• Biomolecules & Therapeutics
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• v.23 no.1
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• pp.19-25
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• 2015

Vascular endothelial growth factor (VEGF) is an important regulator of neovascularization. Hypoxia inducible nitric oxide (NO) enhanced the expression of VEGF and thymosin beta-4 ($T{\beta}4$), actin sequestering protein. Here, we investigated whether NO-mediated VEGF expression could be regulated by $T{\beta}4$ expression in HeLa cervical cancer cells. Hypoxia inducible NO production and VEGF expression were reduced by small interference (si) RNA of $T{\beta}4$. Hypoxia response element (HRE)-luciferase activity and VEGF expression were increased by the treatment with N-(${\beta}$-D-Glucopyranosyl)-N2-acetyl-S-nitroso-D, L-penicillaminamide (SNAP-1), to generate NO, which was inhibited by the inhibition of $T{\beta}4$ expression with $T{\beta}4$-siRNA. In hypoxic condition, HRE-luciferase activity and VEGF expression were inhibited by the treatment with $N^G$-monomethyl-L-arginine (L-NMMA), an inhibitor to nitric oxide synthase (NOS), which is accompanied with a decrease in $T{\beta}4$ expression. VEGF expression inhibited by L-NMMA treatment was restored by the transfection with pCMV-$T{\beta}4$ plasmids for $T{\beta}4$ overexpression. Taken together, these results suggest that $T{\beta}4$ could be a regulator for the expression of VEGF via the maintenance of NOS activity.

• Proceedings of the Korean Environmental Health Society Conference
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• 2005.06a
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• pp.341-344
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• 2005

The expression of the glutathione S-transferase (GST), whose induction accounts for antioxidant defense system, is regulated by activation of CCAAT/enhancer binding protein ${\beta}$ ($C/EBP{\beta}$), Sick house syndrome (SHS) presents healthy damage owing to the indoor environment of a building. Toluene has been implicated in one of the important causes of SHS. The present study investigated the effects of toluene treatment on the induction of GSTA2 gene and its mechanism. H411E cells treated with toluene, and GSTA2 expression was determined by immunoblot analysis. The translocation of $C/EBP{\beta}$ was assessed by immunocytochemical assays. $C/EBP{\beta}$ DNA binding activity was determined by electrophoretic mobility shift assays. The role of the C/EBP binding site in the induction of the GSTA2 gene was assessed by luciferase reporter-gene activity. Toluene induced GSTA2 protein expression. In toluene-treated cells, $C/EBP{\beta}$ translocated to the nucleus and bound to the consensus sequence of C/EBP (TTGCGCAA). Toluene treatment increased luciferase reporter-gene activity in cells transfected with the C/EBP-containing regulatory region of the GSTA2 gene. Oxidative stress is believed to play an important role in the induction of GSTA2 gene by toluene This study shows that toluene-induced GSTA2 gene expression is dependent upon nuclear translocation and binding of $C/EBP{\beta}$ to the C/EBP response element in the GSTA2 gene promoter.

• International Journal of Industrial Entomology and Biomaterials
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• v.24 no.2
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• pp.49-55
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• 2012

We previously isolated 9 clones that show stronger signal compared to B. mori cytoplasmic actin gene (BmA3) by using a dot blot hybridization. In this study, we focused on one clone among these clones which has high amino acid homology with elongation factor ${\alpha}$ gene of B. mori. This clone, named $bEF1{\alpha}$ (B. mori elongation factor ${\alpha}$) was ubiquitously expressed in all tissues and developmental stage of B. mori. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-702/+38) in the 5'-flanking region of $bEF1{\alpha}$ gene, which has about 20 fold more intensive promoter activity than BmA3 promoter. Moreover, the $bEF1{\alpha}$ promoter was normally regulated in Bm5, Sf9, and S2 cells. Therefore, we suggest that $bEF1{\alpha}$ promoter may be used more powerful and effectively for transgene expression in various insects containing B. mori as a universal promoter.

