• The Journal of the Korean bone and joint tumor society
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• v.15 no.2
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• pp.111-121
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• 2009

Background: Osteosarcoma is one of the most common primary malignant tumors of bone occurring mainly in children and adolescents. Although surgery combined with chemotherapy has markedly improved patient survival during the last years, the use of anticancer drugs is still associated with serious problem, such as the frequent acquisition of drug-resistant phenotypes and occurrence of "secondary malignancies". Several solid tumors display enhanced expression of matrix metalloproteinases (MMPs), and recently clinical trials have been initiated on MMP-inhibitors. On the other hand, bisphosphonates (BPs) are inhibitors of bone resorption, and widely used to treat osteoclast-mediated bone diseases. Also they appear to possess direct antitumor activity. Methods: One osteosarcoma cell line (U2OS) was treated with ibandronate (0, 0.1, 1, $10{\mu}M$) for 48 hours. Cell viabilities were determined using MTT assay, the mRNA levels of MMP-2 and MT1-MMP were detected by reverse-transcription polymerase chain reaction, the amount of MMP-2 and MT1-MMP protein were measured by Westernblot, the activities of MMP-2 were observed by Gelatin zymography, and Matrigel invasion assays were used to investigate the invasive potential of osteosarcoma cell lines before and after ibandronate treatment. Results: The invasiveness of U2OS cell line was reduced dose-dependently following 48 hour treatment of up to $10{\mu}M$ of the ibandronate at which concentration no cytotoxicity occurred. Furthermore, the gelatinolytic activities and protein and mRNA levels of MMP-2 and MT1-MMP were also suppressed by increasing ibandronate concentrations. Conclusion: Given that MMP-2 is instrumental in tumor cell invasion, it is very likely that the reduction in osteosarcoma cell invasion by ibandronate is a consequence, at least in part, of suppressed expression of both MMP-2 and MT1-MMP. Isolation of a molecule (s) responsible for the bisphosphonate inhibition of tumor cell invasion would pave the way for the development of a new generation of metastasis inhibitors.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.30 no.1
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• pp.63-71
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• 2004

The human skin is constantly exposed to environmental irritants such as ultraviolet, smoke, chemicals. Free radicals and reactive oxygen species (ROS) caused by these environmental facts play critical roles in cellular damage. These irritants are in themselves damaging to the skin structure but they also participate the immensely complex inflammatory reaction. The purpose of this study was to investigate the skin cell protective effect of Juniperus chinensis xylem extract on the UV and SLS-induced skin cell damages. We tested free radical and superoxide scavenging effect in vitro. We found that Juniperus chinensis xylem extracts had potent radical scavenging effect by 98％ at 100 $\mu\textrm{g}$/mL. Fluorometric assays of the proteolytic activities of matrix metalloproteinase-l(MMP-1, collagenase) were performed using fluorescent collagen substrates. UV A induced MMP-1 synthesis and activity were analyzed by enzyme-linked immunosorbent assay (ELISA) and gelatin-based zymography in skin fibroblasts. The extract of Juniperus chinensis showed strong inhibitory effect on MMP-1 activities by 97％ at 100 $\mu\textrm{g}$/mL and suppressed the UVA induced expression of MMP-1 by 79％ at 25 $\mu\textrm{g}$/mL. This extract also showed strong inhibition on MMP-2 activity in UVA irradiated fibroblast by zymography. We also examined anti-inflammatory effects by the determination test of proinflammatory cytokine, interleukin 6 in HaCaT keratinocytes. In this test Juniperus chinensis decreased expression of interleukin 6 about 30％. Expression of prostaglandin E$_2$, (PGE$_2$) after UVB irradiation was measured by competitive enzyme immunoassay (EIA) using PGE$_2$ monoclonal antibody. At the concentrations of 5-50 $\mu\textrm{g}$/mL of the extracts, the production of PGE$_2$ by HaCaT keratinocytes (24 hours after 10 mJ/$\textrm{cm}^2$ UVB irradiation) was significantly inhibited in culture supernatants (p〈0.05). The viability of cultured HaCaT keratinocytes was significantly reduced at the doses of above 10 mJ/$\textrm{cm}^2$ of UVB irradiation, but the presence of these extracts improved cell viability comparing to control after UVB irradiation. We also investigated the protective effect of this extract in sodium lauryl sulfate (SLS)-induced irritant skin reactions from 24 hour exposure. Twice a day application of the extract for reducing local inflammation in human skin was done. Irritant reactions were assessed by various aspects of skin condition, that is, erythema (skin color reflectance) and transepidermal water loss (TEWL). After 5 days the extract was found to reduce SLS-induced skin erythema and improve barrier regeneration when compared to untreated symmetrical test site. In conclusion, our results suggest that Juniperus chinensis can be effectively used for the prevention of UV and SLS-induced adverse skin reactions such as radical production, inflammation and skin cell damage.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.30 no.3 s.47
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• pp.393-397
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• 2004

