• Archives of Pharmacal Research
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• 제25권6호
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• pp.903-909
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• 2002

Research in the cytokine field has grown exponentially in recent years, and the validity of such studies relies heavily on the appropriate measurement of levels of cytokines in various biological samples. Transforming growth factor (TGF)-$\beta$, a hormonally active polypeptide found in normal and transformed tissue, is a potent regulator of cell growth and differentiation. The most widely used bioassay for TGF-$\beta$ is the inhibition of the proliferation of mink lung epithelial cells. Though detection of [$^3$H]thymidine incorporation is more sensitive than the MTT assay, it presents some disadvantages due to the safety and disposal problems associated with radioisotopes. In this study, we attempted to ascertain the experimental conditions which could be used for measuring the in vitro biological activity of TGF-$\beta$ in a safer and more sensitive way compared with the currently available methods. We compared the commonly used method, the MTT assay, to the XTT assay using different parameters including cell number, incubation time and the wave length used for detecting the product. We examined the anti-proliferative activities of TGF-$\beta$ in three different cell lines: Mv-1-Lu mink lung epithelial cells, MCF10A human breast epithelial cells and H-ras-transformed MCF10A cells. Herein, we present an experimental protocol which provides the most sensitive method of quantifying the biological activity of TGF-$\beta$, with a detection limit of as low as 10 pg/ml: Mv-1-Lu or H-ras MCF10A cells ($1{\times}10^5/well$) were incubated with TGF-$\beta$ at $37^{\circ}C$ in a humidified $CO_2$ incubator for 24 hr followed by XTT treatment and determination of absorbance at 450 or 490 nm. Our results may contribute to the establishment of an in vitro bioassay system, which could be used for the satisfactory quantitation of TGF-$\beta$.

• Toxicological Research
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• 제32권3호
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• pp.225-230
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• 2016

Zingiber officinale Roscoe has been widely used as a folk medicine to treat various diseases, including cancer. This study aims to re-examine the therapeutic potential of co-administration of natural products and cancer chemotherapeutics. Candidate material for this project, ${\alpha}$-zingiberene, was extracted from Zingiber officinale Roscoe, and ${\alpha}$-zingiberene makes up $35.02{\pm}0.30%$ of its total essential oil. ${\alpha}$-Zingiberene showed low $IC_{50}$ values, $60.6{\pm}3.6$, $46.2{\pm}0.6$, $172.0{\pm}6.6$, $80.3{\pm}6.6$ (${\mu}g/mL$) in HeLa, SiHa, MCF-7 and HL-60 cells each. These values are a little bit higher than $IC_{50}$ values of general essential oil in those cells. The treatment of ${\alpha}$-zingiberene produced nucleosomal DNA fragmentation in SiHa cells, and the percentage of sub-diploid cells increased in a concentration-dependent manner in SiHa cells, hallmark features of apoptosis. Mitochondrial cytochrome c activation and an in vitro caspase-3 activity assay demonstrated that the activation of caspases accompanies the apoptotic effect of ${\alpha}$-zingiberene, which mediates cell death. These results suggest that the apoptotic effect of ${\alpha}$-zingiberene on SiHa cells may converge caspase-3 activation through the release of mitochondrial cytochrome c into cytoplasm. It is considered that anti-proliferative effect of ${\alpha}$-zingiberene is a result of apoptotic effects, and ${\alpha}$-zingiberene is worth furthermore study to develop it as cancer chemotherapeutics.

• Biomolecules & Therapeutics
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• 제23권1호
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• pp.71-76
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• 2015

Peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$) was identified as a cell-intrinsic regulator of Th17 cell differentiation. Th17 cells have been associated with several autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), inflammatory bowel disease (IBD), and collagen-induced arthritis. In this study, we confirmed $PPAR{\gamma}$-mediated inhibition of Th17 cell differentiation and cytokine production at an early stage. Treatment with ciglitazone, a $PPAR{\gamma}$ ligand, reduced both IL-$1{\beta}$-mediated enhancement of Th17 differentiation and activation of Th17 cells after polarization. For Th17 cell differentiation, we found that ciglitazone-treated cells had a relatively low proliferative activity and produced a lower amount of cytokines, regardless of the presence of IL-$1{\beta}$. The inhibitory activity of ciglitazone might be due to decrease of CCNB1 expression, which regulates the cell cycle in T cells. Hence, we postulate that a pharmaceutical $PPAR{\gamma}$ activator might be a potent candidate for treatment of Th17-mediated autoimmune disease patients.

