Young Mi Ha

Modulation of age-related NF-κB activation by dietary zingerone via MAPK pathway.

Zingerone, a major component found in ginger root, has been known as anti-mutagenic and anti-carcinogenic activities that are often associated with its anti-oxidative and anti-inflammatory activities. In recent studies, we examined molecular mechanism of zingerone treatment on pro-inflammatory NF-kappaB activation via the redox-related NIK/IKK and MAPK pathways. Action mechanism of zingerone on NF-kappaB signaling was investigated in aged rat kidney and endothelial cells. The results showed that zingerone had not only the antioxidant effect by constitutive suppression of ROS, but also anti-inflammatory effects by suppression of nuclear factor (NF)-kappaB activation in aged rat. In addition, zingerone treatment suppressed gene activation of pro-inflammatory enzymes, COX-2 and iNOS, which were upregulated with aging through NF-kappaB activation and IKK/MAPK signaling pathway. These experiments strongly indicate that zingerone treatment exerts a beneficial efficacy by suppressing both oxidative stress and age-related inflammation through the modulation of several key pro-inflammatory genes and transcription factors. Thus, the significance of our findings is that the zingerone treatment may provide some preventive measure against chronic inflammatory conditions that underlie many age-related inflammatory diseases, such as metabolic syndrome, cardiovascular disease, dementia, arthritis, diabetes, osteoprosis, and cancers.

Ginsenoside Rd inhibits the expressions of iNOS and COX-2 by suppressing NF-κB in LPS-stimulated RAW264.7 cells and mouse liver

Ginsenoside Rd is a primary constituent of the ginseng rhizome and has been shown to participate in the regulation of diabetes and in tumor formation. Reports also show that ginsenoside Rd exerts anti-oxidative effects by activating anti-oxidant enzymes. Treatment with ginsenoside Rd decreased nitric oxide and prostaglandin E2 (PGE2) in lipopolysaccharides (LPS)-challenged RAW264.7 cells and in ICR mouse livers (5 mg/kg LPS; LPS + ginsenoside Rd [2, 10, and 50 mg/kg]). Furthermore, these decreases were associated with the down-regulations of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 and of nuclear factor (NF)-κB activity in vitro and in vivo. Our results indicate that ginsenoside Rd treatment decreases; 1) nitric oxide production (40% inhibition); 2) PGE2 synthesis (69% to 93% inhibition); 3) NF-κB activity; and 4) the NF-κB-regulated expressions of iNOS and COX-2. Taken together, our results suggest that the anti-inflammatory effects of ginsenoside Rd are due to the down-regulation of NF-κB and the consequent expressional suppressions of iNOS and COX-2.

Inhibitory Effect of mTOR Activator MHY1485 on Autophagy: Suppression of Lysosomal Fusion

Autophagy is a major degradative process responsible for the disposal of cytoplasmic proteins and dysfunctional organelles via the lysosomal pathway. During the autophagic process, cells form double-membraned vesicles called autophagosomes that sequester disposable materials in the cytoplasm and finally fuse with lysosomes. In the present study, we investigated the inhibition of autophagy by a synthesized compound, MHY1485, in a culture system by using Ac2F rat hepatocytes. Autophagic flux was measured to evaluate the autophagic activity. Autophagosomes were visualized in Ac2F cells transfected with AdGFP-LC3 by live-cell confocal microscopy. In addition, activity of mTOR, a major regulatory protein of autophagy, was assessed by western blot and docking simulation using AutoDock 4.2. In the result, treatment with MHY1485 suppressed the basal autophagic flux, and this inhibitory effect was clearly confirmed in cells under starvation, a strong physiological inducer of autophagy. The levels of p62 and beclin-1 did not show significant change after treatment with MHY1485. Decreased co-localization of autophagosomes and lysosomes in confocal microscopic images revealed the inhibitory effect of MHY1485 on lysosomal fusion during starvation-induced autophagy. These effects of MHY1485 led to the accumulation of LC3II and enlargement of the autophagosomes in a dose- and time- dependent manner. Furthermore, MHY1485 induced mTOR activation and correspondingly showed a higher docking score than PP242, a well-known ATP-competitive mTOR inhibitor, in docking simulation. In conclusion, MHY1485 has an inhibitory effect on the autophagic process by inhibition of fusion between autophagosomes and lysosomes leading to the accumulation of LC3II protein and enlarged autophagosomes. MHY1485 also induces mTOR activity, providing a possibility for another regulatory mechanism of autophagy by the MHY compound. The significance of this study is the finding of a novel inhibitor of autophagy with an mTOR activating effect.

Design and synthesis of 5-(substituted benzylidene)thiazolidine-2,4-dione derivatives as novel tyrosinase inhibitors.