L-Carnitine $({\beta}-hydroxy-{\gamma}-trimethyl-ammoniumbutyric{\;}acid)$ is a small water-soluble molecule important in mammalian fat metabolism. It is essential for the normal oxidation of fatty acids by the mitochondria, and is involved in the trans-esterification and excretion of acyl-CoA esters. In this paper, to investigate the relationship between aging and L-carnitine, we investigated the effects of in vitro matrix-metalloproteinase (MMP) inhibition and activity and expression of UYA-induced MMPs in human skin fibroblasts. Also, we studied to develop as anti-aging cosmetics with L-carnitine. Fluorometric assays of the proteolytic activities of MMP-1 (collagenase) were performed using fluorescent collagen substrates. ELISA (enzyme linked immune sorbent assay), gelatin-substrate zymography, RT-PCR ELISA techniques were used for the effects of L-carnitine on MMP expression, activity, and MMP mRNA expression in UVA irradiated fibroblast $(5\;J/cm^2)$, respectively. In addition, we performed clinical study with L-carnitine cream. L-carnitine inhibited the activities of MMP-1 in a dose-dependent manner and the $IC_{50}$ values calculated from semi-log plots were 2.45 mM, and L-carnitine showed strong inhibition on MMP-2 (gelatinase) activity in UVA irradiated fibroblast by zymography. Also, UVA induced MMP-1, 2 expression was reduced $43\%,\;53\%$ by treated with L-carnitine at 1.25 mM, and MMP-1 mRNA expression was reduced dose-dependent manner. Therefore L-carnitine was able to significantly inhibit the MMP activity, and regulate MMP expression in protein and mRNA level. The results of clinical study showed that $1.0\%$ L-carnitine treated group reduced wrinkle significantly compared with placebo treated group (P<0.05). All these results suggest that L-carnitine may be useful as new anti-aging cosmetics for protection against UVA induced Mm expression and activity.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.30 no.2
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• pp.227-233
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• 2004

