Indomethacin inhibits melanogenesis via down-regulation of Mitf mRNA transcription.

Non-steroidal anti-inflammatory drugs (NSAIDs) exhibit several divergent biological effects. In this study, we investigated the effect of indomethacin on melanin synthesis using B16F1 melanoma cells. Indomethacin inhibited α-melanocyte stimulating hormone (α-MSH)-enhanced melanin synthesis in a dose-dependent manner. Western blotting analysis revealed that indomethacin significantly suppressed tyrosinase and Mitf protein levels. In a luciferase reporter assay, we found that indomethacin reduced tyrosinase promoter activity. Moreover, real-time RT-PCR analysis showed that indomethacin lowered mRNA levels of melanogenic genes, including Mitf. Together, our findings indicate that indomethacin inhibits melanogenesis via the suppression of Mitf transcription.

2-Ethoxybenzamide stimulates melanin synthesis in B16F1 melanoma cells via the CREB signaling pathway.

Non-steroidal anti-inflammatory drugs are frequently used for the treatment of inflammation, pain, and fever. In this study, we found that 2-ethoxybenzamide (ETZ) significantly enhanced melanin synthesis in B16F1 melanoma cells, and also induced melanosome formation. Therefore, we investigated the mechanism by which ETZ up-regulated melanin synthesis. Western blot analysis demonstrated that ETZ increased melanogenic protein levels, except that for TRP-2. Moreover, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR analyses showed that ETZ enhanced the mRNA levels of melanogenic genes, including microphthalmia-associated transcription factor and melanocortin 1 receptor. We also observed phosphorylation of cAMP response element-binding protein (CREB) following ETZ treatment. However, ETZ did not affect intracellular cAMP levels. ERK was also activated by ETZ treatment, and melanin content was enhanced upon treatment with the specific ERK inhibitor PD98059. Together, our results indicate that ETZ induces melanin synthesis via CREB phosphorylation.

Dual effects of acetylsalicylic acid on ERK signaling and Mitf transcription lead to inhibition of melanogenesis.

Acetylsalicylic acid (ASA) is widely used as an analgesic/antipyretic drug. It exhibits a wide range of biological effects, including preventative effects against heart attack and stroke, and the induction of apoptosis in various cancer cells. We previously found that ASA inhibits melanogenesis in B16 melanoma cells. However, the mechanisms of how ASA down-regulates melanin synthesis remain unclear. Here, we investigated the effect of ASA on melanogenic pathways, such as extracellular signal-regulated kinase (ERK) and microphthalmia-associated transcription factor (Mitf) transcription. ASA significantly inhibited melanin synthesis in a dose-dependent manner without oxidative stress and cell death. Semi-quantitative reverse transcription-polymerase chain reaction analysis showed that the inhibitory effect of ASA might be due to the inhibition of Mitf gene transcription. Interestingly, ASA also induced ERK phosphorylation. Additionally, treatment with PD98059, a specific ERK phosphorylation inhibitor, abolished the anti-melanogenic effect of ASA. These results suggest that the depigmenting effect of ASA results from down-regulation of Mitf, which is induced by both the induction of ERK phosphorylation and the inhibition of Mitf transcription.

Effect of monochromatic visible light on intracellular superoxide anion production and mitochondrial membrane potential of B16F1 and B16F10 murine melanoma cells.

We have investigated the effect of visible light on animal cells using light-emitting diodes to emit monochromatic visible light (red, yellow, green and blue light). To explore the relevant mechanism of apoptosis, we assessed the intracellular superoxide anion production and mitochondrial membrane potential (ΔΨm) of B16F1 and B16F10 murine melanoma cells after monochromatic light irradiation. Blue light caused ΔΨm depolarization subsequent to elevation of intracellular superoxide production. However, red and yellow light had no affect on both cell lines. Green light induced ΔΨm collapse only in B16F1 melanoma cells. ΔΨm is a key indicator of mitochondrial function, therefore its disruption causes mitochondria-dependent apoptosis. Thus, blue light causes mitochondrial dysfunction and subsequent cell death.

Complete sequencing and characterization of 21,243 full-length human cDNAs.

As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

Adverse Effect of Blue Light on DNA Integrity Is Accelerated by 5-Aminolevulinic Acid in HaCaT Human Keratinocyte Cells and B16F1 Murine Melanoma Cells.

