• Maxillofacial Plastic and Reconstructive Surgery
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• v.32 no.3
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• pp.218-228
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• 2010

Components of dental resin-based restorative materials are reported to leach from the filling materials even after polymerization. Hydroquinone (HQ) is one of the major monomers used in the dental resin and is known as a carcinogen. Thus, carcinogenic risk of HQ leaching from the dental resin becomes a public health concern. The present study attempted to examine the carcinogenic potentials of HQ on the human epithelial cell, which is the target cell origin of the most of oral cancers. Cytotoxicity of HQ was observed above 50${\mu}M$ as measured by LDH assay, indicating a relatively low toxicity of this substance in human epithelial cells. The parameters of neoplastic cellular transformation such as cell saturation density, soft agar colony formation and cell aggregation were analyzed to examine the carcinogenic potential of HQ. The study showed that 2-week exposure of HQ showed the tendency of increase in the saturation density and the significant enhancement of soft agar colony formation at the highest dose, 50 ${\mu}M$ only. It is suggested that HQ has a weak potential of carcinogenicity. When cells were treated with HQ and TPA, a well-known tumor promoter, the parameters of neoplastic cellular transformation was significantly increased. This result indicates that the potential risk of carcinogenicity from HQ is largely dependent upon the presence of promoter. Exposure of 50 ${\mu}M$ HQ increased the time-dependent apoptosis as measured by the ELISA kit. This concentration coincides with a dose of neoplastic transformation, indicating a possible link between apoptosis and HQ-induced cellular transformation. Hydroquinone generated Reactive Oxygen Species (ROS) which was evidenced by the treatment of antioxidants such as trolox and N-acetyl cysteine and the GSH depleting agent, BSO. Antioxidants blocked the generation of ROS and the GSH depleting agent, BSO dramatically increased the ROS production. Since HQ is known to increase ROS production thru activation of transcriptional factor such as c-Myb and Pim-1, it is speculated that ROS generation by HQ plays a role in the activation of oncogene, which may lead to neoplastic transformation. In addition, ROS is involved in the alteration of signal transduction, which regulates the apoptosis in many cellular systems. Thus, ROS-mediated apoptosis may be involved in the HQ-induced carcinogenic processes. Protein kinase C (PKC) is known to play pivotal roles in neoplastic transformation of cells and its high expression is often found in a variety of types of tumors including oral cancer. PKC translocation of PKC-${\alpha}$ was observed following HQ exposure. Altered signaling system may also play a role in the transformation process. Taken together, HQ leached from the dental resin does not pose a significant threat as a cancer causing agent, but its carcinogenic potential can be significantly elevated in the presence of promoter. The mechanism of HQ-induced carcinogenesis involved ROS generation, apoptosis and altered signaling pathway. The present study will provide a valuable data to estimate the potential risk of HQ as a carcinogen and understand mechanism of HQ-induced carcinogenesis in human epithelial cells.

• Development and Reproduction
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• v.2 no.1
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• pp.9-20
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• 1998

It has been wel known that phospholipase C(PLC) plays an important role in the intracellular signaling in a variety of cell types. However, involvement of PLC in mouse oocyte maturation and preimplantation embryo development remains unknown. The present study examined the expression patterns of the mouse PLC \beta 1 and \gamma 1 during oocyte maturatio and preimplantation embryo development study examined the expression patterns of the mouse PLC \beta 1 and \gamma 1 during oocyte maturation and preimplantation embryo development by the competitive reverse transcription-polymerase chain reaction (RT-PCR method). PLC \gamma 1 mRNA (0.1 fg) was readily detected in germinal vesicle (GV)-stage oocyte and its level was reduced as meiotic resumption proceeded. PLC-\beta 1 mRNA (<0.1 fg) as detected at low level at GV-stage oocytes and scarcely detected at germinal vescle breakdown (GVBD)-stage oocytes. After fertilization, both PLC \beta 1 and \gamma 1 mRNA levels began to increase at morula-stage embryos (0.2 fg) and were more prominent in blastocyst-stage embryos(1 fg). to elucidate the possible involvement of PLC via protein kinase C(PKC) pathway during oocyte maturation and preimplantation embryo development , the effects of sphingosine (PKC inhibitor), sn-$diC_{8}$(PKC activator) anc U73122 (PLC ingibitor) were examined. Treatment of GV-stage oocytes with sphingosine (20 \mu M) facilitated the meiotic resuption by 10-20 over the control within 1 h as judged by GVBD, whereas U73122 failed to show any significant effect. U73122 (10 \mu M) effectively blocked the compaction of morula, while sn-$diC_{8}$(50 \mu M). In summary, the present study shows that the mouse PLC \beta 1 and \gamma 1 are expressed in a developmental stage-specific manner and PLC-PKC pathway may be involved in early preimplantation embryo development.