Chronic exposure to solar radiation, particularly ultraviolet (UV) light, causes a variety of adverse reactions on human skin, such as sunburn, photoaging and photocarcinogenesis. Free radicals and reactive oxygen species (ROS) caused by UV exposure or other environmental facts play critical roles in cellular damage. And, repeated-UV irradiation activated the expression of the matrix metalloproteinase (MMP) and induced skin irritation. Therefore, the development of effective and safe photoprotectants that can reduce and improve the skin damage has been required. The purpose of this study was to investigate the photo-protective effect of several chinese medical plants (Juniperus chinensis) on the UV -induced skin cell damages. We tested free radical and superoxide scavenging effect in vitro. Fluorometric assays of the proteolytic activities of MMP-1 (collagenase) were performed using fluorescent collagen substrates. UVA induced MMP-1 synthesis and activity were analyzed by enzyme-linked immunosorbent assay (ELISA) and gelatin-based zymography in skin fibroblasts. We also examined anti-inflammatory effects by the determination test of proinflammatory cytokine, interleukin 6 in HaCaT keratinocytes. Expression of prostaglandin E$_2$ (PGE$_2$) after UVB irradiation was measured by competitive enzyme immunoassay(EIA) using PGE$_2$ monoclonal antibody. In the human skin we tested anti-irritation effect on the SLS-induced damage skin after appling the extract containing emulsion. We found that Juniperus chinensis extract had potent radical scavenging effect by 98% at 100$\mu\textrm{g}$/mL. The extract of Juniperus chinensis showed strong inhibitory effect on MMP-1 activities by 97% at 100 $\mu\textrm{g}$/mL and suppressed the UVA induced expression of MMP-1 by 79% at 25$\mu\textrm{g}$/mL. This extract also showed strong inhibition on MMP-2 activity in UVA irradiated fibroblast by zymography. In the test of proinflammatory cytokines of human keratinocytes Juniperus chinensis extract decreased expression of interleukin 6 about 30%. The amount of PGE$_2$ by HaCaT keratinocytes was significantly increased at the doses of above 10 mJ/$\textrm{cm}^2$ of UVB (p < 0.05). At the concentrations of 3.2-25$\mu\textrm{g}$/mL of this extract, the production of PGE$_2$ by HaCaT keratinocytes (24 h after 10mJ/$\textrm{cm}^2$ UVB irradiation) was significantly inhibited in culture supernatants (p < 0.05). In SLS-induced skin irritation model in vivo, we found to reduce skin erythema and improve barrier recovery after appling Juniperus chinensis extract containing emulsion when compared to irritated non-treated and placebo-treated skin. Our results suggest that Juniperus chinensis extract can be effectively used for the prevention of UV and SLS-induced adverse skin reactions and applied as anti-aging and anti-irritation cosmetics.

• Tuberculosis and Respiratory Diseases
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• v.50 no.4
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• pp.426-436
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• 2001

Background : Chronic obstructive pulmonary disease(COPD) is one of the major contributors to morbidity and mortality among the adult population. Cigarette smoking(CS) is undoubtedly the single most important factor in the pathogenesis of COPD. However, its mechanism is unclear. The current hypothesis regarding the pathogenesis of COPD postulates that an imbalance between proteases and antiproteases leads to the destructive changes in the lung parenchyma. This study had two aims. First, to evaluate the effect of CS exposure on histologic changes of the lung parenchyme, and second, to evaluate the effect of CS exposure on the expression of the gelatinolytic enzymes in BAL fluid cells in guinea pigs. Methods : Two groups of five guinea pigs were exposed to the whole smoke of 20 commercial cigarettes per day, 5 hours/day, 5 days/week, for 6weeks, and 12 weeks, respectively, using a smoking apparatus. Five age-matched guinea pigs exposed to room air were used as controls. Five or more sections were microscopically extamined(${\times}400$) and the number of cellular infiltration of the alveolar wall was measured in order to evaluate the effect of CS exposure on the histologic changes of lung parenchyme. The statistical significance was analyzed by a linear regression method. To evaluate the expression of the gelatinolytic enzymes in intraalveolar cells, BAL fluid was obtained and the intraalveolar cells were separated by centrifugation (500 g for 10 min at $4^{\circ}C$). Two sets of culture plates were loaded with $1{\times}10^6$ intraalveolar cells. One plate, contained O.1mM EDTA, a inhibitor of matrix metalloproteases(MMPs), and the other plate had no EDTA. Both plates were incubated for 48 hours at $37^{\circ}C$. After incubation, gelatinolytic protease expression in the supernatants was analyzed by gelatin zymography. Results : At the end of CS exposure, the level of blood carboxy Hb had increased significantly(4.1g/dl in control group, 24g/dl immediately after CS exposure, 18g/dl 30 min after CS exposure, 15g/dl 1 hour after CS exposure). Alveolar inflammatory cells were identified in the CS exposed guinea pigs. The number of alveolar cellular cells observed in a microscopic field ($400{\times}$) was $121.4{\pm}7.2$, $158.0{\pm}20.2$, $196.8{\pm}32.8$, in the control, the 6 weeks, and the 12 weeks group, respectively. The increased extent of inflammatory cellular infiltration of the lung parenchema showed a statistically significant linear relationship with the duration of CS exposure(p=0.001, $r^2=0.675$). Several types of gelatinolytic enzymes in the intraalveolar cells of CS exposed guinea pigs were expressed, of which some were inhibited by EDT A. However, the gelatinolytic enzymes were not expressed in the control groups. Conclusion : CS exposure increases inflammatory cellular infiltration of the alveolar wall and the expression of gelatinolytic proteases in guinea pigs. EDTA inhibits some of the gelatinolytic proteases. These findings suggest a possibility that CS exposure may increase MMP expression in the lungs of guinea pigs.