• Journal of Life Science
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• v.19 no.9
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• pp.1289-1293
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• 2009

Proto-oncogene human cervical cancer oncogene (HCCR) functions as a negative regulator of p53 and contributes to tumorigenesis in various human tissues. However, it is unknown how HCCR contributes to the cellular and biochemical mechanisms of human tumorigenesis. In this study, we showed how the expression of HCCR is modulated. The luciferase activity assay indicated that the HCCR 5'-flanking region at positions -370 to -406 plays an important role in the promoter activity. Computational analysis of this region identified one consensus sequence for the TG-interacting factor (TGIF) located at -390 to -366 (TG). Mobility shift assays (EMSA) revealed that nuclear proteins from K562 bind to the TG site, but not to the mutated TG site. The reporter activity assay with promoter constructs carrying mutated TGIF sequences pGL3-mTGIF significantly increased reporter activities compared to wild type constructs pGL3-$406{\sim}+30$. In this study, we characterized the HCCR promoter and found that HCCR expression was partially regulated by the transcription repressor TGIF, which bound the promoter at positions -390 to -366.

• Current Research on Agriculture and Life Sciences
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• v.32 no.3
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• pp.119-124
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• 2014

Physalis alkekengi var. francheti Hort. is known as an insecticide and traditional remedy for liver related diseases. Therefore, this study investigated the chemopreventive effects of extracts and several solvent fractions (n-hexane, dichloromethane, n-butanol, water) of Physalis alkekengi var. francheti Hort. First, their cytotoxicity and NQO1 activity were measured using an MTT assay, plus a quinone reductase [NAD(P)H dehydrogenase (quinone); NAD(P)H: (quinone acceptor) oxidoreductase, EC 1.6.99.2]-inducing activity assay was performed using cultured murine hepatoma cells (Hepa1c1c7) and its mutant cells(BpRc1). The reduction of electrophilic quinones by NQO1 is an important detoxification pathway and major mechanism of chemoprevention. When compared with the other solvent soluble fractions with different polarities, the dichloromethane fraction of Physalis alkekengi var. francheti Hort. showed a higher NQO1-inducing activity that was also dose-dependent. Moreover, the dichloromethane fraction of Physalis alkekengi var. francheti Hort. induced ARE-luciferase activities in HepG2-C8 cells that were generated by transfecting the ARE-luciferase gene construct, suggesting the Nrf2-ARE-mediated induction of anti-oxidative enzymes. In conclusion, the dichloromethane-soluble fraction of Physalis alkekengi var. francheti Hort. showed a relatively strong induction of detoxifying enzymes, thereby meriting further study to identify the active components and evaluate their potential as cancer preventive agents.

• Tuberculosis and Respiratory Diseases
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• v.48 no.5
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• pp.682-698
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• 2000

Glucocorticoid receptor (GR) functions as a suppressor of inflammation by inhibiting the expression of many cytokine genes activated by NF-${\kappa}B$. The goal of this study is to investigate the mechanism by which GR repress NF-${\kappa}B$ activation in lung epithelial cells. We used A549 and BEAS-2B lung epithelia! cell lines. Using Ig$G{\kappa}$-NF-${\kappa}B$ luciferase reporter gene construct, we found that dexamethasone significantly suppressed TNF-$\alpha$-induced NF-${\kappa}B$ activation and the overexpression of GR showed dose-dependent reduction of TNF-$\alpha$-induced NF-${\kappa}B$ activity in both cell lines. However, DNA binding of NF-${\kappa}B$ induced by TNF-$\alpha$ in electromobility shift assay was not inhibited by dexamethasone. Super shift assay with anti-p65 antibody demonstrated the existence of p65 in NF-${\kappa}B$ complex induced by $\alpha$ Western blot showed that $I{\kappa}B{\alpha}$ degradation induced by TNF-$\alpha$ was not affected by dexamethasone and $I{\kappa}B{\kappa}$ was not induced by dexamethasone, neither. To evaluate p65 specific transactivation, we adopted co-transfection study of Gal4-p65TA1 or TA2 fusion protein expression system together with 5xGal4-luciferase vector. Co-transfection of GR with Gal4-p65TA1 or TA2 repressed luciferase activity profoundly to the level of 10-20% of p65TA1- or TA2-induced transcriptional activity. And this transrepressional effect was abolished by co-transfection of CBP of SRC-1 expression vectors. These results suggest that GR-mediated transrepression of NF-${\kappa}B$ in lung epithelial cells is through competing for binding to limiting amounts of transcriptional coactivators, CBP or SRC-1.

