• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제27권2호
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• pp.129-134
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• 2001

Since NNK is one of the most abundant tobacco-specific alkaloids and a strong carcinogenic nitrosamine, it has been used for evaluating a potential of carcinogenicity in the animal models. The present study has attempted to examine the potential of carcinogenicity of NNK in human epithelial cells, from which the cell type the most of cancers including oral cancer and nasal cavity cancer are originated. The cellular model used for the study is a human keratinocyte cell system immortalized by Ad12-SV40 hybrid virus. The cellular system has successfully been used for the carcinogenicity studies because of its limitless life span, epithelial morphology and nontumorigenicity. When cells were treated with a variety of NNK concentrations, levels of saturation density and soft agar colony formation were increased in a dose-dependent fashion. Colonies of large cell aggregates were above 5 at the higher doses. The results indicate that exposure of human cells with NNK induced loss of contact inhibition and increases of anchorage independence and cellular adhesion, which are typical characteristics of the neoplatically transformed cells. When cells were exposed with 100uM NNK for 2hr, mRNA levels of IL-1 and PAI-2 were increased in a dose-dependent manner, but expression of TGF- 1 was not affected. While expression of growth regulatory factors were altered with a short-term exposure, there was no alteration of these factors in the NNK-transformed cells. However, mRNA levels of fibronectin were increased both in the short-term treatment and in the transformation. The results suggest that altered expression of extracellular matrix such as fibronectin following short-term exposure might be fixed in the genome and these altered properties be continuously transfered throughout the cell division. Western blot analysis showed a translocation of PKC- from cytosolic fraction to the particulate fraction, indicating a possible role of NNK in the signal transduction pathway. The present study provided an evidence that NNK in the smoking may be associated with epithelial origin cancer such as oral and nasal cavity cancers. In addition, this study suggested that altered expression of extracellular matrix and PKC may play an important role in the carcinogenic mechanism of NNK.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제36권6호
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• pp.481-489
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• 2010

Introduction: TLR-5, a member of the toll-like receptor (TLR) family, is a element of the type I transmembrane receptors, which are characterized by an intracellular signaling domain homolog to the interleukin-1 receptor. These receptors recognize microbial components, particularly bacterial flagellin. All-trans retinoic acid (atRA, tretinoin), a natural metabolite of vitamin A, acts as a growth and differentiation factor in many tissues, and is also needed for immune functions. In this study, THP-1 human macrophage-monocytes were used to examine the mechanisms by which atRA regulated the expression of TLR-5. Because the molecular mechanism underlying this regulation at the transcriptional level is also unclear, this study examined which putative transcription factors are responsible for TLR-5 expression by atRA in immune cells. Materials and Methods: This study examined whether atRA induces the expression of TLR-5 in THP-1 cells using reverse transcription-polymerase chain reaction (RT-PCR), and which transcription factors are involved in regulating the TLR-5 promoter in RAW264.7 cells using a reporter assay system. Western blot analysis was used to determine which signal pathway is involved in the expression of TLR-5 in atRA-treated THP-1 cells. Results: atRA at a concentration of 10 nM greatly induced the expression of TLR-5 in THP-1 cells. Human TLR-5 promoter contains three Sp-1/GC binding sites around -50 bp and two NF-kB binding sites at -380 bp and -160 bp from the transcriptional start site of the TLR-5 gene. Sp-1/GC is primarily responsible for the constitutive TLR-5 expression, and may also contribute to NF-kB at -160 bp to induce TLR-5 after atRA stimulation in THP-1 cells. The role of NF-kB in TLR-5 expression was further confirmed by inhibitor pyrrolidine dithiocarbamate (PDTC) experiments, which greatly reduced the TLR-5 transcription by 70-80%. Conclusion: atRA induces the expression of the human TLR-5 gene and NF-kB is a critical transcription factor for the atRA-induced expression of TLR-5. Accordingly, it is conceivable that retinoids are required for adequate innate and adaptive immune responses to agents of infectious diseases. atRA and various synthetic retinoids have been used therapeutically in human diseases, such as leukemia and other cancers due to the antiproliferative and apoptosis inducing effects of retinoids. Therefore, understanding the molecular regulatory mechanism of TLR-5 may assist in the design of alternative strategies for the treatment of infectious diseases, leukemia and cancers.

