• Proceedings of the Korean Society of Applied Pharmacology
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• 2007.11a
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• pp.79-92
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• 2007

Oxidative stress have known to be a risk factor for the degenerative processes and closely related to a lot of diseases. It is well established that antioxidants are good in protection and therapeutic means against oxidative damage. There is increasing interest in natural antioxidants and many natural antioxidants have been found and utilized as the possible protection for various diseases and skin aging. We have screened natural antioxidant agents for cosmeceuticals, nutraceuticals, and drugs as therapeutic and preventive means against oxidative stress, and have developed a number of novel antioxidants from various natural sources. A novel melanin synthesis inhibitor, Melanocin A, isolated from the metabolite of a fungal strain Eupenicillium shearii F80695 inhibited mushroom tyrosinase and melanin biosynthesis of B16 melanoma cells with $IC_{50}$ value of 9.0 nM and MIC value of $0.9\;{\mu}M$, respectively. Melanocin A also exhibited potent antioxidant activity by scavenging of DPPH and superoxide anion radicals. UV was found to increase the level of hydrogen peroxides and other reactive oxygen species (ROS) in skin tissues. This increase in ROS may not only alter the structure and function of many genes and proteins directly but may also modulate their expressions through signal transduction pathways and, ultimately, lead to skin damage. We investigated the effect of Melanocin A on UV-induced premature skin aging. Firstly, the effect of Melanocin A on UV-induced matrix metalloproteinase (MMP)-9 expression in an immortalized human keratinocyte cell line, HaCaT in vitro was investigated. Acute UV irradiation induced MMP-9 expression at both the mRNA and protein levels and Melanocin A suppressed this expression in a dose-dependent manner. We then investigated UV-induced skin changes in hairless mice in vivo by Melanocin A. Chronic exposure of hairless mouse dorsal skin to UV increased skin thickness and induced wrinkle formation and the gelatinase activities of MMP-2 and MMP-9. Moreover, Melanocin A significantly suppressed UV-induced morphologic skin changes and MMP-2 and MMP-9 expression. These results show that Melanocin A can prevent the harmful effects of UV that lead to skin aging. Therefore, we suggest that Melanocin A should be viewed as a potential therapeutic agent for preventing and/or treating premature skin aging. Terrein is a bioactive fungal metabolite isolated from Penicillium species. Terrein has a relatively simple structure and can be easily synthesized. However, the biologic effects of terrein are comparatively unknown. We found for the first time that terrein potently inhibit melanin production in melanocytes and has a strong hypopigmentary effect in a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. Treatment of Mel-Ab cells with terrein (10-100 mM) for 4 days significantly reduced melanin levels in a dose-dependent manner. In addition, terrein at the same concentration also reduced tyrosinase activity. We then investigated whether terrein influences the extracellular signal-regulated protein kinase (ERK) pathway and the expression of microphthalmia-associated transcription factor (MITF), which is required for tyrosinase expression. Terrein was found to induce sustained ERK activation and MITF down-regulation, and luciferase assays showed that terrein inhibits MITF promoter activity in a dose-dependent manner. To elucidate the correlation between ERK pathway activation and a decreased MITF transcriptional level, PD98059, a specific inhibitor of the ERK pathway, was applied before terrain treatment and found to abrogate the terrein-induced MITF attenuation. Terrein also reduced the tyrosinase protein level for at least 72 h. These results suggest that terrain reduces melanin synthesis by reducing tyrosinase production via ERK activation, and that this is followed by MITF down-regulation.

• Biomolecules & Therapeutics
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• v.30 no.2
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• pp.203-211
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• 2022

Melanogenesis is the production of melanin from tyrosine by a series of enzyme-catalyzed reactions, in which tyrosinase and DOPA oxidase play key roles. The melanin content in the skin determines skin pigmentation. Abnormalities in skin pigmentation lead to various skin pigmentation disorders. Recent research has shown that the expression of EMP2 is much lower in melanoma than in normal melanocytes, but its role in melanogenesis has not yet been elucidated. Therefore, we investigated the role of EMP2 in the melanogenesis of MNT1 human melanoma cells. We examined TRP-1, TRP-2, and TYR expression levels during melanogenesis in MNT1 melanoma cells by gene silencing of EMP2. Western blot and RT-PCR results confirmed that the expression levels of TYR and TRP-2 were decreased when EMP2 expression was knocked down by EMP2 siRNA in MNT1 cells, and these changes were reversed when EMP2 was overexpressed. We verified the EMP2 gene was knocked out of the cell line (EMP2 CRISPR/Cas9) by using a CRISPR/Cas9 system and found that the expression levels of TRP-2 and TYR were significantly lower in the EMP2 CRISPR/Cas9 cell lines. Loss of EMP2 also reduced migration and invasion of MNT1 melanoma cells. In addition, the melanosome transfer from the melanocytes to keratinocytes in the EMP2 KO cells cocultured with keratinocytes was reduced compared to the cells in the control coculture group. In conclusion, these results suggest that EMP2 is involved in melanogenesis via the regulation of TRP-2 expression.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.42 no.4
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• pp.403-412
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• 2016

