• Journal of Veterinary Clinics
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• v.33 no.4
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• pp.194-199
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• 2016

The objective of this study was to examine the effect of trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) on the expression of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) pathway in lipopolysaccharide (LPS)-stimulated porcine peripheral blood mononuclear cells (PBMCs). t10c12-CLA was treated with different concentrations in culture medium of LPS$na{\ddot{i}}ve$ and LPS-stimulated PBMCs. The mRNA expressions of prostaglandin $E_2$ ($PGE_2$)-synthase, COX-2 and 5-LOX were measured using quantitative real-time PCR. In addition, the production levels of $PGE_2$ and 5-LOX in culture supernatant from PBMCs with or without LPS were assessed by ELISA. In LPS$na{\ddot{i}}ve$ PBMCs, treatment of t10c12-CLA significantly (p < 0.05) increased the mRNA expressions of PGE2 synthase and 5-LOX compared to vehicle control. Expression of COX-2 mRNA did not show significant difference compared to vehicle control by t10c12-CLA treatment in LPS$na{\ddot{i}}ve$ PBMCs. However, the addition of LPS in PBMCs markedly (p < 0.05) increased the mRNA expression of COX-2, $PGE_2$ synthase and 5-LOX, and also significantly (p < 0.05) enhanced the production of $PGE_2$ and 5-LOX relative to LPS$na{\ddot{i}}ve$ PBMCs, respectively. However, the addition of t10c12-CLA significantly (p < 0.01) suppressed the LPS-induced excessive expression of COX-2, $PGE_2$ synthase, and 5-LOX compared to those of PBMCs treated with LPS alone. The production levels of $PGE_2$ and 5-LOX in culture supernatant from LPS-stimulated PBMCs were also significantly (p < 0.05) inhibited by the treatment of t10c12-CLA compared to LPS alone. These results suggested that t10c12-CLA has an anti-inflammatory effect via dual inhibition of COX-2 and 5-LOX with gene expression and production level in LPS-stimulated porcine PBMCs. Therefore, it was thought that t10c12-CLA can attenuate the inflammatory response by down-regulation of eicosanoids production.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.18
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• pp.8579-8587
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• 2016

Background: The molecular mechanisms linking breast cancer progression and inflammation still remain obscure. The aim of the present study was to investigate the possible association of angiopoeitin like protein 4 (ANGPTL4) and its regulatory factor, hypoxia inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$), with the inflammatory markers nuclear factor kappa B/p65 (NF-${\kappa}B$/P65) and interleukin-1 beta (IL-$1{\beta}$) in order to evaluate their role in inflammation associated breast cancer progression. Materials and Methods: Angiopoietin-like protein 4 (ANGPTL4) mRNA expressions were evaluated using quantitative real time PCR and its protein expression by immunohistochemistry. DNA binding activity of NF-${\kappa}B$/P65 was evaluated by transcription factor binding immunoassay. Serum levels of ANGPTL4, HIF-$1{\alpha}$ and IL-$1{\beta}$ were immunoassayed. Tumor clinico-pathological features were investigated. Results: ANGPTL4 mRNA expressions and serum levels were significantly higher in high grade breast carcinoma ($1.47{\pm}0.31$ and $184.98{\pm}18.18$, respectively) compared to low grade carcinoma ($1.21{\pm}0.32$ and $171.76{\pm}7.58$, respectively) and controls ($0.70{\pm}0.02$ and $65.34{\pm}6.41$, respectively), (p<0.05). Also, ANGPTL4 high/moderate protein expression was positively correlated with tumor clinico-pathological features. In addition, serum levels of HIF-$1{\alpha}$ and IL-$1{\beta}$ as well as NF-${\kappa}B$/P65 DNA binding activity were significantly higher in high grade breast carcinoma ($148.54{\pm}14.20$, $0.79{\pm}0.03$ and $247.13{\pm}44.35$ respectively) than their values in low grade carcinoma ( $139.14{\pm}5.83$, $0.34{\pm}0.02$ and $184.23{\pm}37.75$, respectively) and controls ($33.95{\pm}3.11$, $0.11{\pm}0.02$ and $7.83{\pm}0.92$, respectively), (p<0.001). Conclusion: ANGPTL4 high serum levels and tissue expressions in advanced grade breast cancer, in addition to its positive correlation with tumor clinico-pathological features and HIF-$1{\alpha}$ could highlight its role as one of the signaling factors involved in breast cancer progression. Moreover, novel correlations were found between ANGPTL4 and the inflammatory markers, IL-$1{\beta}$ and NF-${\kappa}B$/p65, in breast cancer, which may emphasize the utility of these markers as potential tools for understanding interactions for axes of carcinogenesis and inflammation contributed for cancer progression. It is thus hoped that the findings reported here would assist in the development of new breast cancer management strategies that would promote patients' quality of life and ultimately improve clinical outcomes. However, large-scale studies are needed to verify these results.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.10
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• pp.4281-4287
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• 2014

