• Tuberculosis and Respiratory Diseases
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• v.50 no.1
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• pp.52-66
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• 2001

Background : NF-${\kappa}B$ is the most important transcriptional factor in IL-8 gene expression. Triptolide is a new compound that recently has been shown to inhibit NF-${\kappa}B$ activation. The purpose of this study is to investigate how triptolide inhibits NF-${\kappa}B$-dependent IL-8 gene transcription in lung epithelial cells and to pilot the potential for the clinical application of triptolide in inflammatory lung diseases. Methods : A549 cells were used and triptolide was provided from Pharmagenesis Company (Palo Alto, CA). In order to examine NF-${\kappa}B$-dependent IL-8 transcriptional activity, we established stable A549 IL-8-NF-${\kappa}B$-luc. cells and performed luciferase assays. IL-8 gene expression was measured by RT-PCR and ELISA. A Western blot was done for the study of $I{\kappa}B{\alpha}$ degradation and an electromobility shift assay was done to analyze NF-${\kappa}B$ DNA binding. p65 specific transactivation was analyzed by a cotransfection study using a Gal4-p65 fusion protein expression system. To investigate the involvement of transcriptional coactivators, we perfomed a transfection study with CBP and SRC-1 expression vectors. Results : We observed that triptolide significantly suppresses NF-${\kappa}B$-dependent IL-8 transcriptional activity induced by IL-$1{\beta}$ and PMA. RT-PCR showed that triptolide represses both IL-$1{\beta}$ and PMA-induced IL-8 mRNA expression and ELISA confirmed this triptolide-mediated IL-8 suppression at the protein level. However, triptolide did not affect $I{\kappa}B{\alpha}$ degradation and NF-$_{\kappa}B$ DNA binding. In a p65-specific transactivation study, triptolide significantly suppressed Gal4-p65T Al and Gal4-p65T A2 activity suggesting that triptolide inhibits NF-${\kappa}B$ activation by inhibiting p65 transactivation. However, this triptolide-mediated inhibition of p65 transactivation was not rescued by the overexpression of CBP or SRC-1, thereby excluding the role of transcriptional coactivators. Conclusions : Triptolide is a new compound that inhibits NF-${\kappa}B$-dependent IL-8 transcriptional activation by inhibiting p65 transactivation, but not by an $I{\kappa}B{\alpha}$-dependent mechanism. This suggests that triptolide may have a therapeutic potential for inflammatory lung diseases.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.11
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• pp.5511-5515
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• 2012

Introduction: The nuclear factor ${\kappa}B$ (NF-${\kappa}B$) is a super family of transcription factors which plays important roles in development and progression of cancer. The present investigation concerns NF-${\kappa}B$ /p65 activity in human breast cancers with overexpression of ER, PR, HER-2/neu, as well as the significance of p65 expression with regard to menopausal status, stage, grade, tumor size, nodal status, and NPI of invasive ductal carcinomas in Eastern India. Materials and Methods: In this hospital based study 57 breast cancer patients attending a Breast Clinic of a reputed institute of Eastern India were assessed for p65 protein expression in breast tumor tissue samples by Western blotting. ER, PR and HER-2/neu expression was determined by immunohistochemistry. Results: NF-${\kappa}B$/p65 was significantly associated with advanced stage, large tumor size (${\geq}5$ cm), high grade, negative ER, negative PR, and positive HER-2/neu. High NF-${\kappa}B$/p65 expression was more frequent in patients with a high NPI ($NPI{\geq}5.4$, 84.6%) compared with low NPI (<5.4, 44.4%) and this association was statistically significant (p = 0.002). Conclusion: NF-${\kappa}B$/p65 overexpression was associated with advanced stage, large tumor size, high grade, and high NPI which are poor prognostic factors linked to enhanced aggressiveness of the disease. NF-${\kappa}B$/p65 expression implies aggressive biological behavior of breast cancer and this study validates significant association of NF-${\kappa}B$ /p65 overexpression with negative estrogen and progesterone receptor status and overexpression of HER-2/neu oncoprotein. In our good clinical practice, patients with NF-${\kappa}B$ positive tumors need to be treated aggressively.