• Journal of Korean Neurosurgical Society
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• v.59 no.2
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• pp.106-116
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• 2016

Objective : Protein disulfide isomerase (PDI) acts as a chaperone on the cell surface, and it has been reported that PDI is associated with the tumor cell migration and invasion. The aims of this study are to investigate the anti-migration effect of bacitracin, which is an inhibitor of PDI, and the associated factor in this process. Methods : U87-MG glioma cells were treated with bacitracin in 1.25, 2.5, 3.75, and 5.0 mM concentrations. Western blot with caspase-3 was applied to evaluate the cytotoxicity of bacitracin. Adhesion, morphology, migration assays, and organotypic brain-slice culture were performed to evaluate the effect of bacitracin to the tumor cell. Western blot, PCR, and gelatin zymography were performed to investigate the associated factors. Thirty glioma tissues were collected following immunohistochemistry and Western blot. Results : Bacitracin showed a cytotoxicity in 3rd (p<0.05) and 4th (p<0.001) days, in 5.0 Mm concentration. The cell adhesion significantly decreased and the cells became a round shape after treated with bacitracin. The migration ability, the expression of phosphorylated focal adhesion kinase (p-FAK) and matrix metalloproteinase-2 (MMP-2) decreased in a bacitracin dose- and time-dependent manner. The U87-MG cells exhibited low-invasiveness in the 2.5 mM, compared with the untreated in organotypic brain-slice culture. PDI was expressed in the tumor margin, and significantly increased with histological glioma grades (p<0.001). Conclusion : Bacitracin, as a functional inhibitor of PDI, decreased the phosphorylated FAK and the secreted MMP-2, which are the downstream of integrin and play a major role in cell migration and invasion, might become one of the feasible therapeutic strategies for glioblastoma.

• Journal of Life Science
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• v.26 no.4
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• pp.439-445
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• 2016

Soybean leaves, a Korean edible plant material, have been reported to prevent the development of osteoporosis and breast cancer. Based on this rational, soybean fallen leaves ethanolic extract (SBFL) was used for the experiment of cell invasion related to metastasis and antioxidant activity. The effect of SBFL on matrix metalloproteinases (MMPs) in human fibrosarcoma cells, HT1080 as well as its anti-oxidant activity was investigated in this study. The effect of SBFL on scavenging activity of reactive oxygen species was evaluated in vitro using lipid peroxidation assay,DPPH radical and reducing power assay. SBFL showed the positive effects on antioxidant activity, compared with vitamin C and vitamin E used as positive controls. Furthermore, SBFL showed cytotoxicity above 16 µg/ml in MTT assay. In particular, it was found that SBFL decreased the activation of MMP-9 stimulated by phorbol 12-myristate 13-acetae (PMA) and phenazine methosulfate (PMS). SBFL treatment increased the expression levels of p-FoxO-1 and SOD-1. Moreover, SBFL inhibited cell invasion stimulated by vascular endothelial growth Factor (VEGF). These results indicate that SBFL could inhibit cell invasion related to the activation of MMP-9 and oxidative stress, suggesting that it could be available as a main ingredient for prevention of metastasis.

