• 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 Korean Society of Grassland and Forage Science
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• v.43 no.4
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• pp.206-215
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• 2023

This study was performed to investigate immune changes by comparing the proportion and function of immune cells in the blood under high-temperature period and convalescence temperature period in Holstein dairy cows. The experiment was conducted using Holstein dairy cows of five animals per group (60 ± 20 months old, 175 ± 78 non-day) from the National Institute of Animal Science at high-temperature period (THI: 76 ± 1.2) and convalescence temperature period (THI: 66 ± 1.3). Complete blood count results showed no change in the number of immune cells between groups. In the analysis using Flow Cytometry of PBMCs, no significant differences were observed among B cells, Helper T cells, cytotoxic T cells, and γδ T cells between groups. However, there was an increase in Th17 cells producing IL-17a, while Th1 cells decreased during the convalescence temperature period. The results of gene expression analysis using qRT-PCR in PBMCs revealed an increase in IL-10 during the convalescence temperature period, while a decrease in HSP70 and HSP90 was observed. In conclusion, the increased expression of IL-10 and the decrease in HSP expression suggest the possibility of a weak recovery from heat stress. However, the lack of observed changes in B cells, T cells, and other immune cells indicates incomplete recovery from heat stress during the convalescence temperature period.

• The Korean Journal of Nuclear Medicine
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• v.38 no.1
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• pp.85-98
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• 2004

Purpose: Cellular uptake of $^{99}mTc$-sestamibi (MIBI) and $^{99}mTc$-tetrofosmin (TF) is low in cancer cells expressing multidrug resistance(MDR) by p-glycoprotein(Pgp) or multidrug related protein(MRP). Verapamil is known to increase cellular uptake of MIBI in MDR cancer cells, but is recently reported to have different effects on tracer uptake in certain cancer cells. This study was prepared to evaluate effects of verapamil on cellular uptake of MIBI and TF in several cancer cells. Materials and Methods: Celluar uptakes of Tc-99m MIBI and TF were measured in erythroleukermia K562 cell, breast cancer MCF7 cell, and human ovarian cancer SK-OV-3 cells, and data were compared with those of doxorubicin-resistant K562(Ad) cells. RT-PCR and Western blot analysis were used for the detection of mdr1 mRNA and Pgp expression, and to observe changes in isotypes of PKC enzyme. Effects of verapamil on MIBI and TF uptake were evaluated at different concentrations upto $200{\mu}M\;at\;1{\times}10^6\;cells/ml\;at\;37^{\circ}C$. Radioactivity in supernatant and pellet was measured with gamma counter to calculate cellular uptake ratio. Toxicity of verapamil was measured with MTT assay. Results: Cellular uptakes of MIBI and TF were increased by time in four cancer cells studied. Co-incubation with verapamil resulted in an increase in uptake of MIBI and TF in K562(Adr) cell at a concentration of $100{\mu}M$ and the maximal increase at $50{\mu}M$ was 10-times to baseline. In contrast, uptakes of MIBI and TF in K562, MCF7, SK-OV3 cells were decreased with verapamil treatment at a concentration over $1{\mu}M$. With a concentration of $200{\mu}M$ verapamil, MIBI and TF uptakes un K562 cells were decreased to 1.5 % and 2.7% of those without verapamil, respectively. Cellular uptakes of MIBI and TF in MCF7 and SK-OV-3 cells were not changed with $10{\mu}M$, but were also decreased with verapamil higher than $10{\mu}M$, resulting 40% and 5% of baseline at $50{\mu}M$. MTT assay of four cells revealed that K562, MCF7, SK-OV3 were not damaged with verapamil at $200{\mu}M$. Conclusion: Although verapamil increases uptake of MIBI and TF in MDR cancer cells, cellular uptakes were further decreased with verapamil in certain cancer cells, which is not related to cytotoxicity of drug. These results suggest that cellular uptakes of both tracers might differ among different cells, and interpretation of changes in tracer uptake with verapamil in vitro should be different when different cell lines are used.

• Nuclear Medicine and Molecular Imaging
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• v.42 no.5
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• pp.383-393
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• 2008