• BMB Reports
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• 제40권2호
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• pp.196-204
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• 2007

Our previous study has shown that sodium selenite can cause apoptosis in acute promyelocytic leukemia-derived NB4 cells in a caspase-dependent manner, but the detailed mechanism is unknown. Here we demonstrate a requirement for extracellular signal-regulated protein kinase (ERK) in mediating sodium selenite -induced apoptosis in NB4 cell. Though no apparent elevation of ERK activity was observed during the apoptosis in NB4 cells caused by 20 μM sodium selenite treatment, PD98059 and U0126, specific chemical inhibitors of the MEK/ERK signaling pathway, were shown to strongly prevent the apoptosis process, while ERK activator TPA enhanced the process. It is also known that p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125 had slight effects on apoptosis. Further study indicated that ERK exerted its proapoptotic effect only at the early stage of apoptosis and played an antiapoptotic role at the later stages. Taken together, our findings suggest that ERK plays an active role in mediating sodium seleniteinduced apoptosis in NB4 cells .

• BMB Reports
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• 제42권7호
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• pp.444-449
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• 2009

Different neurodegenerative disorders like prion disease, is caused by protein misfolding conformers. Reverse-transfected cytosolic prion protein (PrP) and PrP expressed in the cytosol have been shown to be neurotoxic. To investigate the possible mechanism of neurotoxicity due to accumulation of PrP in cytosol, a PrP mutant lacking the signal and GPI (CytoPrP) was introduced into the SH-SY5Y cell. MTT and trypan blue assays indicated that the viability of cells expressing CytoPrP was remarkably reduced after treatment of MG-132. Obvious apoptosis phenomena were detected in the cells accumulated with CytoPrP, including loss of mitochondrial transmembrane potential, increase of caspase-3 activity, more annexin V/PI-double positive-stained cells and reduced Bcl-2 level. Moreover, DNA fragmentation and TUNEL assays also revealed clear evidences of late apoptosis in the cells accumulated CytoPrP. These data suggest that the accumulation of CytoPrP in cytoplasm may trigger cell apoptosis, in which mitochondrial relative apoptosis pathway seems to play critical role.

• 동의생리병리학회지
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• 제17권6호
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• pp.1393-1403
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• 2003

The genetic effects of restraint stress challenge on HPA axis and the therapeutic effect of Boshimgeonbi-Tang on the stress were studied with cDNA microarray analyses on hypothalamus using an immobilization-stress mouse as stress model. Male CD-1 mice were restrained in a tightly fitted and ventilated vinyl holder for 2hours once a day, and this challenge was repeated for seven consecutive days. The body weights of the immobilization-stress mice were diminished about 25 percent degree as compared to normal ones. Seven days later, total RNA was extracted from the organs of the mouse, body-labeled with CyDye/sup TM/ fluorescence dyes (Amersham Bioscience Co., NJ), and then hybridized to cDNA microarray chip. Scanning and analyzing the array slides were carried out using GenePix 4000 series scanner and GenePix Pro/sup TM/ analyzing program, respectively. The expression profiles of 109 genes out of 6000 genes on the chip were significantly modulated in hypothalamus by the immobilization stress. Energy metabolism-, lipid metabolism-, apoptosis- and signal transduction-related genes were transcriptionally activated whereas DNA repair-, protein biosynthesis-, and structure integrity-related genes were down-regulated in hypothalamus. The 58 genes were up-regulated by the mRNA expression folds of 1.5 to 7.9. and the 51 genes were down-regulated by 1.5 - 3.5 fold. The 20 genes among them were selected to confirm the expression profiles by RT-PCR. The mRNA expression levels of Tnfrsf1a (apoptosis), Calm2 (cell cycle), Bag3 (apoptosis), Hspe1 (protein folding), Aatk (apoptosis), Dffa (apoptosis), Itgb1 (cell adhesion), Vcam1 (cell adhesion), Fkbp5 (protein folding), BDNF (neuron survival) were restored to the normal one by the treatment of Boshimgeonbi-Tang.