In continuing our search for novel tyrosinase inhibitors, a series of 5-(substituted benzylidene)thiazolidine-2,4-diones were rationally designed and synthesized, and their inhibitory effects on mushroom tyrosinase activity were evaluated. Twelve target compounds 2a-2l were designed and synthesized based on the structural characteristics of N-phenylthiourea, a tyrosinase inhibitor, and tyrosine and L-DOPA, the natural substrates of tyrosinase. Among them, (Z)-5-(4-hydroxybenzylidene)thiazolidine-2,4-dione (2a) and (Z)-5-(3-hydroxy-4-methoxybenzylidene)thiazolidine-2,4-dione (2f) exhibited much higher tyrosinase inhibitory activities, with IC(50) values of 13.36 and 9.87 μM, respectively, than kojic acid (IC(50) = 24.72 μM). Kinetic analysis of tyrosinase inhibition revealed that 2a and 2f are competitive inhibitors of mushroom tyrosinase. In addition, through prediction of the potato catechol oxidase tertiary structure and simulation of docking with compounds 2a and 2f using DOCK6, we found that these inhibitors likely bind to the active site of the enzyme. Docking simulation results suggested that 2a and 2f have high binding affinities with potato catechol oxidase. In addition, compounds 2a and 2f effectively inhibited tyrosinase activity and reduced melanin levels in B16 cells treated with α-melanocyte-stimulating hormone (α-MSH). These data strongly suggest that compounds 2a and 2f suppress the production of melanin via the inhibition of tyrosinase activity.

Evaluation of in vitro and in vivo anti-melanogenic activity of a newly synthesized strong tyrosinase inhibitor (E)-3-(2,4 dihydroxybenzylidene)pyrrolidine-2,5-dione (3-DBP).

BACKGROUND Tyrosinase inhibitors have become increasingly important because of their ability to inhibit the synthesis of the pigment melanin. A search for new agents with strong tyrosinase activity led to the synthesis of the tyrosinase inhibitor (E)-3-(2,4-dihydroxybenzylidene)pyrrolidine-2,5-dione (3-DBP). METHODS The inhibitory effect of 3-DBP on tyrosinase activity and melanin production was examined in murine melanoma B16F10 cells. Additional experiments were performed using HRM2 hairless mice to demonstrate the effects of 3-DBP in vivo. RESULTS The novel compound, 3-DBP, showed an inhibitory effect against mushroom tyrosinase (IC50=0.53 μM), which indicated that it was more potent than the well-known tyrosinase inhibitor kojic acid (IC50=8.2 μM). When tested in B16F10 melanoma cells treated with α-melanocyte stimulating hormone (α-MSH), 3-DBP also inhibited murine tyrosinase activity, which in turn induced a decrease in melanin production in these cells. The anti-melanogenic effect of 3-DBP was further verified in HRM2 hairless mice. The skin-whitening index (L value) of HRM2 hairless mice treated with 3-DBP before irradiation with UVB was greater than that of UVB-irradiated mice that were not treated with 3-DBP. GENERAL SIGNIFICANCE The newly synthesized 3-DBP has a potent inhibitory effect on tyrosinase. In addition to an in vitro investigation of the effects of 3-DBP on tyrosinase, in vivo studies using an HRM2 hairless mouse model demonstrated the anti-melanogenic potency of 3-DBP. Our newly synthesized 3-DBP showed efficient tyrosinase inhibitory effect in vivo and in vitro. Our finding suggests that 3-DBP can be an effective skin-whitening agent.

Analogs of 5-(substituted benzylidene)hydantoin as inhibitors of tyrosinase and melanin formation

BACKGROUND Many tyrosinase inhibitors find application in cosmetics and pharmaceutical products for the prevention of the overproduction of melanin in the epidermis. A series of 5-(substituted benzylidene)hydantoin derivatives 2a-2k were prepared, and their inhibitory activities toward tyrosinase and melanin formation were evaluated. METHODS The structures of the compounds were established using (1)H and (13)C NMR spectroscopy and mass spectral analyses. All the synthesized compounds were evaluated for their mushroom tyrosinase inhibition activity. RESULTS The best results were obtained for compound 2e which possessed hydroxyl group at R(2) and methoxy group at R(3), respectively. We predicted the tertiary structure of tyrosinase, simulated its docking with compound 2e and confirmed that this compound interacts strongly with mushroom tyrosinase residues as a competitive tyrosinase inhibitor. In addition, we found that 2e inhibited melanin production and tyrosinase activity in B16 cells. CONCLUSIONS Compound 2e could be considered as a promising candidate for preclinical drug development in skin hyperpigmentation applications. GENERAL SIGNIFICANCE This study will enhance understanding of the mechanism of tyrosinase inhibition and will contribute to the development of effective drugs for use hyperpigmentation.