Background: Methylation of tumor suppressor genes has been investigated in all kinds of cancer. Tumor specific epigenetic alterations can be used as a molecular markers of malignancy, which can lead to better diagnosis, prognosis and therapy. Therefore, the aim of this study was to evaluate the association between gene hypermethylation and expression of fragile histidine triad (FHIT), glutathione S-transferase P1 (GSTP1) and p16 genes and various clinicopathologic characteristics in primary non-small cell lung carcinomas (NSCLC). Materials and Methods: The study included 28 primary non-small cell lung carcinomas, where an additional 28 tissue samples taken from apparently normal safety margin surrounding the tumors served as controls. Methylation-specific polymerase chain reaction (MSP) was performed to analyze the methylation status of FHIT, GSTP1 and p16 while their mRNA expression levels were measured using a real-time PCR assay with SYBR Green I. Results: The methylation frequencies of the genes tested in NSCLC specimens were 53.6% for FHIT, 25% for GSTP1, and 0% for p16, and the risk of FHIT hypermethylation increased among patients with NSCLC by 2.88, while the risk of GSTP1 hypermethylation increased by 2.33. Hypermethylation of FHIT gene showed a highly significant correlation with pathologic stage (p<0.01) and a significant correlation with smoking habit and FHIT mRNA expression level (p<0.05). In contrast, no correlation was observed between the methylation of GSTP1 or p16 and smoking habit or any other parameter investigated (p>0.05). Conclusions: Results of the present study suggest that methylation of FHIT is a useful biomarker of biologically aggressive disease in patients with NSCLC. FHIT methylation may play a role in lung cancer later metastatic stages while GSTP1 methylation may rather play a role in the early pathogenesis.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.10
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• pp.4353-4358
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• 2014

Background: PI3/AKT and NF-kB signaling pathways are constitutively active in acute myeloid leukemia and cross-talk between the two has been shown in various cancers. However, their role in acute myeloid leukemia has not been completely explored. We therefore used cell penetrating inhibitor peptides to define the contributions of AKT and NF-kB to survival and multi drug resistance (MDR) in HL-60 cells. Materials and Methods: Inhibition of AKT and NF-kB activity by AKT inhibitor peptide and NBD inhibitor peptide, respectively, resulted in decreased expression of mRNA for the MDR1 gene as assessed by real time PCR. In addition, treatment of HL-60 cells with AKT and NBD inhibitor peptides led to inhibition of cell viability and induction of apoptosis in a dose dependent manner as detected by flow cytometer. Results: Finally, co-treatment of HL-60 cells with sub-optimal doses of AKT and NBD inhibitor peptides led to synergistic apoptotic responses in AML cells. Conclusions: These data support a strong biological link between NF-kB and PI3-kinase/AKT pathways in the modulation of antiapoptotic and multi drug resistant effects in AML cells. Synergistic targeting of these pathways using NF-kB and PI3-kinase/AK inhibitor peptides may have a therapeutic potential for AML and possibly other malignancies with constitutive activation of these pathways.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.10
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• pp.4197-4201
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• 2014

Minor trauma to the uterine cervix is supposed to induce local immunity to prevent cervical lesions caused by human papillomavirus (HPV) infection. This study aimed to investigate the local cervical immunity in women with low grade squamous intraepithelial lesion (LSIL) and effects of abrasion after cryosurgery or Pap smear. One hundred women with LSIL and known results of HPV detection were recruited. HPV positive women were randomly divided according to abrasion into cryotherapy and Pap smear observation groups. Cervical tissues and cervico-vaginal lavage (CVL) were collected at 6 and 12 months after allocation. The levels of cytokines at first recruitment were compared with cytokine levels at 6 months after abrasions. The mRNA of IFN-${\gamma}$, TNF-${\alpha}$ and IL-10 in cervical tissues and these cytokines secreted in CVL were determined using real time PCR and ELISA, respectively. Anti-HPV16 IgG and IgA antibodies in CVL were assessed by western blotting. At first recruitment of women with LSIL (100 cases), IL-10 mRNA and cytokine in HPV positive group (60 cases) was significantly higher than negative group (40 cases). IFN-${\gamma}$ and TNF-${\alpha}$ mRNA level in both groups were comparable but their secretions in CVL were significantly increased in HPV negative group. After abrasion for 6 months in HPV-positive women, all mRNA and secreted cytokines were changed, but no significant difference was observed between cryotherapy and observation groups. When individuals were compared between first recruitment and after abrasion for 6 months, IFN-${\gamma}$ mRNA and anti-HPV16 L1 IgA antibodies were significantly increased in the cryotherapy group. The results suggest that modulation of local cervical immunities by abrasion might promote different effects in clearance of HPV-related cytological abnormalities.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.4
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• pp.203-209
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• 2011