Purpose: Cancer specific killing can be achieved by therapeutic gene activated by cancer specific promotor. Expression of sodium iodide symporter (NIS) gene causes transportation and concentration of iodide into the cell, therefore radioiodine treatment after NIS gene transfer to cancer cell could be a form of radionuclide gene therapy. luciferase (Luc) gene transfected cancer cell can be monitored by in vivo optical imaging after D-luciferin injection. Aims of the study are to make vector with both therapeutic NIS gene driven by AFP promoter and reporter Luc gene driven by CMV promoter, to perform hepatocellular carcinoma specific radiodiodine gene therapy by the vector, and assessment of the therapy effect by optical imaging using luciferase expression. Materials and Methods: A Vector with AFP promoter driven NIS gene and CMV promoter driven Luc gene (AFP-NIS-CMV-Luc) was constructed. Liver cancer cell (HepG2, Huh-7) and non liver cancer cell (HCT-15) were transfected with the vector using liposome. Expression of the NIS gene at mRNA level was elucidated by RT-PCR. Radioiodide uptake, perchlorate blockade, and washout tests were performed and bioluminescence also measured by luminometer in these cells. In vitro clonogenic assay with 1-131 was performed. In vivo nuclear imaging was obtained with gamma camera after 1-131 intraperitoneal injection. Results: A Vector with AFP-NIS-CMV-Luc was constructed and successfully transfected into HepG2, Huh-7 and HCT-15 cells. HepG2 and Huh-7 cells with AFP-NIS-CMV-Luc gene showed higher iodide uptake than non transfected cells and the higher iodide uptake was totally blocked by addition of perchlorate. HCT-15 cell did not showed any change of iodide uptake by the gene transfection. Transfected cells had higher light output than control cells. In vitro clonogenic assay, transfected HepG2 and Huh-7 cells showed lower colony count than non transfected HepG2 and Huh-7 cells, but transfected HCT-15 cell did not showed any difference than non transfected HCT-15 cell. Number of Huh-7 cells with AFP-NIS-CMV-Luc gene transfection was positively correlated with radioidine accumulation and luciferase activity. In vivo nuclear imaging with 1-131 was successful in AFP-NIS-CMV-Luc gene transfected Huh-7 cell xenograft on nude mouse. Conclusion: A Vector with AFP promoter driven NIS and CMV promoter driven Luc gene was constructed. Transfection of the vector showed liver cancer cell specific enhancement of 1-131 cytotoxicity by AFP promoter, and the effect of the radioiodine therapy can be successfully assessed by non-invasive luminescence measurement.

• Journal of Korean Medicine Rehabilitation
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• v.21 no.4
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• pp.23-40
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• 2011

