S1P reduced melanin synthesis via S1P(3) receptor-mediated ERK and RSK-1 activation, and subsequent MITF dual phosphorylation and degradation.

Indole-3-carbinol and ultraviolet B induce apoptosis of human melanoma cells via down-regulation of MITF.

We investigated the mechanism of indole-3-carbinol (13C)/ultraviolet B (UVB)-induced apoptosis using SK-MEL-2 and SK-MEL-5 human melanoma cells. 13C/UVB significantly reduced the viability of SK-MEL-2 cells, whereas it had little influence on SK-MEL-5 cells. Correspondingly, cell cycle analysis showed that 13C/UVB induced a clear increase in the sub-G0/G1 phase in SK-MEL-2 cells. Furthermore, 13C/UVB activated caspase-9, caspase-8, caspase-3, and Bid and caused the cleavage of poly(ADP-ribose) polymerase (PARP) in SK-MEL-2 cells. In contrast, 13C/UVB showed no effects on the apoptotic signaling pathways in SK-MEL-5 cells. Moreover, we found that 13C down-regulated the microphthalmia-associated transcription factor (MITF) in SK-MEL-2 cells, but not in SK-MEL-5 cells. Next, to investigate the involvement of MITF in 13C/UVB-induced apoptosis, MITF silencing was conducted using small interfering RNA (siRNA) for MITF in SK-MEL-5 cells. Interestingly, 13C/UVB dramatically decreased the viability of MITF-down-regulated SK-MEL-5 cells. These results indicate that MITF plays a critical role in melanoma cell survival.

Sphingosylphosphorylcholine inhibits melanin synthesis via pertussis toxin-sensitive MITF degradation.

OBJECTIVES: Sphingolipids act as structural components in cell membranes, and form lipid intermediates that have functional roles as signalling molecules in various cellular processes. Our previous findings have suggested that sphingolipid metabolites are deeply involved in the regulation of melanogenic processes. In this study we aimed to examine sphingosylphosphorylcholine-mediated signalling pathways related to melanogenesis. METHODS: We determined the hypopigmenting effects and the related signalling pathways of sphingosylphosphorylcholine in Mel-Ab cells. In particular, we analysed the involvement of the G-protein-coupled receptor in sphingosylphosphorylcholine-induced MITF degradation. KEY FINDINGS: Western blotting revealed that sphingosylphosphorylcholine induced the activation of extracellular signal-regulated kinase (ERK), as well as Akt. Moreover, the specific Akt pathway inhibitor LY294002 blocked the hypopigmenting effect of sphingosylphosphorylcholine and abrogated the sphingosylphosphorylcholine-mediated down-regulation of microphthalmia-associated transcription factor (MITF), showing that the Akt pathway is involved in sphingosylphosphorylcholine-mediated melanin inhibition. Treatment with the proteasome inhibitor MG132 blocked the decrease in MITF by sphingosylphosphorylcholine, but sphingosylphosphorylcholine did not decrease the level of MITF mRNA, indicating that the reduction in the level of MITF results from MITF degradation. Furthermore, pre-incubation of Mel-Ab cells with pertussis toxin completely abolished the hypopigmenting effects and the activation of ERK and Akt by sphingosylphosphorylcholine, suggesting that the effects of sphingosylphosphorylcholine are mainly dependent on the G-protein-coupled receptor). CONCLUSIONS: Together, these results suggest that sphingosylphosphorylcholine reduces melanin synthesis via pertussis toxin-sensitive ERK and Akt activation, and subsequent MITF degradation.

Hypopigmentary action of dihydropyranocoumarin D2, a decursin derivative, as a MITF-degrading agent.

In this study, the decursin derivative dihydropyranocoumarin D2 (1) was selected for its effects on melanogenesis using a spontaneously immortalized mouse melanocyte cell line (Mel-Ab). The results showed that 1 effectively inhibited melanin synthesis in a concentration-dependent manner, but that it did not inhibit tyrosinase in a cell-free system. In addition, the changes in ERK, Akt, and microphthalmia-associated transcription factor (MITF) in response to treatment with 1 were assessed. The results revealed that ERK was dramatically up-regulated and MITF was down-regulated in response to treatment with 1, but that Akt was unchanged. Therefore, the effects of 1 on melanogenesis were examined in the absence or presence of PD98059 (a specific inhibitor of the ERK pathway). PD98059 restored hypopigmentation and the down-regulation of MITF induced by 1. Finally, MITF down-regulation by 1 was clearly restored by both chloroquine, a lysosomal proteolysis inhibitor, and MG132, a proteasome inhibitor.