• Biomolecules & Therapeutics
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• 제23권2호
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• pp.180-188
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• 2015

This study investigated the possible effects and molecular mechanisms of diallyl disulfide (DADS) against cyclophosphamide (CP)-induced hemorrhagic cystitis (HC) in rats. Inflammation response was assessed by histopathology and serum cytokines levels. We determined the protein expressions of nuclear transcription factor kappa-B (NF-${\kappa}B$), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), oxidative stress, urinary nitrite-nitrate, malondialdehyde (MDA), and 8-hydroxy-2'-deoxyguanosine (8-OHdG). Finally, we studied the involvement of mitogen-activated protein kinases (MAPKs) signaling in the protective effects of DADS against CP-induced HC. CP treatment caused a HC which was evidenced by an increase in histopathological changes, proinflammatory cytokines levels, urinary nitrite-nitrate level, and the protein expression of NF-${\kappa}B$, COX-2, iNOS, TNF-${\alpha}$, p-c-Jun N-terminal kinase (JNK), and p-extracellular signal regulated kinase (ERK). The significant decreases in glutathione content and glutathione-S-transferase and glutathione reductase activities, and the significant increase in MDA content and urinary MDA and 8-OHdG levels indicated that CP-induced bladder injury was mediated through oxidative DNA damage. In contrast, DADS pretreatment attenuated CP-induced HC, including histopathological lesion, serum cytokines levels, oxidative damage, and urinary oxidative DNA damage. DADS also caused significantly decreased the protein expressions of NF-${\kappa}B$, COX-2, iNOS, TNF-${\alpha}$, p-JNK, and p-ERK. These results indicate that DADS prevents CP-induced HC and that the protective effects of DADS may be due to its ability to regulate proinflammatory cytokines production by inhibition of NF-${\kappa}B$ and MAPKs expressions, and its potent anti-oxidative capability through reduction of oxidative DNA damage in the bladder.

• 한국임상수의학회지
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• 제32권5호
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• pp.464-468
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• 2015

6살의 중성화한 수컷 말티즈는 운동실조로 인하여 내원하였다. 본 증례는 일반방사선 촬영, 컴퓨터단층촬영, 자기공명영상을 실시하여 낭종의 위치, 크기, 척수와의 관련성 및 동반된 질환을 알 수 있었다. 자기공명영상 결과 T1W과 FLAIR 영상에서는 hypointense한 영역, T2W 영상에서는 hyperintense한 영역이 L1~2 척수의 등쪽으로 나타났다. 이는 CSF와 동일한 신호로 나타났으며, 등쪽에서 척수를 압박하여 배쪽으로 이동시켰다. 낭종의 길이는 25 mm, 두께는 4 mm로 나타났다. 무증상일 경우는 치료가 필요 없으나, 본 증례는 신경학적 이상이 나타나 수술적 치료를 실시하였다.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.186-186
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• 1994

Many agonists have been known to activate the hydrolysis of membrane phospholipids through the bindings with corresponding receptors on the various cells. Diacylglycerol and inositol 1,4,5-trisphosphate(IP3) generated by the action of phosphoinositide-specific phospholipase C (PI-PLC) are well known second messengers for the activation of protein kinase C and the mobilization of Ca2+ in many cells. Three types of PI-PLC isozyme (${\alpha}$,${\gamma}$, and $\delta$) and several subtrpes for each type have been identified from mammalian sources by purification of enzymes and cloning of their cDNAs. Each type PI-PLC isozyme is coupled to different receptors and mediators, for example, ${\beta}$-types are coupled to the seven-transmembrane-receptors via Gq family of G-proteins and ${\beta}$-types directly to the receptor tyrosine kinases. Specific modulators for the signaling pathway through each type of PI-PLC should be very useful as potential potential candidates for lend substances in developing novel drugs. To establish the sensitive and convenient screening systems for searching modulators on PI-PLC mediated signaling, two kinds of approaches have been tried. (1) Establishment of in vitro assay condition for each type of PI-PLC isozyme: Overexpression by using vaccinia virus and purification of each isozyme was carried out for the preparation of large amounts of enaymes. Optimum and sensitive assay condition for the measurements of PI-ELC activities were established. (2) Development of the cell lines in which each type of PI-PLC is permanently overexpressed: A fibroblast cell line (3T3${\gamma}$1-7) in which PI-PLC-${\gamma}$1 was overexpressed by using pZip-neo expression vector was developed and used for the measurement of PDGF-induced IP3 formation. The responses for IP3 formed in 3T3${\gamma}$1-7 cells by the treatment of PDGF is 8 times more sensitive than those in control cells. 3T3${\gamma}$l-7 cell is useful for the screening of the inhibitors on the PDGF-induced cellular responses from large number of samples in a small volume(50 ${\mu}$l) and short time(5-15 min). Using these systems, we screened hundreds of herb-extracts for the inhibition of PDGF-induced IP3 formation and selected several extracts that showed the inhibition as the candidates for isolation and characterization of active substances. The determination of the acting point of selected extracts or fractions in the PDGF signaling pathway has been analyzing.