Objectives: This study was designed to examine the effects of extracts of Ojeoksangamibang($W{\check{u}}j\bar{i}s\check{a}nji\bar{a}w\grave{e}if\bar{a}ng$) on the lipid lowering, anti-oxidation and concentration of proinflammatory cytokines and was investigated on hyperlipidemic rats. Methods: Male rats weighing $182.39{\pm}4.71g$ were fed high fat diet for 8 weeks and 36 rats(above 400 g) were divided into 4 groups. Each of 9 rats was divided a control group and experimental groups. We fed a control group of rats a basal diet and administered normal saline(100 mg/kg, 1 time/1 day) for 4 weeks. And we fed each experimental group of rats basal diet and administered an extract of Ojeoksangamibang($W{\check{u}}j\bar{i}s\check{a}nji\bar{a}w\grave{e}if\bar{a}ng$) extracts(100 mg/kg, 200mg/kg, 300 mg/kg, 300 mg/kg, 1 time/1 day) for 4 weeks. At the end of the experiment, the rats were sacrificed to determine their chemical composition. We measured lipid of plasma and liver, concentration of proinflmmatory cytokines, anti-oxidative activity and $TNF-{\alpha}$, Apo-B, Apo-E and leptin gene expression. Results: 1. Concentration of plasma free fatty(FFA) showed no significant difference in all the treatment groups. Concentration of plasma triglyceride(TG) showed a significant decrement in the 300 mg/kg in Ojeoksangamibang($W{\check{u}}j\bar{i}s\check{a}nji\bar{a}w\grave{e}if\bar{a}ng$) groups than that of control group. 2. Concentration of plasma total cholesterol showed a significant decrement in the 200 and 300 mg/kg in Ojeoksangamibang($W{\check{u}}j\bar{i}s\check{a}nji\bar{a}w\grave{e}if\bar{a}ng$) groups than that of control group. Concentration of plasma low density lipoprotein(LDL)-cholesterol showed a Significant decrement in the 300 mg/kg in Ojeoksangamibang($W{\check{u}}j\bar{i}s\check{a}nji\bar{a}w\grave{e}if\bar{a}ng$) groups than that of control group. Concentration of plasma high density lipoprotein(HDL)-cholesterol showed a significant increment in the 300 mg/kg in Ojeoksangamibang($W{\check{u}}j\bar{i}s\check{a}nji\bar{a}w\grave{e}if\bar{a}ng$) group. 3. Concentration of liver total cholesterol showed a tendence to decrease in Ojeoksangamibang($W{\check{u}}j\bar{i}s\check{a}nji\bar{a}w\grave{e}if\bar{a}ng$) groups. Concentration of liver TG showed a significant decrement in all Ojeoksangamibang groups than that of control group. 4. Concentration of plasma and liver thiobarbituric acid reactive substance(TBARS) showed a tendence to decrease in Ojeoksangamibang($W{\check{u}}j\bar{i}s\check{a}nji\bar{a}w\grave{e}if\bar{a}ng$) groups. 5. The values of glutathione peroxidase(GSH-Px), superoxide dismutase(SOD) and catalase(CAT) activity showed a significant increment in all Ojeoksangamibang($W{\check{u}}j\bar{i}s\check{a}nji\bar{a}w\grave{e}if\bar{a}ng$) groups than that of control group. 6. The values of plasma aspartate aminotransferase(AST) and alanine aminotransferase(ALT) activity showed no significant different in all treatment group. 7. Concentration of plasma $interleukin(IL)-1{beta}$ showed no significant difference in all the treatment groups. Concentration of plasma IL-6 showed a significant decrement in the 300 mg/kg in Ojeoksangamibang($W{\check{u}}j\bar{i}s\check{a}nji\bar{a}w\grave{e}if\bar{a}ng$) group than that of control group. Concentration of plasma tumor necrosis $factor-{\alpha}(TNF-{\alpha})$ a siginifant decrement in the 200 and 300 mg/kg in Ojeoksangamibang($W{\check{u}}j\bar{i}s\check{a}nji\bar{a}w\grave{e}if\bar{a}ng$) group than that of control group. However the concentration of plasma IL-10 in the 300 mg/kg Ojeoksangamibang($W{\check{u}}j\bar{i}s\check{a}nji\bar{a}w\grave{e}if\bar{a}ng$) groups showed a significant increment than that of control group. 9. In the analysis of reverse transcription-polymerase chain reaction(RT-PCR), gene expression of $TNF-{\alpha}$, Apo-B and Apo-E in the Ojeoksangamibang($W{\check{u}}j\bar{i}s\check{a}nji\bar{a}w\grave{e}if\bar{a}ng$) groups showed a lower expression than that of control group. However the gene expression of leptin showed no difference in the treatment groups. 10. The ratio of $TNF-{\alpha}$, Apo-B, and Apo-E per ${\beta}-actin$ expression in the Ojeoksangamibang($W{\check{u}}j\bar{i}s\check{a}nji\bar{a}w\grave{e}if\bar{a}ng$) groups showed a significant decrement than that of control group. However The ratio of leptin expression per ${\beta}-actin$ expression showed no significant difference among all the treatment groups. Conclusions: According to above results, in lowering lipid effect, anti-oxidation and control of pro-inflammatory cytokines production, Ojeoksangamibang($W{\check{u}}j\bar{i}s\check{a}nji\bar{a}w\grave{e}if\bar{a}ng$) gives effect.

• Tuberculosis and Respiratory Diseases
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• v.58 no.6
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• pp.590-599
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• 2005

Object : Cigarette smoking is a major cause of mucus hypersecretion, which is a pathophysiological feature of many inflammatory airway diseases. Mucins, which are an important part of the airway mucus, are synthesized from the Muc gene in airway epithelial cells. However, the signaling pathways for cigarette smoke-induced mucin synthesis are unknown. The aim of this study was to determine the signal pathway for smoking induced Muc5ac gene expression. Methods : A549 cells were cultured and transiently transfected with the Muc5ac promoter fragment. These cells were stimulated with 5% cigarette smoke extract (CSE) alone or with CSE after a pretreatment with various signal transduction pathway inhibitors (AG1478, PD98059 and SB203580). The Muc5ac promoter activity was examined using the luciferase reporter system, and the level of phosphorylated EGFR, ERK1/2, p38 MAPK and JNK were all examined using Western blot analysis. Muc5ac mRNA expression was also examined using reverse transcriptase polymerase chain reactions (RT-PCR). Results : 1. The peak level of luciferase activity of the Muc5ac promoter was observed at 5% concentration and after 3 hours of incubation with the CSE. The level of EGFR phosphorylation and the luciferase activity of the transfected cells caused by the CSE were significantly suppressed by AG1478 or PD98059 (P<0.01). 2. CSE phosphorylated ERK1/2 or p38 MAPK but not JNK. The Muc5ac mRNA expression level was increased by the CSE but that was suppressed by PD98059 or AG1478. 3. The CSE-induced phosphorylation of ERK1/2 was blocked by PD98059 and that of p38 MAPK was blocked by either PD98059 or SB203580. Either PD98059 or SB203580 suppressed the luciferase activity of the transfected cells (P<0.0001). Conclusion : The Muc5ac mRNA expression level was increased by the CSE. The increased CSE-induced transcriptional activity was mediated via EGF receptor activation, which led to ERK1/2 and p38 MAPK phosphorylation.