Differential expression of p63 isoforms in normal skin and hyperproliferative conditions.

BACKGROUND: The p63 is regarded as a potential stem cell marker. METHODS: Expression of p63 isoforms was examined in normal skin and hyperproliferative conditions including psoriasis and artificial skin equivalents (SEs). Rapidly adhering (RA) and slowly adhering (SA) cells were isolated, and Western blotting was performed. RESULTS: Expression of p63 (4A4) and p63 (H-137) is similar in all conditions, although there is some variation in psoriasis. However, expression of p63alpha (C-12) is markedly different. In normal skin, p63alpha (C-12)-positive cells were scattered in whole epidermis. But in psoriasis, p63alpha (C-12)-positive cells were observed at the tips of rete ridges. In SEs, p63alpha (C-12)-positive cells were not well observed. Western blot results showed that the RA cells express p63 (4A4) and p63 (H-137) strongly compared with SA or nonadhering (NA) cells. In contrast, SA or NA cells strongly express p63alpha (C-12). CONCLUSIONS: Results suggest that both p63 (4A4) and p63 (H-137) can detect epidermal stem cells. But, p63 (H-137) seemed to be a better marker because p63 (H-137)-positive cells were more localized at basal layer. In addition, it can be said that p63alpha (C-12) can detect TAp63, which is important in differentiation of epidermis. Furthermore, it is concluded that molecular control of TAp63 is especially disorganized in hyperproliferative condition including psoriasis and SEs.

Long-term suppression of tyrosinase by terrein via tyrosinase degradation and its decreased expression.

Previously, we reported that a fungal metabolite, terrein, decreases melanin synthesis via downregulation of microphthalmia-associated transcription factor (MITF). In the present study, we further investigated the long-term hypopigmenting action of terrein in a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. Treatment with terrein at a concentration of 50 mum strongly decreased melanogenesis in a time-dependent manner. Interestingly, the decreased tyrosinase protein levels lasted for at least 7 days, even though the MITF protein levels were restored after 3 days of treatment. In accordance with the results of Western blot analyses, the tyrosinase mRNA levels were found to be continuously decreased for at least 7 days, even though recovery of the MITF mRNA levels began after 3 days of terrein treatment. Therefore, we evaluated tyrosinase downregulation to determine if it is caused by proteasomal degradation. We found that the reduction in tyrosinase levels that was induced by terrein was clearly recovered by MG-132, a proteasome inhibitor. Moreover, ubiquitination of tyrosinase increased following treatment with terrein in the presence of MG-132. Taken together, these results suggest that terrein decreases melanogenesis through ubiquitin-dependent proteasomal degradation as well as via decreased expression of its mRNA.

Tumor apoptosis by indole-3-acetic acid/light in B16F10 melanoma-implanted nude mice.

Recently, we reported that UVB-activated indole-3-acetic acid (IAA) induces the apoptosis of G361 human melanoma cells. In the present study, we used IAA and visible light combinations to treat B16F10 melanoma-implanted nude mice using an experimental intense pulsed light (IPL) therapy model. We first investigated whether activated IAA by horseradish peroxidase (HRP) or UVB causes apoptosis of B16F10 melanoma cells. IAA/HRP or IAA/UVB combination lead to apoptosis of B16F10 cells, as reported in other cell lines. Interestingly, IAA alone was not cytotoxic. These findings suggested the potential use of IAA in the treatment of melanoma. For the future clinical use, we also tested whether visible light has the same effects like UVB and found that visible light also activates IAA to produce free radicals and that IAA/visible light decreased cell viability significantly. Based on these results, IAA/IPL combination was tried whether it can induce apoptosis in vivo status. TUNEL staining showed that IAA/IPL treatment induced apoptosis of tumor cells. In addition, the expressions of p53, Fas, and PARP were upregulated in the IAA/IPL-treated group than in untreated control, demonstrating that IAA/IPL treatment caused apoptosis in melanoma-implanted nude mice. In conclusion, we showed that IAA/IPL induces melanoma regression in B16F10 melanoma-implanted nude mice. These results suggest the potential use of IAA/IPL in the treatment of malignant melanoma.

The hypopigmentary action of KI-063 (a new tyrosinase inhibitor) combined with terrein.