• 동의생리병리학회지
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• 제18권4호
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• pp.990-994
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• 2004

Cancer is an intractable disease for humans to overcome. Recently natural products or Oriental prescriptions have been on the spotlight to develop anticancer agents with little side-effects and good efficacy. KamikeKyuktang has been used for the treatment of cancer in Oriental medicine. However, its anti-cancer mechanism still remains unclear. KMKKTE is an ethanol extract of KamikeKyuktang composed of 12 medicinal herbs. Anti-proliferative effects of KMKKT was investigated on Lewis lung carcinoma cell (LLC) and A549 (human lung cancer cells). Half-maximal inhibition of the LLC and A549 cell proliferation by KMKKTE was found approximately 125㎍/㎖ and 250㎍/㎖, respectively. It also effectively inhibited the proliferation of HUVEC cells treated by bFGF and VEGF up to 30% of control at 125㎍/㎖ and the cell migration to 80% at 25 ㎍/㎖ in concentration dependent manner. Tube formation of HUVEC cells on matrigel also was significantly suppressed from 25㎍/㎖ of KMKKTE. Taken together, these results demonstrate that KMKKTE has antiangiogenic activity and be applied to angiogenesis dependent cancers.

• 동의생리병리학회지
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• 제18권4호
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• pp.1071-1077
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• 2004

Mudanpi (Cortex Moutan Radicis; the root cortex of Paeonia suffruticosa Andrews) is an important Chinese crude drug used in many oriental prescriptions. 1,2,3,4,6-Penta-O-galloyl-beta-D-glucose (PGG), a major component of this crude drug, has been shown to possess potent antioxidant, anti-mutagenic and anti-proliferative effects. In this study, I examined whether PGG could protect Neuro 2A cells, a kind of neuronal cell lines, from oxidative damage through the induction of HO-1 expression and HO activity. Exposure of Neuro 2A cells to PGG (10-50μM) resulted in a concentration- and time-dependent induction of HO-1 mRNA, and protein expressions and heme oxygenase activity. PGG protected the cells from hydrogen peroxide-induced cell death. The protective effect of PGG on hydrogen peroxide-induced cell death was abrogated by zinc protoporphyrin IX (ZnPP IX), a HO inhibitor. These results indicate that PGG is a potent inducer of HO-1 and HO-1 induction is responsible for the PGG-mediated cytoprotection against oxidative damage.

• 생약학회지
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• 제35권2호통권137호
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• pp.164-170
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• 2004

The flavonoids are a very large and important group of polyphenolic natural products, which are united by their derivatization from the heterocycle, flavone. They are distributed in higher plants and occur widely in the fruits and vegetables that make up the human diet. They exhibit a wide range of biological properties, including antitumor, antiinflammatory, hepatoprotective, antimicrobial, insecticidal and estrogenic activities. They are also major components of many plant drugs and it is possible that they contribute to the curative properties. For the purpose of developing anticancer agent of natural origin, we have evaluated forty four kinds of naturally occurring flavonoids for the inhibitory activity upon the proliferation of cultured human tumor cells such as A549 (non small cell lung), SK-OV-3 (ovary), SK-MEL-2 (melanoma), XF498 (central nerve system) and HCT-15 (colon) in vitro.

• 대한한의학회지
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• 제27권4호
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• pp.48-56
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• 2006

Objectives : This study is aimed to elucidate anti-hepatoma activity of Coptis Chinensis Extract (CCE) and evaluate its effect on proliferation of human hepatoma Hep G2 cells. Methods : To identify CCE and control the quality, we performed fingerprinting by high-performance thin layer chromatography (HPTLC). To investigate effects of CCE on anti-hepatoma activity, we measured cytotoxicity against Hep G2 cells compared with treatment of paclitaxel and 5-fluorouracil (5-FU). To examine the mechanism of inhibitory effect of CCE on Hep G2 cell proliferation, cell cycle distribution was evaluated using fluorescent activated cell sorter (FACS) Result : CCE showed a significant effect that arrests Hep G2 cells at the G2/M phase of the cell cycle. CCE combined with paclitaxel inhibited synergistically cell growth of Hep G2 cells. Conclusion : CCE may present anticancer effects through inhibition of hepatocellular carcinoma (HCC) cell proliferation via G2/M arrest, and may be a useful anticancer agent for HCC.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1267-1274
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• 2004