• Biomolecules & Therapeutics
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• 제10권2호
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• pp.71-77
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• 2002

Heme oxygenase-1 (HO-1) is the inducible from of the rate-limiting enzyme of heme degradation; it regulates the cellular contents of heme. HO-1 is up-regulated by various stimuli including oxidative stress so that it is thought to participate in general cellular defense mechanisms against oxidative stress in mammalian cells. To investigate the role of the cAMP-dependent protein kinase A (PKA) signaling pathway on nitrogen oxidative stress-induced HO-1 gene expression, RAW 264.7 cell cultures were treated with sodium nitroprusside (SNP). SNP increased the expression of HO-1 mRNA and protein, time- and concentration-dependently. Treatment with H89, PKA inhibitor, but not LY83583, guanylate cyclase inhibitor, significantly diminished the HO-1 expression by SNP, indicating that cAMP plays a crucial role in the induction of HO-1. Incubation with cAMP-elevating agents, such as forskolin or isoproterenol resulted in up-regulation of the expression of HO-1. Forskolin-induced expression of HO-1 was inhibited by H89. Furthermore, propranolol, $\beta$-adrenoceptor blocker, inhibited the isoproterenol-induced HO-1 expression, supporting the importance of cAMP in the induction of HO-1 expression. Higenamine-S, but not higenamineR, enhanced the HO-1 expression induced by SNP. Furthermore, cellular toxicity induced by hydrogen peroxide was attenuated by the presence of SNP, which was further increased by the presence of ZnPPIX, HO-1 inhibitor. Collectively, these results strongly suggest that up-regulation of HO-1 expression in RAW 264.7 cells involves PKA signal pathway.

• Journal of Microbiology and Biotechnology
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• 제18권6호
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• pp.1170-1178
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• 2008

The cyclopentenone prostaglandins (cyPGs) prostaglandin $A_1$ ($PGA_1$) and 15-deoxy-${\Delta}^{12,14}$-prostaglandin $J_2$ (15d-$PGJ_2$) have been reported to exhibit antiinflammatory activity in activated monocytes/macrophages. However, the effects of these two cyPGs on the expression of cytokine genes may differ. In this study, we investigated the mechanism of action of $PGA_1$ in lipopolysaccharide (LPS)-induced expression of inter leu kin (IL)-10 mRNA in mouse peritoneal macrophages. 15d-$PGJ_2$ inhibited expression of LPS-induced IL-10, whereas $PGA_1$ increased LPS-induced IL-10 expression. This synergistic effect of $PGA_1$ on LPS-induced IL-10 expression reached a maximum as early as 2 h after simultaneous $PGA_1$ and LPS treatment ($PGA_1$/LPS), and did not require new protein synthesis. The synergistic effect of $PGA_1$ was inhibited by GW9662, a specific peroxisome proliferator-activated receptor ${\gamma}(PPAR{\gamma})$ antagonist, and Bay-11-7082, a NF-${\kappa}B$ inhibitor. The extracellular signal-regulated kinases (ERK) inhibitor PD98059 increased the expression of $PGA_1$/LPS-induced IL-10 mRNA, rather than inhibiting the IL-10 expression. Moreover, $PGA_1$ inhibited LPS-induced ERK phosphorylation. The synergistic effect of $PGA_1$ on LPS-induced IL-10 mRNA and protein production was inhibited by p38 inhibitor PD169316, and $PGA_1$ increased LPS-induced p38 phosphorylation. In the case of stress-activated protein kinase/c-Jun $NH_2$-terminal kinase (SAPK/JNK), the SAPK/JNK inhibitor SP600125 did not inhibit IL-10 mRNA synthesis but inhibited the production of IL-10 protein remarkably. These results suggest that the synergistic effect of $PGA_1$ on LPS-induced IL-10 expression is NF-${\kappa}B$-dependent and mediated by mitogen-activated protein (MAP) kinases, p38, and SAPK/JNK signaling pathways, and also associated with the $PPAR{\gamma}$ pathway. Our data may provide more insight into the diverse mechanisms of $PGA_1$ effects on the expression of cytokine genes.