The tight junction, one of Intercellular junctions, performs a variety of biological functions by bonding adjacent cells, including the barrier function to control the movement of the electrolyte and water. Recent studies have revealed that unusual expression of tight junction-related genes have been shown to be related in cancer development and progression. Recently, there are many reports that control of tight junction proteins expression is closely related to the skin moisture. In this study, we are focusing on the regulating mechanism of tight junction-associated genes by the steviol and its derivatives. Steviol, used as a sweetner, is known to chemical compound isolated from stevia plant. The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay was carried out in HaCaT cells (human keratinocyte cell line) in order to determine the cytotoxicity. As a result, while steviol showing cytotoxicity from $250{\mu}M$, steviol derivatives are not cytotoxic more than $250{\mu}M$ concentration. We have observed a change in the tight junction protein via quantitative real-time PCR. Claudin 8 among tight junction proteins is only significantly reduced up to 30% in the presence of steviol. In addition, cell migration was inhibited by steviol, not by stevioside and rebaudioside. Finally, we could observe that steviol, not stevioside and rebaudioside, is able to increase the skin barrier permeability through the transepithelial electric resistance (TEER) measurements. These results suggest that the steviol and its derivatives are specifically acts on the tight junction related gene expression, but steviol derivatives are more suitable as a cosmetic material.

• Journal of Life Science
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• v.25 no.9
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• pp.961-969
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• 2015

Epigallocatechin gallate (EGCG), the main catechin in green tea, has been shown to have some beneficial effects against various human diseases, including diabetes, neurodegenerative disorders, cancer, cardiovascular disease and obesity. To investigate the mechanism of the suppressive effects of EGCG on inflammatory response in macrophages, alterations on the levels of nitric oxide (NO) regulatory factors and inflammatory cytokines were measured in lipopolysaccharide (LPS)-activated RAW264.7 cells. No significant toxicity was detected in RAW264.7 cells treated with 100–400 μM EGCG. Moreover, the optimal concentration of LPS was determined to be 1 μg/ml based on the results of cell viability assay, NO assay and IL-6 enzyme-linked immunsorbent assay (ELISA). Furthermore, NO levels decreased significantly by 68.2% in the 400 μM EGCG/LPS treated group, while the level of inducible nitric oxide synthase (iNOS) expression decreased by 12-17% in the 200 and 400 μM EGCG/LPS treated group. A significant decrease in transcription of pro-inflammatory cytokines (TNF- α and IL-1β) and anti-inflammatory cytokine (IL-10) was also detected in the EGCG/LPS treated group. However, IL-6 transcript and protein was maintained at a constant level when in the LPS treated group relative to the EGCG/LPS treated group. Overall, these results suggest that the differential regulation of inflammatory cytokines is an important factor influencing the suppressive effects of EGCG against LPS-activated inflammatory response in RAW264.7 cells.

• Journal of Plant Biotechnology
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• v.31 no.3
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• pp.239-248
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• 2004

This experiment was carried out to confirm that phytochrome regulates anthocyanin bio-synthesis during cell suspension culture system of grape or not. In suspension culture of grape, maximum accumulation of anthocyanin was observed at the stationary phase under continuous white light condition. From mono-chromatic light interruption for 24h at the 4th or 7th day on the suspension cultured cells, the anthocyanin accumulation was highly enhanced at the light interruption at 7th day than 4th day under all monochromatic light treatment. However, the cell growth patterns were not affected by any light treatment. In the darkness, the anthocyanin synthesis was very low but remarkably increased by blue light or red light irradiation. However, the increase of anthocyanin accumulation by blue or red light was suppressed by far-red light in the suspension cells of grape. This suppression by far-red light on the anthocyanin synthesis also observed on the cells treated red or far-red light alternatively. These results implied that phytochrome regulation system may be involved in the anthocyanin biosynthesis of the suspension grape cells. By RNA expression analysis, chalcone synthase (CHS) gene was expressed highly by blue and red light but low by far-red light. The synergistic increase of CHS gene expression was also observed at the treatment of blue light followed by red for 24h. This result may explain the increase of anthocyanin accumulation in B/R treatment. Although the expression of phytochrome gene (PHYA or PHYB) was not highly increased by all light treatment (blue, red, and far-red light) the expression of both PHYA gene and PHYB gene was increased a little in cells treated red or far-red light. In grape suspension cells, the red light enhanced the anthocyanin synthesis, whereas the far-red light was suppressed. Although it was not confirmed whether or not phytochrome gene is activated in anthocyanin accumulating grape cells, we believed that anthocyanin biosynthesis in grape cells may be regulated under phytochrome signal transduction system.