Resorcinol derivatives are known to inhibit melanin synthesis. In this study, resorcinol derivatives were synthesized and screened for their activity on melanogenesis. KI-063 (a tyrosinase inhibitor) was examined for its effects on melanogenesis using a spontaneously immortalized mouse melanocyte cell line (Mel-Ab). In a cell-free system, KI-063 directly inhibited tyrosinase, the rate-limiting melanogenic enzyme. Moreover, in a cell system, it inhibited melanin synthesis in a concentration-dependent manner. In addition, KI-063 inhibited the activity of cellular tyrosinase. Thus, this study examined the effects of a combination of KI-063 with terrein, an agent that down-regulates microphthalmia-associated transcription factor. The data suggest that KI-063 has an additive effect in combination with terrein. Thus, the suppression of tyrosinase activity by KI-063 and the inhibition of tyrosinase production by terrein appear to be an optimal combination for skin whitening.

Terrein inhibits keratinocyte proliferation via ERK inactivation and G2/M cell cycle arrest.

Terrein, a fungal metabolite, has been recently shown to have a strong antiproliferative effect on skin equivalents. In the present study, we further investigated the effects of terrein on the possible signalling pathways involved in the growth inhibition of human epidermal keratinocytes by examining the regulations of extracellular signal-regulated protein kinase (ERK) and of the Akt pathway by terrein. It was observed that ERK was inactivated by terrein and that keratinocyte proliferation was inhibited, whereas Akt was unaffected. The inhibition of the ERK pathway by U0126 (a specific ERK inhibitor) also had a dose-dependent antiproliferative effect on human keratinocytes. These results indicate that ERK inhibition is involved in keratinocyte growth inhibition by terrein. Moreover, flow cytometric analysis showed that terrein inhibits DNA synthesis, as evidenced by a reduction in the S phase and an increase in the G2/M phase of the cell cycle. Thus, we next examined changes in the expressions of G2/M cell cycle-related proteins. Terrein was found to downregulate cyclin B1 and Cdc2 without Cdc2 phosphorylation, but upregulated p27(KIP1) (p27), a known inhibitor of cyclin-dependent kinase. These results suggest that terrein reduces human keratinocyte proliferation by inhibiting ERK and by decreasing the expressions of cyclin B1 and Cdc2 complex.

Additive effects of heat and p38 MAPK inhibitor treatment on melanin synthesis.

It has been reported that the activation of extracellular signal-regulated kinase (ERK) reduces melanin synthesis. Recently, we also found that heat treatment induces ERK activation and inhibits melanogenesis in Mel-Ab cells (a mouse melanocyte cell line). In addition, it was reported that p38 MAPK (mitogen-activated protein kinase) inhibition blocks melanogenesis. Thus, we investigated the effects of heat and of the p38 MAPK inhibitor, SB203580, on melanogenesis. In this study, we found that heat treatment activates ERK and reduces melanin production in human melanocytes, and that this is accompanied by a reduction in tyrosinase activity. To regulate the ERK and p38 MAPK pathways simultaneously, we combined heat treatment and SB203580 and measured melanin synthesis. The results obtained showed that heat treatment and SB203580 reduced melanin synthesis more effectively than heat or SB203580 alone. We conclude that ERK activation and p38 MAPK inhibition can work in an additive manner to decrease melanogenesis.

A new 2-imino-1,3-thiazoline derivative, KHG22394, inhibits melanin synthesis in mouse B16 melanoma cells.

During our on-going attempts to develop a new skin-whitening agent, we identified a novel candidate compound KHG22394, a 2-imino-1,3-thiazoline derivative. Our data show that KHG22394 significantly inhibits melanin production in a dose-dependent manner, but that it does not directly inhibit tyrosinase, the rate limiting melanogenic enzyme. It has been reported that the activation of extracellular signal-regulated kinase (ERK) reduces melanin synthesis by downregulating microphthalmia-associated transcription factor (Mitf). Thus, we examined the effects of KHG22394 on the ERK pathway and found that it induced ERK and 90 kDa ribosomal S6 kinase (RSK-1) activation. Moreover, alpha-melanocyte-stimulating hormone (alpha-MSH) is known to increase melanin biosynthesis by increasing tyrosinase production, and here, we found that alpha-MSH-induced Mitf and tyrosinase increases were inhibited in B16 melanoma cells treated with KHG22394. These findings suggest that the hypopigmentary effect of KHG22394 results from the downregulation of Mitf and subsequently of tyrosinase, although KHG22394 did not inhibit tyrosinase activity directly. Our findings indicate that 2-imino-1,3-thiazoline derivatives are potential skin whitening agents.