Colostrum contains various kinds of cytokines including TGF-$\beta$ that has potent regulatory effects on cells of the immune system. We compared the levels of TGF-$\beta$1 and TGF-$\beta$2 in bovine and human colostrum. Based on the isoform-specific ELISA, bovine colostrum collected on day 1 post-delivery retained $53.71{\pm}29.55\;ng/ml$ of TGF-$\beta$1 and $40.41{\pm}21.78\;{\mu}g/ml$ of TGF-$\beta$2 (n=4), while in human, $381.45{\pm}158.24\;ng/ml$ of TGF-$\beta$1 and $41.47{\pm}9.63\;ng/ml$ of TGF-$\beta$2 (n=5). Thus, dominant TGF-$\beta$ isoforms were completely opposite between human and bovine colostrum samples. The concentrations of both isoforms declined as lactation proceeded. Biological activities of the colostrum samples were determined using an MV1LU cell line. Consistent with the result from the immunoassay, TGF-$\beta$1 in human and TGF-$\beta$2 in bovine colostrum were responsible for the anti proliferative activity against MV1LU cells. Furthermore, bovine colostrum increased IgA secretion by LPS-stimulated mesenteric lymph node (MLN) cells, and this effect was abrogated by either anti­TGF-$\beta$2 antibody or combined anti-TGF-$\beta$1/$\beta$2 antibody, but not by anti- TGF-$\beta$1 antibody alone. Similarly, TGF-$\beta$2 in bovine colostrum enhanced the Ig germ line (GL) promoter activity, which is the earliest event toward IgA isotype switching. Taken together, these results suggest that TGF-$\beta$ isoforms, differentially expressed in human and bovine colostrum, may promote IgA isotype production in the neonatal intestine.

• Reproductive and Developmental Biology
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• 제38권2호
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• pp.85-91
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• 2014

Despite many researches related with in-vitro culture of porcine spematogonial stem cells (SSCs), adherent culture system widely used has shown a limitation in the maintenance of porcine SSC self-renewal. Therefore, in order to overcome this obstacle, suspension culture, which is known to have numerous advantage over adherent culture, was applied to the culture of porcine SSCs. Porcine SSCs retrieved from neonatal testes were suspension-cultured for 5 days or 20 days, and characteristics of suspension-cultured porcine SSCs including proliferation, alkaline phosphatase (AP) activity, and self-renewal-specific gene expression were investigated and compared with those of adherent-cultured porcine SSCs. As the results, the suspension-cultured porcine SSCs showed entirely non-proliferative and significantly higher rate of AP-positive cells and expression of self-renewal-specific genes than the adherent-cultured porcine SSCs. In addition, long-term culture of porcine SSCs in suspension condition induced significant decrease in the yield of AP staining-positive cells on post-day 10 of culture. These results showed that suspension culture was inappropriate to culture porcine SSCs, because the culture of porcine SSCs in suspension condition didn't stimulate proliferation and maintain AP activity of porcine SSCs, regardless of culture periods.

• Journal of Periodontal and Implant Science
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• 제31권1호
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• pp.135-148
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• 2001

Gingival fibroblasts are major cellular component of gingiva. However, the molecular mechanisms of senescence of human gingival fibroblasts are unknown. Human fibroblasts undergo replicative senescence in vitro after a limited number of population doublings. A reduced rate of proliferation is a prominent phenomenon observed in senescent fibroblasts. This phenomenon is controled by cell cycle regulatory proteins. The purpose of present study was to investigate the effect of replicative senescence on cell cycle progression and to find out its molecular mechanisms in human gingival fibroblasts. Replicative senescence of gingival fibroblasts were induced by subsequent cultures that were repeated up to 18 passage. In the present study, I examined change of cell proliferation, cell activity, cell viability and cell cycle progression during the replicative process. Also, I examined expression of cell cycle regulatory proteins which was estimated by western blot analysis. Cell proliferation, cell activity and cell viability of gingival fibroblasts were notably decreased with increase of population doubling level(PDL). S phase was decreased and G1 phase was increased with increase of PDL. Western blot analysis showed that levels of P16, p21 and p53 of senescent gingival fibroblasts(PDL41, PDL58) were higher than young fibroblasts(PDL27) and cdk4 were lower than young fibroblasts(PDL27). In conclusion, these results suggest that proliferative function of human gingival fibroblasts may be decreased by replicative senescence and its molecular mechanisms may be activatied with p16, p21, p53 and pRB, and repressed wtih cdk4.