Objective: This study was performed to compare the efficiency of slow freezing and vitrification based on survival, development to blastocysts, and cell numbers of blastocysts. Changes in embryonic gene expression in fresh and frozen-thawed embryos were also examined. Methods: Eight-cell stage embryos were collected from superovulated female BDF1 mice. The collected embryos were randomly divided into three groups. One group was maintained as fresh controls (n=42), one was frozen by slow freezing (n=43), and one was cooled by vitrification (n=43). After thawing or cooling, survival rates, development to blastocyst, and cell numbers and inner cell mass (ICM) cell numbers of blastocysts were compared with those of the control group. The expressions of eight genes ($Rbm3$, $Birc5$, $Sod1$, $Sod2$, $Cirbp$, $Caspase3$, $Trp53$, $Hsp70.1$) were examined by real time-quantitative polymerase chain reaction in the fresh and frozen-thawed embryos. Results: There were no significant differences in the slow freezing and vitrification groups' survival rate after thawing (88.4% vs. 88.4%), development to blastocyst (100% vs. 97.4%), cell numbers ($107.0{\pm}21.0$ vs. $115.0{\pm}19.7$), or ICM cell numbers of blastocysts ($11.3{\pm}5.2$ vs. $11.1{\pm}3.7$). Cell numbers of blastocysts were significantly ($p$ <0.05) lower in the frozen-thawed embryos than the fresh embryos. There were no significant differences in the slow freezing and the vitrification groups' expressions of the eight genes. The expressions of $CirbP$ and $Hsp70.1$ were higher in the frozen-thawed embryos than in the fresh embryos but there were no significant differences. Conclusion: These results suggest that there were no significant differences between embryos that underwent slow freezing and vitrification.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.4
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• pp.193-202
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• 2011

Objective: We found previously that $interferon$ $regulatory$ factor ($Irf$)-1 is a germinal vesicle (GV)-selective gene that highly expressed in GV as compared to metaphase II oocytes. To our knowledge, the function of $Irf-1$ in oocytes has yet to be examined. The present study was conducted to determine the relationship between retinoic acid (RA) and RA-mediated expression of $Irf-1$ and the mouse oocyte maturation. Methods: Immature cumulus-oocyte-complexes (COCs) were collected from 17-day-old female mice and cultured $in$ $vitro$ for 16 hours in the presence of varying concentrations of RA (0-10 ${\mu}M$). Rate of oocyte maturation and activation was measured. Gene expression was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and cytokine secretion in the medium was measured by Bio-Plex analysis. Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results: The rates of oocyte maturation to metaphase II and oocyte activation increased significantly with RA treatment (10 nM-1 ${\mu}M$). With 100 nM RA treatment, lowest level of $Irf-1$ mRNA and cumulus cell's apoptosis was found. Among 23 cytokines measured by Bio-Plex system, the substantial changes in secretion of tumor necrosis factor-${\alpha}$, macrophage inflammatory protein-$1{\beta}$, eotaxin and interleukin-12 (p40) from COCs in response to RA were detected. Conclusion: We concluded that the maturation of oocytes and $Irf-1$ expression are negatively correlated, and RA enhances the developmental competence of mouse immature oocytes $in$ $vitro$ by suppressing apoptosis of cumulus cells. Using a mouse model, results of the present study provide insights into improved culture conditions for $in$ $vitro$ oocyte maturation and relevant cytokine production and secretion in assisted reproductive technology.