Light-activated indole-3-acetic acid induces apoptosis in g361 human melanoma cells.

Indole-3-acetic acid (IAA) activation by horseradish peroxidase (HRP) has been suggested as a new cancer therapy. Interestingly, we found that ultraviolet B UVB radiation also can activate IAA and produce free radicals in a dose-dependent manner. In this study, we attempted to identify the free radicals generated by UVB-irradiated IAA (IAAUVB), and to determine whether IAAUVB can induce the apoptosis of G361 human melanoma cells. Since IAA/HRP produces reactive oxygen species (ROS), we examined whether IAAUVB-generated radicals include ROS. Our results show that IAAUVB-induced free radical production is not inhibited by catalase, superoxide dismutase, or sodium formate, indicating that ROS are not generated by IAAUVB. On the other hand, IAAUVB caused lipid peroxidation, and this was blocked by Trolox, a water-soluble vitamin E derivative. Moreover, we found that IAAUVB caused apoptotic cell death and that this was inhibited by a low temperature. We further investigated IAAUVB-mediated apoptotic pathways, and found that IAAUVB causes caspase-8, Bid, caspase-3 activation, and poly (ADP-ribose) polymerase (PARP) cleavage. In addition, these apoptotic pathways were also blocked by low temperature. From these results, we propose that IAAUVB-induced free radicals cause human melanoma cell apoptosis via a death receptor-mediated apoptotic pathway.

Indole-3-acetic acid/horseradish peroxidase-induced apoptosis involves cell surface CD95 (Fas/APO-1) expression.

Recently, we showed that a combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) produces hydrogen peroxide (H2O2), and that this leads to the apoptosis of G361 human melanoma cells. In the present study, flow cytometric analysis confirmed that H2O2 is involved the IAA/HRP-induced apoptotic process. We also found that IAA/HRP increases cell surface CD95 (Fas/APO-1) expression, and that this is blocked by catalase treatment. Furthermore, blocking CD95 with a neutralizing antibody significantly restored IAA/HRP-induced apoptosis. In addition, the IAA/HRP-induced activations of CD95 downstream molecules, i.e., caspase-8, Bid, and caspase-3, were also inhibited by catalase. Moreover, a caspase-8 inhibitor significantly blocked IAA/HRP-induced apoptosis. These results indicate that IAA/HRP-induced apoptosis involves a CD95-initiated death receptor signaling pathway initiated by hydrogen peroxide.

Differential regulation of melanosomal proteins after hinokitiol treatment.

BACKGROUND: Melanogenesis is regulated by a series of enzymes under the control of microphthalmia-associated transcription factor (MITF). OBJECTIVE: The aim of this study was to examine melanosome-associated protein levels in Mel-Ab cells after hinokitiol treatment. METHODS: We measured melanin contents and analyzed melanosome-associated protein levels using Western blot and RT-PCR analysis. RESULTS: Hinokitiol markedly inhibited melanin synthesis and also reduced the protein levels of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP-1), tyrosinase-related protein 2 (TYRP-2) and MITF in Mel-Ab cells. In addition, hinokitiol significantly increased the phosphorylations of extracellular signal-regulated kinases 1 and 2 (ERK1/2). Furthermore, reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that TYR and MITF mRNA levels were significantly decreased but that levels of TYRP-1 and TYRP-2 mRNA were unaffected by hinokitiol treatment. These results suggest that hinokitiol-induced ERK phosphorylation reduces MITF and TYR transcription, and mediates the action of hinokitiol on melanogenesis. Interestingly, the mRNAs of TYRP-1 and TYRP-2 were unaffected, although the protein levels of TYRP-1 and TYRP-2 were down-regulated. Thus, the effects of hinokitiol on the transcription of TYR may differ from its effects on TYRP-1 and TYRP-2. CONCLUSION: Therefore, we suggest that TYRP-1 and TYRP-2 may be regulated by post-translational degradation after hinokitiol treatment.

The fixation of living skin equivalents.