• Herbal Formula Science
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• v.28 no.3
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• pp.255-269
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• 2020

Objectives : Hemistepta lyrata Bunge (Bunge) is a wild herb that has been used for managing fever and wound in Korean Traditional Medicine. The present study explored the effects of H. lyrata extract on liver X receptor (LXR) α-dependent lipogenic genes in hepatocyte-derived cells. Methods : After HepG2 cells or Huh7 cells were pre-treated with 1-10 ㎍/mL of H. lyrata extract or its fractionated extract for 0.5 h, the cells were subsequently exposed to LXR ligand for 6-24 h. Cell viability, LXR response element (LXRE)-driven luciferase activity, sterol regulatory element binding protein-response element (SREBP-RE)-driven luciferase activity, SREBP-1c expression, and mRNA levels of LXRα and its-dependent target genes were determined. In addition, LC-MS/MS analysis was conducted to explore major compounds in H. lyrata-chloroform fractionated extract #4 (HL-CF4). Results : Of various H. lyrata extracts tested, chloroform extract and its fractionated extract #4, HL-CF4, significantly decreased T0901317-mediated SREBP-1c expression. In addition, HL-CF4 significantly reduced LXRE atransactivation and LXRα mRNA expression without any cytotoxicity. Moreover, HL-CF4 prevented the SREBP-RE-driven luciferase activity and mRNA levels of fatty acid synthase and stearoyl-CoA desaturase-1 induced by T0901317. Results from LC-MS/MS analysis at positive/negative mode indicated that HL-CF4 contained several compounds showing m/z 197.1176 (C₁₁H₁₇O₃), 693.2913/227.1069 (C₃₈H₄₅O₁₂/C₁₅H₁₅O₂), 203.1797 (C₁₅H₂₃), 181.1225 (C₁₁H₁₇O₂), 591.2957 (C₃₅H₄₃O₈), 379.1040 (C₁₈H₁₉O₉), 409.1509 (C₂₀H₂₅O₉), 309.1348 (C₁₆H₂₁O₆), 391.1404 (C₂₀H₂₃O₈), and 669.2924/389.1248 (C₃₆H₄₅O₁₂/C₂₀H₂₁O₈). Conclusion : Based on its inhibition of the LXRα-dependent signaling pathway, H. lyrata chloroform extract and HL-CF4 have prophylactic potentials for managing non-alcoholic fatty liver.

• Korean Journal of Pharmacognosy
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• v.34 no.3 s.134
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• pp.228-232
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• 2003

For effective management of atopic dermatitis, it is important to introduce a therapeutic agent although having the fewest side effects, has the greatest anti- inflammatory effect. In the course of screening anti-inflammatory agents, we obtained BSASM composed of several plant extracts. This study was designed to investigate anti-inflammatory and immunomodulatory effects of BSASM. As a first step, $NF-{\kappa}B$ luciferase reporter assay was performed to know the involvement of BSASM in the production of proinflammatory cytokines because $NF-{\kappa}B$ element has been known to play a major role in expression of cytokine genes such as interleukin-8 (IL-8) or tumor necrosis $factor-{\alpha}\;(TNF-{\alpha})$. LPS (lipolysaccharide)-induced $NF-{\kappa}B$ activation was inhibited by BSASM. In addition, we found the fact that BSASM inhibits LPS-induced produced production of IL-8 and $TNF-{\alpha}$ proinflammatory cytokines, indicating BSASM has anti-inflammatory effect. In interleukin-2 (IL-2) luciferase reporter assay in Jurkat T cells, BSASM reduced PHA (Phytohemagglutinin)-induced IL-2 luciferase activity, suggesting the possibility that BSASM might also have an immunomodulatory function in T cell-mediated immune response. Based on these results, we suggest the possibility that BSASM can be introduced to improve symptom of immune-related skin diseases, namely, atopic dermatitis.