• Food Science and Biotechnology
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• v.15 no.6
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• pp.975-979
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• 2006

The extracts of Oenanthe javanica were evaluated for their effects on the expression of cyclooxygenase-2 (COX-2), which is mediated by the translocation of the p65 subunit into the nucleus. Fractions of ethyl acetate and chloroform from 80% ethanol extracts of O. javanica exhibited inhibitory effects on the secretion of tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) from lipopolysaccharide (LPS)-stimulated peritoneal macrophages; however, the aqueous- and hexane-fractions showed no significant effect. The ethyl acetate- and chloroform-fractions also reduced the COX-2 enzyme levels after 24-hr treatment. RT-PCR showed that the mRNA levels of COX-2 decreased following treatment with these fractions, suggesting that COX-2 expression is transcriptionally regulated by these extracts. We examined the effects of the chloroform- and ethyl acetate-fractions on the cytosolic activation of nuclear factor-${\kappa}B$ ($NF-{\kappa}B$, p65 subunit) and on the degradation of inhibitor-${\kappa}B{\alpha}$ ($I-{\kappa}B{\alpha}$) in order to determine the mechanism of COX-2 regulation. The LPS-stimulated activation of the p65 subunit was significantly blocked upon the addition of $50\;{\mu}g/mL$ of these fractions, and the cytosolic $I-{\kappa}B{\alpha}$ degradation process was simultaneously inhibited. These findings suggest that the inhibition of COX-2 expression by the ethyl acetate-and chloroform-fractions may result from the inhibition of p65 translocation by blocking the degradation of $I-{\kappa}B{\alpha}$; this may be the mechanistic basis for the anti-inflammatory effects of O. javanica.

• Journal of Veterinary Clinics
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• v.28 no.2
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• pp.190-195
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• 2011

Nuclear factor ${\kappa}B$ (NF-${\kappa}B$) is a nuclear transcription factor that modulates the expression of inflammatory cytokines such as tumor necrosis factor (TNF)-${\alpha}$. trans-10, cis-12 (t10c12)-conjugated linoleic acid (CLA) participates in the inhibition of TNF-${\alpha}$ production upon lipopolysaccharide (LPS)-stimulation. However, in our previous study, t10c12-CLA enhanced the production of TNF-${\alpha}$ by LPS-unstimulated porcine peripheral blood mononuclear cells (PBMCs) and RAW 264.7 macrophages in vitro. To resolve this apparent contradiction, we hypothesized that the effect of t10c12-CLA on TNF-${\alpha}$ production depends on NF-${\kappa}B$ activation induced by LPS stimulation. To test this hypothesis, we assessed the in vitro effect of t10c12-CLA on TNF-${\alpha}$ production and NF-${\kappa}B$ p65 activity in LPS-stimulated and LPS-unstimulated porcine PBMCs. t10c12-CLA treatment resulted in increased TNF-${\alpha}$ production by LPS-unstimulated PBMCs but decreased TNF-${\alpha}$ production by LPS-stimulated PBMCs. t10c12-CLA increased the degradation of inhibitory ${\kappa}B$ ($I{\kappa}B$)-${\alpha}$ protein and activated NF-${\kappa}B$ p65 in LPS-unstimulated PBMCs, but had the opposite effect in LPS-stimulated PBMCs. Notably, t10c12-CLA enhanced NF-${\kappa}B$ p65 binding activity in LPS-unstimulated PBMCs exposed to caffeic acid phenethyl ester (CAPE), a NF-${\kappa}B$ inhibitor. Conversely, it inhibited NF-${\kappa}B$ p65 binding activity in LPS-stimulated PBMCs exposed to CAPE. These results suggest that t10c12-CLA may have different actions under different physiological conditions, and that its effect may be associated with a change in NF-${\kappa}B$ p65 activity.

• 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.

• Tuberculosis and Respiratory Diseases
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• v.46 no.2
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• pp.185-194
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• 1999

Background: NF-$\kappa$B is a characteristic transcriptional factor whose functional activity is determined by post-translational modification of protein and subsequent change of subcellular localization. The involvement of the NF-$\kappa$B family of the transcription factors in the control of such vital cellular functions as immune response, acute phase reaction, replication of certain viruses and development and differentiation of cells has been clearly documented in many previous studies. Several recent observations have suggested that the NF-$\kappa$B might also be involved in the carcinogenesis of some hematological and solid tumors. Investigating the possibility that members of the NF-$\kappa$B family participate in the molecular control of malignant cell transformation could provide invaluable information on both molecular pathogenesis and cancer-related gene therapy. Method: To determine the expression patterns and functional roles of NF-$\kappa$B family transcription factors in human lung cancer cell lines NCI-H792, NCI-H709, NCI-H226 and NCI-H157 were analysed by western blot, using their respective antibodies. The nuclear and the cytoplasmic fraction of protein extract of these cell lines were subsequently obtained and NF-$\kappa$B expression in each fraction was again determined by western blot analysis. The type of NF-$\kappa$B complex present in the cells was determined by immunoprecipitation. To detect the binding ability of cell-line nuclear extracts to the KB consensus oligonucleotide, electrophoretic mobility shift assay(EMSA) was performed. Results: In the cultured human lung cancer cell lines tested, transcription factors of the NF-$\kappa$B family, namely the p50 and p65 subunit were expressed and localized in the nuclear fraction of the cellular extract by western blot analysis and immunocytochemistry. Immunoprecipitation assay showed that in the cell, the p50 and p65 subunits made NF-$\kappa$B complex. Finally it was shown by Electrophoretic Mobility Shift Assay(EMSA) that nuclear extracts of lung cancer cell lines are able to bind to NF-$\kappa$B consensus DNA sequences. Conclusion: These data suggest that in human lung cancer cell lines the NF-$\kappa$B p50/p65 complex might be activated. and strengthen the hypothesis that NF-$\kappa$B family transcription factors might be involved in the carcinogenesis of human lung cancer.