Several studies have suggested the potential benefits of 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT). 5-ALA is a precursor of heme, which generates reactive oxygen species (ROS) following photoirradiation. Some reports indicate that blue light induces intracellular ROS production. In the present study, we elucidated the effects of blue light and 5-ALA on DNA integrity in B16F1 murine melanoma and human keratinocyte HaCaT cells using a variety of comet assay techniques. Co-treatment with blue light and 5-ALA significantly decreased cell viability in both cell lines. A neutral comet assay was performed to assess DNA double-strand break (DSB) formation and blue light and 5-ALA caused DSBs. We also performed an alkali comet assay to detect single-strand breaks (SSB) and alkali labile sites (ALS). The results indicated that 5-ALA accelerated blue light-induced SSB formation. In addition, modified comet assays were done using two types of enzymes to evaluate oxidative DNA damages. The results indicated that blue light and 5-ALA generated oxidized purine and pyrimidines in both cell lines. In summary, co-treatment with 5-ALA and photoirradiation may cause unexpected DNA damage in cells and tissues.

Blue light exposure enhances oxidative stress, causes DNA damage, and induces apoptosis signaling in B16F1 melanoma cells.

Studies have shown that visible light, specifically blue light, adversely affects cells, tissues, organs, and organisms. We investigated the effect of blue light on apoptosis, DNA integrity, and transcription of apoptotic and melanogenic genes using B16F1 melanoma cells. In this study, cells were irradiated with 2-50 W/m2 blue light (465 nm) for several time duration. Exposure to blue light decreased cell viability, but the pan-caspase inhibitor Z-VAD-FMK rescued blue light-induced cell death. Blue light also inhibited cell proliferation and arrested the cell cycle. Blue light-irradiated cells displayed several apoptotic features, like depolarized mitochondrial membranes and enhanced caspase-3 activity. Furthermore, blue light induced strand breaks in the genomic DNA in a dose- and time-dependent manner but did not induce the formation of cyclobutene pyrimidine dimers. The cell cycle inhibitor p21 and the pro-apoptotic gene Bax were upregulated in blue light-exposed cells, whereas the anti-apoptotic gene Bcl-2 and the apoptosis inhibitor survivin were downregulated. The key enzyme in melanin synthesis, tyrosinase, was upregulated after high-intensity (50 W/m2) blue light exposure and downregulated after low-intensity (0.2 W/m2) blue light exposure. Our study demonstrates that blue light triggers apoptosis and some of its effects are similar to those of ultraviolet radiation.

The inhibitory effect of non-steroidal anti-inflammatory drugs (NSAIDs) on the monophenolase and diphenolase activities of mushroom tyrosinase.

In the present work, we investigated the effect of non-steroidal anti-inflammatory drugs (NSAIDs) on the monophenolase and diphenolase activity of mushroom tyrosinase. The results showed that diflunisal and indomethacin inhibited both monophenolase and diphenolase activity. For monophenolase activity, the lag time was extended in the presence of diflunisal. In the presence of indomethacin, the lag time did not change. IC(50) values of monophenolase activity were estimated to be 0.112 mM (diflunisal) and 1.78 mM (indomethacin). Kinetic studies of monophenolase activity revealed that both diflunisal and indomethacin were non-competitive inhibitors. For diphenolase activity, IC(50) values were estimated to be 0.197 mM (diflunisal) and 0.509 mM (indomethacin). Diflunisal and indomethacin were also found to be non-competitive diphenolase inhibitors.

Salicylamide Enhances Melanin Synthesis in B16F1 Melanoma Cells.

Salicylamide, a non-steroidal anti-inflammatory drug (NSAID), is used as an analgesic and antipyretic agent. We have previously shown that several NSAIDs have anti-melanogenic properties in B16F1 melanoma cells. In contrast, we have found that salicylamide enhances melanin contents in B16F1 melanoma cells; however, the underlying mechanism is not known. Therefore, we investigated the mechanism through which salicylamide stimulates melanogenesis. Interestingly, salicylamide enhanced diphenolase activity in a cell-free assay. Western blotting and real-time RT-PCR revealed that salicylamide increased tyrosinase expression via transcriptional activation of the Mitf gene. Together, our results indicate that salicylamide could be used as an anti-hypopigmentation agent for skin and/or hair.

Effect of pyrroloquinoline quinone (PQQ) on melanogenic protein expression in murine B16 melanoma.