• Horticultural Science & Technology
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• v.29 no.6
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• pp.623-632
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• 2011

To increase the anti-carcinogens phenylethylisothiocyanate (PEITC), myrosinase (MYR), and glutathione S-transferase (GST), genes related to PEITC pathway were isolated and the gene expressions were regulated by Agrobacterium transformation. Isolated cDNAs, MYR, and GST genes were 1,647 bp and 624 bp, respectively, and the protein expression was confirmed through pET system. Thereafter, we constructed a sense-oriented over-expressing myrosinase (pBMY) and RNAi down-regulated GST (pJJGST) binary vectors for the Chinese cabbage transformation. After the transformation, thirteen over-expressing transgenic Chinese cabbage plants (IMS) with pBMY and five down-regulated ones (IGA) with pJJGST were selected by PCR analysis. Selected $T_0$ transgenic plants were generated to $T_1$ plants by self-pollination. Based on the Southern blot analysis on these $T_1$ transgenic plants, 1-4 copies of T-DNA were transferred to Chinese cabbage genome. Thereafter, RNA expression level of myrosinase gene or GST gene was analyzed through real-time RT PCR of IMS, IGA, and non-transgenic inbred lines. In case of IMS lines, myrosinase gene was increased 1.03-4.25 fold and, in IGA lines, GST gene was decreased by 26.42-42.22 fold compared to non-transgenic ones, respectively. Analysis of PEITC concentrations using GC-MS it showed that some IMS lines and some IGA lines increased concentrations of PEITC up to 4.86 fold and up to 3.89 fold respectively compared to wild type. Finally in this study IMS 1, 3, 5, 12, and 15 and IGA 1, 2, and 4 were selected as developed transgenic lines with increasing quantities of anti-carcinogen PEITC.

• Microbiology and Biotechnology Letters
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• v.32 no.1
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• pp.72-77
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• 2004

Previously, we synthesized a novel Cyclin-dependent kinase inhibitor, MCS-5A. Also, we investigated the involvement of cell cycle regulatory events during MCS-5A-mediated apoptosis in HL-60(+p16/-p53) cells with up-regulation of p16 protein expression. In contrast, apoptosis was not observed in A549(-p16/+p53) cells. Therefore we propose that $p16^{INK4A}$ is a key enzyme for inducing apoptosis. In the present studies, we have explored the mechanism of $p16^{INK4A}$ -mediated cytotoxicity and the role of p16.sup INK4A/ overexpression in the induction of apoptosis in human tumor cells. The tumor suppressor gene $p16^{INK4A}$ is known as a cyclin-dependent kinase inhibitor (CKI) and cell cycle regulator. We expressed wild type $p16^{INK4A}$ in pcDNA3.1 vector and then transfected into non-small cell lung cancer (NSCLC) cell expressing different statue of p16$^{INK4A}$, p53 gene〔A549(-p16/+p53), H1299(-p16/-p53) and HeLa(+pl6/+p53) cell line〕. TUNEL assay (including propidium iodide staining following transfection of these cell line with pcDNA3.1-pl6) indicate that p16$^{INK4A}$-mediated cytotoxicity was associated with apoptosis. This is supported by studies demonstrating an induction of caspase 3 cleavage due to the transfection of A549, H1299 and HeLa cells with pcDNA3.1-pl6. These results suggest that p16$^{INK4A}$ has a new function of inducing apoptosis which is not related with the function of tumor suppressor gene p53.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.8
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• pp.1197-1206
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• 2016

Adipose tissue in the loin muscle area of beef cattle as a marbling factor is directly associated with beef quality. To elucidate whether properties of proteins involved in depot specific adipose tissue were sex-dependent, we analyzed protein expression of intramuscular adipose tissue (IMAT) and omental adipose tissue (OMAT) from Hanwoo cows, steers, and bulls of Korean native beef cattle by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis, quantitative polymerase chain reaction (PCR) and western blot analysis. Two different adipose depots (i.e. intramuscular and omental) were collected from cows (n = 7), steers (n = 7), or bulls (n = 7). LC-MS/MS revealed a total of 55 and 35 proteins in IMAT and OMAT, respectively. Of the 55 proteins identified, 44, 40, and 42 proteins were confirmed to be differentially expressed in IMAT of cows, steers, and bulls, respectively. In OMAT of cows, steers, and bulls, 33, 33, and 22 were confirmed to be differentially expressed, respectively. Tropomyosin (TPM) 1, TPM 2, and TPM3 were subjected to verification by quantitative PCR and western blot analysis in IMAT and OMAT of Hanwoo cows, steers, and bulls as key factors closely associated with muscle development. Both mRNA levels and protein levels of TPM1, TPM2, and TPM3 in IMAT were lower in bulls compared to in cows or steers suggesting that they were positively correlated with marbling score and quality grade. Our results may aid the regulation of marbling development and improvement of meat quality grades in beef cattle.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.194-194
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• 2017