• Analytical Science and Technology
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• v.28 no.6
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• pp.361-369
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• 2015

The precise mechanism underlying the therapeutic efficacy of an extraction powder of Angelica gigas (AGE) for the treatment of degenerative osteoarthritis was investigated in primary cultured rabbit chondrocytes and in a monosodium-iodoacetate (MIA)-induced osteoarthritis rat model. The treatment with AGE (50 μg/mL) effectively inhibited NF-B activation. The anti-inflammatory mechanism was clarified by gelatin zymography and western blotting measurements of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) activities. The AGE (50 μg/mL) treatment significantly reduced MMP-9 activity. The constituents of AGE— decursinol, decursin, and decursinol angelate—were determined by LC-MS/MS after a 24 hr treatment of rabbit chondrocytes. The contents of the major products, decursin and decursinol angelate, were 3.62±0.47 and 2.14 ±0.36 μg/mg protein, respectively in AGE-treated (50 μg/mL) rabbit chondrocytes. An in vivo animal study on rats fed a diet containing 25, 50, and 100 mg/kg AGE for 3 weeks revealed a significant inhibition of the MMPs in the MIA-induced rat articular cartilage. The genetic expression of arthritic factors in the articular cartilage was examined by RT-PCR of collagen Type I, collagen Type II, aggrecan, and MMP (MMP3, MMP-9, MMP13). Specifically, AGE up-regulated the expression of collagen Type I, collagen Type II, and aggrecan and inhibited MMP levels at all tested concentrations. Collectively, AGE showed a strong specific site of action on MMP regulation and protected against the degeneration of articular cartilage via cellular regulation of MMP expression both in vitro and in vivo.

• Korean Journal of Food Science and Technology
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• v.43 no.2
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• pp.213-219
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• 2011

The metastatic effect of Eupatorium japonicum extract (EJE) on MDA-MB-231 human breast cancer cells was investigated. MDA-MB-231 cells were treated with various concentrations of EJE (0, 5, 10 and $20{\mu}g/mL$). EJE inhibited cell migration, invasion and adhesion of MDA-MB-231 cells in dose-dependent manners. Gelatin zymography exhibited that EJE significantly down regulated secretion of matrix metalloproteinase (MMP)-9 and MMP-2. EJE decreased the protein levels of tissue inhibitor of metalloproteinase (TIMP)-1 but increased TIMP-2 levels. Additionally, EJE reduced the protein and mRNA levels of urokinase-type plasminogen activator (uPA), vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM). In several solvent fractions of EJE, the hexane fraction markedly decreased MDAMB-231 cell migration. Thus, these finding suggest that EJE may be a potential antimetastatic agent, which can considerably inhibit the metastatic and invasive capacity of breast cancer cells.

• Journal of the Korean Society of Food Science and Nutrition
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• v.26 no.2
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• pp.285-290
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• 1997

The insulin-like growth factors(IGFs) are bound to several binding proteins(IGFBPs) that appear to regulate IGF transport, receptor binding, and its action. The concentration of these peptides are altered by catabolic conditions. To determine IGF-I and IGFBP levels in noninsulin-dependent diabetes mellitus (NIDDM), sera was obtained from 5 patients and 7 controls. Serum levels of IGF-I in NIDDM were lower than those in either of the controls. By western immunoblot analysis, especially IGFBP-1 levels are increased, whereas IGFBP-3 levels decreased and their fragments was increased in NIDDM serum. IGFBP-3 proteolytic activity in NIDDM sera was inhibited by phenylmethylsulfonylfluoride (PMSF), aprotinin, and ethylenediaminetetraacetic acid(EDTA). This pattern of inhibition was consistent with a metal-dependent serine protease. By gelatin zymography, these proteolytic enzymes were identified as the size of 97 and 69 kDa. IGFBP-1, which is primarily insulin regulated, was increased in NIDDM and may modulate circulating IGF-I levels by regulating capillary passage of IGF-I. IGFBP-3 proteolysis markedly reduces its affinity for the IGFs, particularly for IGF-I. This accelerates their kinetics of dissociation, thereby increasing the proportions of IGF-I in free form and its availability to the cells.