• Clinical and Experimental Pediatrics
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• v.53 no.2
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• pp.228-234
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• 2010

Purpose : Although intravenous immunoglobulin (IVIG) treatment is an effective first-line treatment for Kawasaki disease, 10-20% of the patients develop persistent fever or coronary artery complications. Medical records of Kawasaki disease patients were reviewed to assess the characteristic laboratory findings of IVIG nonresponsiveness. Methods : We reviewed the clinical records of 118 children with Kawasaki disease who were treated at the Chonnam National University Hospital from March 2003 to February 2008. The laboratory findings of the IVIG-responder group (n=110) and the IVIG-nonresponder group (n=8) were compared at admission day and at 48 hours and 14 days after IVIG administration. Results : At admission, the level of creatine kinase (CK) was lower (P =0.03) and that of total protein was higher (P <0.01) in the nonresponders than in the responders. At 48 hours after IVIG administration, the white blood cell (WBC) count (P =0.04) and neutrophil% (P <0.01) was higher in the nonresponders than in the responders. The neutrophil% (P <0.01) and CK (P =0.01) level at admission was lower than that at 48 hours after IVIG administration in the responders; this decrease was not as apparent in the nonresponders. Conclusion : IVIG nonresponders have lower CK and higher total protein levels at admission and higher WBC count and neutrophil% at 48 hours after IVIG administration. The decrease in the neutrophil% and CK level between at admission and at 48 hours after IVIG administration is remarkably higher in responders than in nonresponders.

• Radiation Oncology Journal
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• v.25 no.4
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• pp.227-232
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• 2007

Purpose: To investigate the flavopiridol effect on radiation-induced apoptosis and expression of apoptosisrelated genes of human laryngeal and lung cancer cells. Materials and Methods: A human laryngeal cancer cell line, AMC-HN3 and a human lung cancer cell line, NCI-H460, were used in the study. The cells were divided into four groups according to the type of treatment: 1) control groups; 2) cells that were only irradiated; 3) cells treated only with flavopiridol; 4) cells treated with flavopiridol and radiation simultaneously. The cells were irradiated with 10 Gy of X-rays using a 4 MV linear accelerator. Flavopiridol was administered to the media at a concentration of 100 nM for 24 hours. We compared the fraction of apoptotic cells of each group 24 hours after the initiation of treatment. The fraction of apoptotic cells was detected by measurement of the sub-G1 fractions from a flow cytometric analysis. The expression of apoptosis-regulating genes, including cleaved caspase-3, cleaved PARP (poly (ADP-ribose) polymerase), p53, p21, cyclin D1, and phosphorylated Akt (protein kinase B) were analyzed by Western blotting. Results: The sub-G1 fraction of cells was significantly increased in the combination treatment group, as compared to cells exposed to radiation alone or flavopiridol alone. Western blotting also showed an increased expression of cleaved caspase-3 and cleaved PARP expression in cells of the combination treatment group, as compared with cells exposed to radiation alone or flavopiridol alone. Treatment with flavopiridol down regulated cyclin 01 expression of both cell lines but its effect on p53 and p21 expression was different according to each individual cell line. Flavopiridol did not affect the expression of phophorylated Akt in both cell lines. Conclusion: Treatment with flavopiridol increased radiation-induced apoptosis of both the human laryngeal and lung cancer cell lines. Flavopiridol effects on p53 and p21 expression were different according to the individual cell line and it did not affect Akt activation of both cell lines.