Synthesis of novel azo-resveratrol, azo-oxyresveratrol and their derivatives as potent tyrosinase inhibitors

Ten azo compounds including azo-resveratrol (5) and azo-oxyresveratrol (9) were synthesized using a modified Curtius rearrangement and diazotization followed by coupling reactions with various phenolic analogs. All synthesized compounds were evaluated for their mushroom tyrosinase inhibitory activity. Compounds 4 and 5 exhibited high tyrosinase inhibitory activity (56.25% and 72.75% at 50 μM, respectively). The results of mushroom tyrosinase inhibition assays indicate that the 4-hydroxyphenyl moiety is essential for high inhibition and that 3,5-dihydroxyphenyl and 3,5-dimethoxyphenyl derivatives are better for tyrosinase inhibition than 2,5-dimethoxyphenyl derivatives. Particularly, introduction of hydroxyl or methoxy group into the 4-hydroxyphenyl moiety diminished or significantly reduced mushroom tryosinase inhibition. Among the synthesized azo compounds, azo-resveratrol (5) showed the most potent mushroom tyrosinase inhibition with an IC(50) value of IC(50)=36.28 ± 0.72 μM, comparable to that of resveratrol, a well-known tyrosinase inhibitor.

Characterization of a novel tyrosinase inhibitor, (2RS,4R)-2-(2,4-dihydroxyphenyl)thiazolidine-4-carboxylic acid (MHY384)

BACKGROUND We synthesized (2RS,4R)-2-(2,4-dihydroxyphenyl)thiazolidine-4-carboxylic acid (MHY384) as a potential tyrosinase inhibitor and investigated its antityrosinase activity. METHODS The structure of MHY384 was established using (1)H and (13)C NMR spectroscopy and mass spectral analyses. To investigate dual mechanisms of action of MHY384 for the inhibition of melanin synthesis, we confirmed the inhibitory effect of tyrosinase catalytic activity of MHY384. Then, we confirmed the inhibitory effect of MHY384 on transcription of tyrosinase mRNA through alpha-MSH-induced cAMP-PKA-MITF signaling. In addition, we supported the inhibitory mechanism of MHY384 against tyrosinase using a kinetic study and docking programs. RESULTS To determine how MHY384 regulates melanogenesis, we measured melanin levels and expression of the genes for microphthalmia-associated transcription factor (MITF) and tyrosinase in α-melanocyte-stimulating hormone (α-MSH)-induced B16F10 melanoma cells. MHY384 potently inhibited tyrosinase activity and melanin production in B16F10 melanoma cells. Through docking models, we were able to construct the tertiary structure of mushroom tyrosinase and simulate its docking with MHY384. The result supports that MHY384 strongly interacts with tyrosinase residues in the active site and it can directly inhibit tyrosinase. To investigate additional mechanisms of action of MHY384, we confirmed that the inhibition of tyrosinase activity was found to be due to the modulation of the expression of tyrosinase and its transcription factor, MITF, through cAMP, which regulates protein kinase A. CONCLUSIONS This study strongly indicates that the depigmenting effect of MHY384 results from the down-regulation of MITF and tyrosinase through direct tyrosinase inhibition. GENERAL SIGNIFICANCE Our findings suggest that MHY384 can be an effective skin-whitening agent.

Inhibitory effects of 6-(3-hydroxyphenyl)-2-naphthol on tyrosinase activity and melanin synthesis

As a part of an ongoing project searching for new skin-lightening agents, the inhibitory property of 6-(3-Hydroxyphenyl)-2-naphthol (HPN) on melanogenesis was investigated. The inhibitory action of HPN (IC50=15.2 μM) on mushroom tyrosinase was revealed. To further explore the action of HPN on melanogenesis, the inhibition of tyrosinase and melanin levels were measured in B16 melanoma cells (B16 cells). Results show that HPN inhibited tyrosinase activity and reduced melanin in B16 cells. Therefore, our data indicate HPN as a new candidate for depigmentation reagents.

Modulation of NF-κB and FOXOs by baicalein attenuates the radiation-induced inflammatory process in mouse kidney

Abstract The bioactive flavonoid baicalein has been shown to have radioprotective activity, although the molecular mechanism is poorly understood in vivo. C57BL/6 mice were irradiated with X-rays (15 Gy) with and without baicalein treatment (5 mg/kg/day). Irradiation groups showed an increase of NF-κB-mediated inflammatory factors with oxidative damage and showed inactivation of FOXO and its target genes, catalase and SOD. However, baicalein suppressed radiation-induced inflammatory response by negatively regulating NF-κB and up-regulating FOXO activation and catalase and SOD activities. Furthermore, baicalein inhibited radiation-induced phosphorylation of MAPKs and Akt, which are the upstream kinases of NF-κB and FOXOs. Based on these findings, it is concluded that baicalein has a radioprotective effect against NF-κB-mediated inflammatory response through MAPKs and the Akt pathway, which is accompanied by the protective effects on FOXO and its target genes, catalase and SOD. Thus, these findings provide new insights into the molecular mechanism underlying the radioprotective role of baicalein in mice.