BACKGROUND: Increased production and accumulation of melanin leads to various hyperpigmentation disorders. Melanin synthesis is regulated by melanogenic proteins such as tyrosinase, tyrosinase-related protein (TRP)-1 and -2, and their transcription factors. OBJECTIVE: In this study, we assessed the effects of PQQ on melanogenic protein expression of murine B16 melanoma cells. METHODS: We assessed melanin production of PQQ-treated B16 melanoma cells. Furthermore, we investigated the effect of PQQ on the activity of melanogenic enzymes and their expression using Western blot and semi-quantitative RT-PCR analyses. RESULTS: In the present study, PQQ inhibited melanin synthesis in cultured melanoma cells stimulated by either alpha-melanocyte stimulating hormone (alpha-MSH) or 3-isobutyl-1-methylxanthine (IBMX). To elucidate the mechanism of the effect of PQQ on melanogenesis, we performed Western blotting for melanogenic proteins, such as tyrosinase, TRP-1, and TRP-2. PQQ inhibited tyrosinase expression, however, it did not inhibit TRP-2 expression. Used as the stimulant for melanogenesis, both alpha-MSH and IBMX gave the same results for melanogenic protein expression. Semi-quantitative RT-PCR analysis showed that the depigmentation effect of PQQ might be due to the inhibition of tyrosinase gene transcription but not the inhibition of microphthalmia-associated transcription factor (Mitf). CONCLUSION: This report indicates that PQQ is a possible anti-melanogenic agent and might be effective against hyperpigmentation disorders.

Depigmenting effect of catechins.

The aim of the present work was to clarify the anti-melanogenic mechanism of the catechin group. In this study, we used (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-catechin (C), and gallic acid (GA). The catechin group inhibited melanin synthesis in B16 melanoma cells. To elucidate the anti-melanogenic mechanism of the catechin group, we performed Western blotting analysis for crucial melanogenic protein, namely tyrosinase. The catechin group inhibited tyrosinase expression. These results indicate that the catechin group is a candidate anti-melanogenic agent and that it might be effective in hyperpigmentation disorders.

Depigmenting mechanisms of all-trans retinoic acid and retinol on B16 melanoma cells.

We assessed the effects of ATRA and retinol on melanogenesis in murine B16 melanoma cells. In the present study, ATRA and retinol inhibited melanin synthesis in melanoma cells stimulated by alpha-melanocyte stimulating hormone (alpha-MSH) or 3-isobutyl-1-methylxanthine (IBMX). To elucidate the target points of ATRA and retinol on melanogenesis, we performed western blotting for melanogenic proteins, such as tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. ATRA inhibited the expression of tyrosinase and TRP-1, and retinol inhibited the expression of tyrosinase, in a dose-dependent manner. Neither ATRA nor retinol inhibited TRP-2 expression. There were no differences in melanogenic protein expression between the two stimulants tested, alpha-MSH and IBMX. Therefore, the depigmenting effect of ATRA and retinol might be due to inhibition of the signaling pathway between cAMP and tyrosinase transcription bound to tyrosinase expression. These results indicate that ATRA and retinol are candidate anti-melanogenic agents that they might be effective in hyperpigmentation disorders.

Depigmenting mechanism of NSAIDs on B16F1 melanoma cells.

The aim of the present work was to clarify the anti-melanogenic mechanism of non-steroidal anti-inflammatory drugs (NSAIDs). Mefenamic acid, diclofenac, and nimesulide were used in this study, and these drugs inhibit melanin synthesis in B16F1 melanoma cells. To elucidate the anti-melanogenic mechanism of NSAIDs, we performed western blotting analysis for melanogenic proteins, such as tyrosinase, TRP-1, and TRP-2. All NSAIDs used in this study inhibited tyrosinase protein level. Semi-quantitative RT-PCR analysis showed that the depigmentation effect of mefenamic acid and nimesulide might be due to the inhibition of tyrosinase gene transcription. These results indicate that NSAIDs inhibit α-MSH-enhanced melanin synthesis, and are candidate anti-melanogenic agents since they might be effective in hyperpigmentation disorders.

Down-regulation of tyrosinase expression by acetylsalicylic acid in murine B16 melanoma.

Acetylsalicylic acid (aspirin; ASA) is widely used as an analgesic/antipyretic drug. ASA exhibits a wide range of biological effects, including preventative effects against heart attack, stroke, and the development of some types of cancer. However, the effects of ASA on melanogenesis are not well known. Therefore, we investigated the effect of ASA on melanin production using B16 murine melanoma cells and demonstrated a new biological effect of ASA. In the presence of alpha-melanocyte stimulating hormone (alpha-MSH), B16 melanoma cells are stimulated to enhance melanin synthesis. ASA (2 mM) inhibited alpha-MSH-enhanced melanin synthesis in melanoma more strongly than other well-known anti-melanogenic agents such as arbutin (2 mM) and kojic acid (200 microM). Interestingly, ASA did not inhibit the catalytic activity of mushroom tyrosinase (concentration range 0.5-4.0 mM). To clarify the target of ASA action in melanogenesis, we performed Western blotting for tyrosinase, which is a key melanogenic enzyme. ASA inhibited tyrosinase expression in a dose-dependent manner. Therefore, the depigmenting effect of ASA might be due to inhibition of tyrosinase expression or enhancement of tyrosinase degradation. This study suggests that ASA is a candidate anti-melanogenic agent and it might be effective in hyperpigmentation disorders.