• Journal of Life Science
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• v.23 no.5
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• pp.689-697
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• 2013

In the United States, about 40,000 new cases of oral cancer are diagnosed each year and nearly 7,800 patients died from it in 2012. Omega-3 polyunsaturated fatty acids have been found to have anticancer effects in a variety of cancer cell lines and animal models, but their effect in oral cancer remains unclear. This study was designed to examine the effect of docosahexaenoic acid (DHA, a kind of omega-3 fatty acid) on oral cancer cells and the molecular mechanism of its action. We found that exposure of squamous cell carcinoma-4 (SCC-4) and squamous cell carcinoma-9 (SCC-9) human oral cancer cells to DHA induced growth inhibition in a dose- and time-dependent manner. Meanwhile, in addition to the elevated levels of apoptotic markers, such as cleaved PARP, subG1 portion and TUNEL-positive nuclei, DHA led to autophagic vesicle formation and an increase in autophagic flux, indicating the involvement of both apoptosis and autophagy in the inhibitory effects of DHA on oral cancer cells. Further experiments revealed that the apoptosis and autophagy induced by DHA were linked to inhibition of mammalian target of rapamycin (mTOR) signaling by AKT inhibition and AMP-activated protein kinase (AMPK) activation in SCC-9 cells. Together, our results suggest that DHA induces apoptosis- and autophagy-associated cell death through the AMPK/AKT/mTOR signaling pathway in oral cancer cells. Thus, utilization of omega-3 fatty acids may represent a promising therapeutic approach for chemoprevention and treatment of human oral cancer.

• Korean Journal of Microbiology
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• v.41 no.1
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• pp.67-73
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• 2005

Photoautotrophic bacteria are promising candidates for the production of poly(3-hydroxybutyrate) (PHB) since they can address the critical problem of substrate costs. In this study, we isolated 25 Tn5-inserted mutants of the Synechocystis sp. PCC 6803 which showed enhanced PHB accumulation compared to the wild-type strain. After 5-days cultivation under nitrogen-limited mixotrophic conditions, the intracellular levels of PHB content in these mutants reached up to $10-30\%$ of dry cell weight (DCW) comparable to $4\%$ of DCW in the wild-type strain. Using the method of inverse PCR, the affected genes of the mutants were mapped on the completely known genome sequence of Synechocystis sp. PCC 6803. As a result, the increased PHB accumulation in 5 mutants were found to be resulted from defects of genes coding for NADH-ubiquinone oxidoreductase, O-succinylbenzoic-CoA ligase, photosystem II PsbT protein or histidine kinase, which are involved in photosystem in thylakoid inner membrane of the cell. The values of $NAD(P)H/NAD(P)^+$ ratio in the cells of these mutants were much higher than that of the wild-type strain as measured by using pulse-amplitude modulated fluorometer, suggesting that PHB synthesis could be enhanced by increasing the level of cellular NAD(P)H which is a limiting substrate for NADPH-dependent acetoacetyl-CoA reductase. From these results, it is likely that NAD(P)H would be a limiting factor for PHB synthesis in Synechocystis sp. PCC 6803.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.1
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• pp.13-23
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• 2010

Objective: The present study was carried out to induce differentiation of human embryonic stem cells (hESCs) into germ cells and to establish a culture condition for single hESCs dissociated by enzyme. Methods: Embryonic body (EB) was formed by hanging drop culture for 3 days from hESCs colony. The EBs were cultured in the medium supplemented with retionic acid (RA) or/and bone morphogenetic protein-4 (BMP4) for 14 days to differentiate into germ cells. Germ cell specific markers, c-kit and VASA were used for immunohistochemistry of EB. Human ESCs colonies were dissociated into single cells by Collagenase, Tryple and Accutase, and then colony formation rate of the single cells was examined. Rho-associated kinase inhibitor (ROCK inhibitor, Y27632) was added into the culture medium of single cells to reduce the apoptotic damage during the dissociation. Results: Single cells dissociated with Tryple or Accutase showed higher colony formation rates compared to the cells dissociated with Collagenase. Seeding of $5{\times}10^3$ cells/well (4 well dish) was efficient to obtain high colony formation rate compared to other concentrations of seeding cell. Addition of Y27632 significantly increased the colony formation rate of the single cells dissociated by Tryple. Immunohistochemistry of EB with c-kit and VASA markers showed a weak fluorescence signals compared to the signals from the testicular tissue. Conclusion: Dissociation with Tryple was useful to obtain healthy single cells and addition of Y27632 was beneficial for survival and colony formation of the single cells. Unlike other studies, we just observed a dim fluorescence staining of the germ cell markers, probably caused by the short-term culture for the differentiation of EB compared to other studies.