• Tuberculosis and Respiratory Diseases
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• v.52 no.5
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• pp.485-496
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• 2002

Background : The NF-${\kappa}B$ transcription factors control various biological processes including the immune response, acute phase reaction and cell cycle regulation. NF-${\kappa}B$ complexes are retained in the cytoplasm in the basal state and various stimuli cause a translocation of the NF-${\kappa}B$ complexes into the nucleus where they bind to the ${\kappa}B$ elements and regulate the transcription of the target genes. Recent reports also suggest that NF-${\kappa}B$ proteins are involved in oncogenesis, tumor growth and metastasis. High expression of NF-${\kappa}B$ expression was reported in many cancer cell lines and tissues. The constitutive activation of NF-${\kappa}B$ was also reported in several cancer cell lines supporting its role in cancer development and survival. The anti-apoptotic action of NF-${\kappa}B$ is important for cancer survival. NF-${\kappa}B$ also controls the expression of several proteins that are important for cellular adhesion (ICAM-1, VCAM-1) suggesting a role in cancer metastasis. In lung cancer, high expression levels of the NF-${\kappa}B$ subunit p50 and c-Rel were reported. In fact, high expression does not mean a high activity, and the activation pattern of NF-${\kappa}B$ in lung cancer has not been reported. Materials and Methods : In this study, the NF-${\kappa}B$ nuclear binding activity in the basal and TNF-${\alpha}$ stimulated states were exmined in various lung cancer cell lines and compared with the normal bronchial epithelial cell line. Twelve lung cancer cell lines including the non-small cell and small cell lung cancer cell lines (A549, NCI-H358, NCI-H441, NCI-H552, NCI-H2009, NCI-H460, NCI-H1229, NCI-H1703, NCI-H157, NCI-H187, NCI-H417, NCI-H526) and BEAS-2B bronchial epithelial cell line were used. To evaluate the NF-${\kappa}B$ expression and DNA binding activity, western blot analysis and an electrophoretic mobility shift assay with the nuclear protein extracts. Results : The basal expressions of the p65 and p50 subunits were observed in the BEAS-2B cell line and all lung cancer cell lines except for NCI-H358 and NCI-H460. The expression levels of p65 and p50 were increased 30 minutes after stimulation with TNF-${\alpha}$ in BEAS-2B and in 10 lung cancer cell lines. In the NCI-H358 and NCI-H460 cell lines, p65 expression was not observed in the basal and stimulated states and the two p50 related protein levels were higher after stimulation with TNF-${\alpha}$ These new proteins were smaller than p50 and are thought to be variants of p50. In the basal state, NF-${\kappa}B$ was nearly activated in the BEAS-2B and all lung cancer cell lines. The DNA binding activity of the NF-${\kappa}B$ complexes was markedly higher after stimulation with TNF-${\alpha}$ In the BEAS-2B and all lung cancer cell line except for NCI-H358 and NCI-H460, the activated NF-${\kappa}B$ complex was a p65/p50 heterodimer. In the NCI-H358 and NCI-H460 lung cancer cell lines, the NF-${\kappa}B$ complex was variant of a p50/p50 homodimer. Conclusion : The NF-${\kappa}B$ activation pattern in the lung cancer cell lines and the normal bronchial epithelial cell lines was similar except for the activation of a variant of the p50/p50 homodimer in some lung cancer cell linse.