Histologic evaluations and immunohistochemical characterizations are important in studies of artificial organs such as skin equivalents. However, tissue compact organization is not easy to obtain when the artificial organ is constructed in vitro. Thus, appropriate fixation methods must be selected according to the compactness of the artificial organ and tissue engineering methodologies. The effects of several fixatives-Carnoy, Bouin's solution, formalin, paraformaldehyde, and paraformaldehyde-glutaraldehyde-were examined to select the best fixation method for preserving the structural and molecular markers of skin equivalents. Formalin-based fixatives ware found to be relatively free of the histologic problems (e.g., tissue shrinkage, poor structural preservation, weak stainability, and nonspecific immunolocalization) presented by the soft tissue fixatives (i.e., Carnoy or Bouin's solution). Unfortunately, the standard concentration of formalin induced detachment of epidermis from dermis, but this was prevented by reducing the concentration of the fixative. These findings suggest that fixation procedures should be selected for particular tissue and specific goals; in particular, they show that the paraformaldehyde-glutaraldehyde combination performed best in terms of preserving the histologic features of skin equivalents.

Sphingosylphosphorylcholine-induced ERK activation inhibits melanin synthesis in human melanocytes.

Sphingosylphosphorylcholine (SPC) is emerging as a potent signaling-lipid mediator. In this study, we investigated the effects of SPC on melanogenesis using cultured human melanocytes. Our results show that SPC significantly inhibits melanin synthesis in a concentration-dependent manner, and further that it reduces the activity of tyrosinase, the rate-limiting melanogenic enzyme. SPC treatment was also found to induce short-thick dendrites in human melanocytes, but not to reduce tyrosinase activity in a cell-free system, whereas kojic acid directly inhibited tyrosinase. These results suggest that SPC reduces pigmentation by indirectly regulating tyrosinase. In further experiments, SPC was found to downregulate microphthalmia-associated transcription factor (MITF) and tyrosinase, and Western blotting showed that SPC induces the activations of extracellular signal-regulated kinase (ERK) and 90 kDa ribosomal S6 kinase (RSK-1). Moreover, the specific ERK pathway inhibitor, PD98059, blocked the hypopigmentation effect of SPC, and abrogated the SPC-mediated downregulation of MITF. These results suggest that the ERK pathway is involved in the melanogenic signaling cascade, and that ERK activation by SPC reduces melanin synthesis via MITF downregulation.

Hydrogen peroxide is a mediator of indole-3-acetic acid/horseradish peroxidase-induced apoptosis.

Recently, we reported that a combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) induces apoptosis in G361 human melanoma cells. However, the apoptotic mechanism involved has been poorly studied. It is known that when IAA is oxidized by HRP, free radicals are produced, and since oxidative stress can induce apoptosis, we investigated whether reactive oxygen species (ROS) are involved in IAA/HRP-induced apoptosis. Our results show that IAA/HRP-induced free radical production is inhibited by catalase, but not by superoxide dismutase or sodium formate. Furthermore, catalase was found to prevent IAA/HRP-induced apoptotic cell death, indicating that IAA/HRP-produced hydrogen peroxide (H2O2) may be involved in the apoptotic process. Moreover, the antiapoptotic effect of catalase is potentiated by NADPH, which is known to protect catalase. On further investigating the IAA/HRP-mediated apoptotic pathway, we found that the IAA/HRP reaction leads to caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage, which was also blocked by catalase. Additionally, we found that IAA/HRP produces H2O2 and induces peroxiredoxin (Prx) sulfonylation. Consequently, our results suggest that H2O2 plays a major role in IAA/HRP-induced apoptosis.

Cytoprotective effect of green tea extract and quercetin against hydrogen peroxide-induced oxidative stress.

In this study, we evaluated the cytoprotective effects of antioxidative substances in hydrogen peroxide (H2O2) treated Mel-Ab melanocytes. Tested substances include selenium, quercetin, green tea (GT) extract, and several vitamins (ascorbic acid, Trolox, and folic acid). Of these, both quercetin and GT extract were found to have strong cytoprotective effects on H2O2-induced cell death. We also examined additive effects, but no combination of two of any of the above substances was found to act synergistically against oxidative damage in Mel-Ab cells. Nevertheless, a multi-combination of GT extract, quercetin, and folic acid appeared to prevent cellular damage in a synergistic manner, which suggests that combinations of antioxidants may be of importance, and that co-treatment with antioxidants offers a possible means of treating vitiligo, which is known to be related to melanocyte oxidative stress.