• Food Science and Preservation
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• v.24 no.8
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• pp.1149-1157
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• 2017

Inflammation is the first response of the immune system to infection or irritation in our body. The use of medicinal plants has been widely applied as an alternative source for drug development. One of marine natural resources, the anti-inflammatory effect of Ishige sinicola ethanol extract (ISEE), was evaluated by using LPS-induced RAW 264.7 cell and mice model. As a result, the production of nitric oxide (NO) and pro-inflammatory cytokines (IL-6, IL-$1{\beta}$, TNF-${\alpha}$) were inhibited with increasing concentration of ISEE without any cytotoxicity. Furthermore, ISEE suppressed the expression of not only inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-${\kappa}B$) p65, and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase (JNK) in a dose-dependent manner. In mice ear edema test, the formation of edema was reduced at the highest dosage of ISEE and the reduction of the number of infiltrated mast cells was observed in histological analysis. These results indicate that ISEE has a potent anti-inflammatory activity and can be used as a pharmaceutical material for many kinds of inflammatory disease.

• Tuberculosis and Respiratory Diseases
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• v.44 no.3
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• pp.601-610
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• 1997

Background : Monocytes/macrophages play a central role in determining the host response during Gram-negative infection through secretion of a variety of mediators after stimulation of LPS. Even though cytokine production has been shown to play an important role in host defense during sepsis, cytokine release may also lead to tissue injury. Thus, regulation of macrophage response to LPS is critical for host survival during Gram-negative sepsis. In animals exposed to nonlethal doses of endotoxin, a characteristic hyporesponsiveness to subsequent administration of endotoxin has been observed. This phenomenon was known as 'LPS tolerance'. However, little information is available regarding the underlying mechanism of LPS tolerance. Method : Peripheral blood monocyte(PBMC) was isolated from peripheral blood of normal volunteers by adhesion purification method. To evaluate the conditions to obtain LPS tolerance, preculture was carried out with LPS at 10ng/ml for 24 hours. For stimulation, culture plates were washed two times and were stimulated with LPS at $1{\mu}g/ml$ for 4, 6 and 26 hours. To assess the underlying mechanisms of LPS tolerance, autologous serum, PMA, anti-CD14 Ab, Indomethacin or $PGF_2$ were added to preculture solution respectively. Cytokine concentrations in culture supernatants were measured using ELISA for TNF-$\alpha$ and IL-8 and mRNA of TNF-$\alpha$ and IL-8 were determined by Northern blot analysis. Results : The exposure of PBMC to low dose of LPS suppressed the cytokine production and mRNA expression of TNF-$\alpha$, but not IL-8. Anti-CD14 Ab partially recovered production of TNF-$\alpha$ which was suppressed by preculture with low dose LPS. The preculture with PMA induces LPS tolerance, as preculture with low dose LPS. Conclusion : LPS tolerance to TNF-$\alpha$ is regulated pretranslationally and is influenced by protein kinase C pathway and CD14.

• Journal of Life Science
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• v.21 no.2
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• pp.183-193
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• 2011

Migration and differentiation of mesenchymal stem cells are crucial for tissue regeneration in response to injury. Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates a variety of biological processes, including proliferation, survival, differentiation and motility. In the present study, we determined the role of S1P in migration and differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs). S1P stimulated migration of BMSCs in a dose- and time-dependent manner, and pre-incubation of the cells with pertussis toxin completely abrogated S1P-induced migration, suggesting involvement of Gi-coupled receptors in S1P-induced cell migration. S1P elicited elevation of intracellular concentration of $Ca^{2+}$ ($[Ca^{2+}]_i$) and pretreatment with VPC23019, an antagonist of $S1P_1/S1P_3$, blocked S1P-induced migration and increase of $[Ca^{2+}]_i$. Small interfering RNA-mediated knockdown of endogenous $S1P_1$ attenuated S1P-induced migration of BMSCs. Furthermore, S1P treatment induced expression of $\alpha$-smooth muscle actin ($\alpha$-SMA), a smooth muscle marker, and pretreatment with VPC23019 abrogated S1P-induced $\alpha$-SMA expression. S1P induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), and pretreatment of cells with SB202190, an inhibitor of p38 MAPK, or adenoviral overexpression of a dominant-negative mutant of the p38 MAPK blocked S1P-induced cell migration and $\alpha$-SMA expression. Taken together, these results suggest that S1P stimulates migration and smooth muscle differentiation of BMSCs through an $S1P_1$-p38 MAPK-dependent mechanism.