• Journal of the Korea Convergence Society
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• v.13 no.1
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• pp.161-166
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• 2022

HIF-2α is a transcription factor activated mainly in hypoxic condition known to play crucial roles in a wide variety of pathophysiological events including cancer, metabolic syndrome, arthritis etc. In this context, a number of N'-aryl isonicotinolyhydrazides, in which known pharmacophores are included, have been selected from commercial chemical library and tested for the inhibitory activities targeting HIF-2α in cultured HTB94 cell. HRE-luciferase and HIF-2α were introduced into the cell by transfection and adenoviri infection, respectively and the reporter gene assay discovered the potency of 2-hydroxy-1-naphthyl structure. Accordingly, the scaffold has been adjusted based on this structure and subjected to anti-HIF-2α activity test, identifying 2 compounds as HIF-2α inhibitors. The activities were confirmed by false positive test. This study has been performed via the convergence of biology and chemistry and the results may be useful for discovering novel inhibitors and HIF-2α biology studies, and contribute to the development of therapeutic agents.

• Tuberculosis and Respiratory Diseases
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• v.65 no.6
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• pp.476-486
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• 2008

Background: TRAIL (TNF-related apoptosis inducing ligand) is a newly identified member of the TNF gene family which appears to have tumor-selective cytotoxicity due to the distinct decoy receptor system. TRAIL has direct access to caspase machinery and induces apoptosis regardless of p53 phenotype. Therefore, TRAIL has a therapeutic potential in lung cancer which frequently harbors p53 mutation in more than 50% of cases. However, it was shown that TRAIL also could activates $NF-{\kappa}B$ in some cell lines which might inhibit TRAIL-induced apoptosis. This study was designed to investigate whether TRAIL can activate $NF-{\kappa}B$ in lung cancer cell lines relatively resistant to TRAIL-induced apoptosis and inhibition of $NF-{\kappa}B$ activation using proteasome inhibitor MG132 which blocks $I{\kappa}B{\alpha}$ degradation can sensitize lung cancer cells to TRAIL-induced apoptosis. Methods: A549 (wt p53) and NCI-H1299 (null p53) lung cancer cells were used and cell viability test was done by MTT assay. Apoptosis was confirmed with Annexin V assay followed by FACS analysis. To study $NF-{\kappa}B$-dependent transcriptional activation, a luciferase reporter gene assay was used after making A549 and NCI-H1299 cells stably transfected with IgG ${\kappa}-NF-{\kappa}B$ luciferase construct. To investigate DNA binding of $NF-{\kappa}B$ activated by TRAIL, electromobility shift assay was used and supershift assay was done using anti-p65 antibody. Western blot was done for the study of $I{\kappa}B{\alpha}$ degradation. Results: A549 and NCI-H1299 cells were relatively resistant to TRAIL-induced apoptosis showing only 20~30% cell death even at the concentration 100 ng/ml, but MG132 ($3{\mu}M$) pre-treatment 1 hour prior to TRAIL addition greatly increased cell death more than 80%. Luciferase assay showed TRAIL-induced $NF-{\kappa}B$ transcriptional activity in both cell lines. Electromobility shift assay demonstrated DNA binding complex of $NF-{\kappa}B$ activated by TRAIL and supershift with p65 antibody. $I{\kappa}B{\alpha}$ degradation was proven by western blot. MG132 completely blocked both TRAIL-induced $NF-{\kappa}B$ dependent luciferase activity and DNA binding of $NF-{\kappa}B$. Conclusion: This results suggest that inhibition of $NF-{\kappa}B$ can be a potentially useful strategy to enhance TRAIL-induced tumor cell killing in lung cancer.