• Nutrition Research and Practice
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• v.17 no.1
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• pp.164-173
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• 2023

BACKGROUND/OBJECTIVES: Hyperglycemia is a major cause of diabetes and diabetesrelated diseases. Sodium butyrate (NaB) is a short-chain fatty acid derivative that produces dietary fiber by anaerobic bacterial fermentation in the large intestine and occurs in foods, such as Parmesan cheese and butter. Butyrate has been shown to prevent obesity, improve insulin sensitivity, and ameliorate dyslipidemia in diet-induced obese mice. Therefore, this study examined the effects and mechanism of NaB on the secretion of inflammatory cytokines induced by high glucose (HG) in THP-1 cells. MATERIALS/METHODS: THP-1 cells were used as an in vitro model for HG-induced inflammation. The cells were cultured under normal glycemic or hyperglycemic conditions with or without NaB (0-25 μM). Western blotting and quantitative polymerase chain reaction were used to evaluate the protein and mRNA levels of nuclear factor-κB (NF-κB), interleukin-6, tumor necrosis factor-α, acetylated p65, acetyl CREB-binding protein/p300 (CBP/p300), and p300 using THP-1 cells. Histone acetyltransferase (HAT), histone deacetylase (HDAC), and pro-inflammatory cytokine secretion activity were analyzed using an enzyme-linked immunosorbent assay. RESULTS: HG significantly upregulated histone acetylation, acetylation levels of p300, NF-κB activation, and inflammatory cytokine release in THP-1 cells. Conversely, the NaB treatment reduced cytokine release and NF-κB activation in HG-treated cells. It also significantly reduced p65 acetylation, CBP/p300 HAT activity, and CBP/p300 gene expression. In addition, NaB decreased the interaction of p300 in acetylated NF-κB and TNF-α. CONCLUSIONS: These results suggest that NaB suppresses HG-induced inflammatory cytokine production through HAT/HDAC regulation in monocytes. NaB has the potential for preventing and treating diabetes and its related complications.

• The Korean Journal of Physiology and Pharmacology
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• v.27 no.4
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• pp.375-381
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• 2023

Numerous studies have revealed the importance of tumor-derived exosomes in rectal cancer (RC). This study aims to explore the influence of tumor-derived exosomal integrin beta-1 (ITGB1) on lung fibroblasts in RC along with underlying mechanisms. Exosome morphology was observed using a transmission electron microscope. Protein levels of CD63, CD9, ITGB1, p-p65 and p65 were detected using Western blot. To determine ITGB1's mRNA expression, quantitative real-time polymerase chain reaction was used. Moreover, levels of interleukin (IL)-8, IL-1β, and IL-6 in cell culture supernatant were measured via commercial ELISA kits. ITGB1 expression was increased in exosomes from RC cells. The ratio of p-p65/p65 as well as levels of interleukins in lung fibroblasts was raised by exosomes derived from RC cells, while was reduced after down-regulation of exosomal ITGB1. The increased ratio of p-p65/p65 as well as levels of pro-inflammatory cytokines caused by exosomes from RC cells was reversed by the addition of nuclear factor kappa B (NF-κB) inhibitor. We concluded that the knockdown of RC cells-derived exosomal ITGB1 repressed activation of lung fibroblasts and the NF-κB pathway in vitro.

• The Korea Journal of Herbology
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• v.26 no.4
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• pp.31-37
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• 2011

Objectives : Chrysanthemum boreale flower is widely distributed in Korea, Japan, China, and Eastern countries. C. boreale flower is also one of the herbs used for the treatment of various inflammatory disease in Korean Medicine. So, this research was designed to study anti-inflammatory effect of C. boreale flower and its mechanism. Methods : We investigated nitro oxide (NO) and prostaglandin $E_2$ ($PGE_2$) production by ELISA. And expressions of inducible nitric oxide synthase (iNOS), Cyclooxygenase-2 (COX-2) and nuclear factor-${\kappa}B$ P50/65 (NF-${\kappa}B$ P50, NF-${\kappa}B$ P65) were measured in RAW 264.7 murine macrophage cells induced by LPS. Results : MeOH ex., EtOAc fr., $CHCl_3$ fr. and Water fr. of C. boreale flower showed anti-inflammatory effect through inhibition of NO and PGE expression respectively. Among them, EtOAc fr. and $CHCl_3$ fr. inhibited production of NO and $PGE_2$ through inhibition of iNOS and COX-2 expression. And MeOH ex., EtOAc fr. and $CHCl_3$ fr. inhibited translocation of NF-${\kappa}B$ P65, NF-${\kappa}B$ P50 by inhibiting phosphrylation of $I{\kappa}B$. Conclusions : MeOH ex. EtOAc fr, $CHCl_3$ fr., and Water fr. of the C. boreale flower have anti-inflammatory activity.