Camelina (Camelina sativa L.) is a potential bio-energy crop that has short life cycle about 90 days and contains high amount of unsaturated fatty acid which is adequate to bio-diesel production. Enhancing environmental stress tolerance is a main issue to increase not only crop productivity but also big mass production. CsRCI2s (Rare Cold Inducible 2) are cold and salt stress related protein that localized at plasma membrane (PM) and assume to be membrane potential regulation factor. These proteins can be divide into C-terminal tail (CsRCI2D/E/F/G) or no-tail group (CsRCI2A/B/C/H). However, function of CsRCI2s are less understood. In this study, physiological responses and functional characterization of CsRCI2s of Camelina under salt stress were analyzed. Full-length CsRCI2s (A/B/E/F) and CsPIP2;1 sequences were confirmed from Camelina genome browser. Physiological investigations were carried out using one- or four-week-old Camelina under NaCl stress with dose and time dependent manner. Transcriptional changes of CsRCI2A/B/E/F and CsPIP2;1 were determined using qRT-PCR in one-week-old Camelina seedlings treated with NaCl. Translational changes of CsRCI2E and CsPIP2;1 were confirmed with western-blot using the antibodies. Water transport activity and membrane potential measurement were observed by cRNA injected Xenopus laevis oocyte. As results, root growth rate and physiological parameters such as stomatal conductance, chlorophyll fluorescence, and electrolyte leakage showed significant inhibition in 100 and 150 mM NaCl. Transcriptional level of CsPIP2;1 did not changed but CsRCI2s were significantly increased by NaCl concentration, however, no-tail type CsRCI2A and CsRCI2B increased earlier than tail type CsRCI2E and CsRCI2F. Translational changes of CsPIP2;1 was constitutively maintained under NaCl stress. But, accumulation of CsRCI2E significantly increased by NaCl stress. CsPIP2;1 and CsRCI2A/B/E/F co-expressed Xenopus laevis oocyte showed decreased water transport activity as 61.84, 60.30, 62.91 and 76.51 % at CsRCI2A, CsRCI2B, CsRCI2E and CsRCI2F co-expression when compare with single expression of CsPIP2;1, respectively. Moreover, oocyte membrane potential was significantly hyperpolarized by co-expression of CsRCI2s. However, higher hyperpolarized level was observed in tail-type CsRCI2E and CsRCI2F than others, especially, CsRCI2E showed highest level. It means transport of $Na^+$ ion into cell is negatively regulated by expression of CsRCI2s, and, function of C-terminal tail is might be related with $Na^+$ ion influx. In conclusion, accumulation of NaCl-induced CsRCI2 proteins are related with $Na^+$ ion exclusion and prevent water loss by CsPIP2;1 under NaCl stress.

• The Korean Journal of Food And Nutrition
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• v.26 no.4
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• pp.806-813
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• 2013

Agrimonia pilosa Ledeb. is a medicinal plant with anti-tumor, anti-oxidant, anti-inflammatory and anti-hyperglycemic activities. However, few studies of the anti-diabetic effect of A. pilosa on insulin resistance status have been performed. In the present study, the anti-diabetic effect of A. pilosa water extract (AP) was determined by investigating its ${\alpha}$-glucosidase inhibitory property, glucose utilization, and uptake, as well as insulin resistance mechanism of action in C2C12 skeletal muscle cells. Compared to positive control (acarbose), AP ($10mg/m{\ell}$) showed a similar ${\alpha}$-glucosidase inhibitory capacity. Glucose uptake was significantly increased by $1{\mu}m$ insulin treatment (p<0.05). However, palmitic acid (FFA, 1 mM) induced muscle insulin resistance and glucose uptake dysfunction. On the other hand, AP ($10{\mu}g/m{\ell}$) was capable of reversing the FFA-induced insulin resistance in C2C12 myotubes. Compared to control, AP ($100{\mu}g/m{\ell}$ without insulin) significantly increased the utilization of glucose (p<0.05) in C2Cl2 myotubes cultured in normal glucose (7 mM). AP treatment significantly increased the relative mRNA and protein expression levels of Akt. In particular, the effect of A. pilosa on the insulin signaling system is associated with the up-regulation of Akt genes and glucose uptake in C2Cl2 myotubes. These results suggest that A. pilosa is useful in the prevention of diabetes and the treatment of hyperglycemic disorders.