• Tuberculosis and Respiratory Diseases
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• v.70 no.5
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• pp.405-415
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• 2011

Background: In an effort to find alternative therapeutic agents to prevent excessive fibrosis as a sequela to complicated parapneumonic effusion or empyema, we examined the effect of tissue plasminogen activator (tPA) as a fibrinolytic agent combined with talc or transforming growth factor (TGF)-${\beta}1$ in a human pleural mesothelial cell line, MeT-5A. Methods: MeT-5A cells were stimulated with various doses of talc, doxycycline or TGF-${\beta}1$ for 24 h and then were treated with tPA for an additional 24 h. Cell viability was measured by MTT assay. The production of interleukin (IL)-8 and vascular endothelial growth factor (VEGF) in the culture supernatants was measured by ELISA. Real-time PCR was carried out for measurement of type I collagen mRNA. Results: MeT-5A cells treated with talc showed a dose-dependent increase in production of IL-8. Talc also increased production of type I collagen mRNA at low doses, but talc did not influence the induction of VEGF. Addition of tPA to talc-stimulated cells showed further increases in the production of IL-8, but tPA did not influence the production of VEGF or type I collagen mRNA. TGF-${\beta}1$ increased the production of both VEGF and collagen type I mRNA, both of which were effectively inhibited by additional tPA treatment in MeT-5A cells. Conclusion: TGF-${\beta}1$ is a potent inducer of collagen synthesis without induction of IL-8 in MeT-5A cells. Addition of tPA after TGF-${\beta}1$ stimulation inhibited further fibrosis by direct inhibition of collagen mRNA synthesis as well as by inhibition of VEGF production.

• Journal of the Korean Society of Food Science and Nutrition
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• v.45 no.1
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• pp.27-34
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• 2016

Garlic has drawn attention as a food material for its anti-oxidative and anti-inflammatory properties as well as for prevention and treatment of cancer. In order to increase efficiency, various aging methods for garlic have been attempted. In particular, thermally processed garlic is known to have higher biological activities due to its various chemical changes during heat treatment. Therefore, in this study, we investigated the anti-oxidative effects of garlic extracts aged at low temperature ($60{\sim}70^{\circ}C$). In the results, 2,2-diphenyl-1-picrylhydrazyl and 2,2-azino-bis (3-ethylbenzo-thiazoline-6-sulfonate) radical scavenging activities and ferric reducing ability of low temperature-aged garlic (LTAG) were similar to those of raw garlic. LTAG also showed decreased lipopolysaccharide (LPS)-induced production of reactive oxygen species, although there were not significant differences among samples. In addition, xanthine oxidase activity was inhibited by LTAG; the 15 days and $60^{\circ}C$ extract showed outstanding inhibition compared with the others. To understand the molecular mechanisms behind the anti-oxidative activity of LTAG, we performed quantitative real-time PCR analysis. The 30 days and $70^{\circ}C$ extract upregulated mRNA expression of antioxidant enzymes such as Cu/Zn-superoxide dismutase (SOD), Mn-SOD, glutathione peroxidase, and catalase in LPS-stimulated RAW 264.7 cells. This result indicates that LTAG can be a functional food as a nature antioxidant and antioxidant substance.

• Korean Journal of Ichthyology
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• v.23 no.1
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• pp.10-20
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• 2011

Genetic determinant for metallothionein (MT), a cysteine-rich protein playing essential roles in metal detoxification and homeostasis, was characterized in the Korean bitterling (Acheilognathus signifer, Cyprinidae), an endemic fish species. The full-length A. signifer MT (AsMT) cDNA (551 bp) is composed of a single open-reading frame (ORF) to encode a polypeptide of 60 amino acids containing 20 cysteine residues whose positions are conserved in most cypriniform MTs. At the genomic level, the AsMT (2,593 bp spanning the 5'-flanking region to the 3'-untranslated region) represented a conserved tripartite (three exons interrupted by two introns) structure with AT-rich introns. The upstream regulatory region (-1,914 bp from the ATG initiation codon) of AsMT displayed various sites and motifs for transcription factors involved in the metal-mediated regulation and stress/immune responses. The AsMT transcript was ubiquitously detected in various organs with variable expression levels, where the ovary and intestine showed the highest expression, while the heart and skeletal muscle represented the lowest level. During an exposure to copper (immersion in $0.5\;{\mu}M$ Cu for 48 h), the levels of AsMT transcripts were significantly elevated in the liver (more than 3.5-fold), moderately in the gill, kidney, and spleen (ranging from 1.5- to 2.5-fold), and barely in the brain and intestine. Results of this study could form a useful basis to explore the metal-related stress physiology of this endangered fish species.