• Tuberculosis and Respiratory Diseases
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• v.55 no.5
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• pp.488-498
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• 2003

Background : Activation of the transcription factor NF-${\kappa}B$ has been shown to protect cells from tumor necrosis factor-alpha, chemotherapy, and radiation-induced apoptosis. NF-${\kappa}B$-dependent cIAP expression is a major antiapoptotic mechanism for that. NF-${\kappa}B$ activation and cIAP expression in A549 lung cancer cells which is relatively resistant to radiation-induced cell death were investigated for the mechanism of radioresistance. Materials and methods : We used A549 lung cancer cells and Clinac 1800C linear accelerator for radiation. Cell viability test was done by MTT assay. NF-${\kappa}B$ activation was tested by luciferase reporter gene assay, Western blot for $I{\kappa}B{\alpha}$ degradation, and electromobility shift assay. For blocking ${\kappa}B$, MG132 and transfection of $I{\kappa}B{\alpha}$-superrepressor plasmid construct were used. cIAP expression was analyzed by RT-PCR and cIAP2 promoter activity was performed using luciferase assay system. Results : MTT assay showed that cytotoxicity even 48 hr after radiation in A549 cells were less than 20%. Luciferas assay demonstrated weak NF-${\kappa}B$ activation of $1.6{\pm}0.2$ fold compared to PMA-induced $3.4{\pm}0.9$ fold. Radiation-induced $I{\kappa}B{\alpha}$ degradation was observed in Western blot and NF-${\kappa}B$ DNA binding was confirmed by EMSA. However, blocking NF-${\kappa}B$ using MG132 and $I{\kappa}B{\alpha}$-superrepressor transfection did not show any sensitizing effect for radiation-induced cell death. The result of RT-PCR for cIAP1 & 2 expression was negative induction while TNF-${\alpha}$ showed strong expression for cIAP1 & 2. The cIAP2 promoter activity also did not show any change compared to positive control with TNF-${\alpha}$. Conclusion : We conclude that activation of NF-${\kappa}B$ does not determine the intrinsic radiosensitivity of cancer cells, at least for the cell lines tested in this study.

• Tuberculosis and Respiratory Diseases
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• v.49 no.3
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• pp.332-342
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• 2000

Background : The importance of pro-inflammatory cytokines, especially tumor necrosis factor $\alpha$ (INF-$\alpha$) and interleukin-1$\beta$ (IL-1$\beta$), have been extensively documented in the generation of inflammatory lung disease. Lung epithelial cells are also actively involved in initiating and maintaining inflammation by producing pro-inflammatory mediators. Understanding the mechanism of pro-inflammatory cytokine expression in lung epithelial cells is crucial to the development of new therapeutic modalities for inflammatory lung disease. Transcription of most pro-inflammatory cytokines is dependent on the activation of NF-${\kappa}B$. However, the relationship between pro-inflammatory cytokine expression and NF-${\kappa}B/I{\kappa}B$ pathway in lung epithelial cells is not clear. Methods : BEAS-2B, A549, Na-H157, NCI-H719 cells were stimulated with IL-$1{\beta}$ or TNF-$\alpha$ at various times, and then IL-8 and TNF-$\alpha$mRNA expressions were assayed by Northern blot analysis. IL-$1{\beta}$ or TNF-$\alpha$-induced NF-${\kappa}B$ activation was assessed by the nuclear translocation of p65 NF-${\kappa}B$ subunit. The degradation of $I{\kappa}B{\alpha}$ and $I{\kappa}B{\beta}$ by IL-$1{\beta}$ or TNF-$\alpha$stimulation was assayed by Western blot analysis. The phosphorylation of $I{\kappa}B{\alpha}$ was evaluated by Western blot analysis after pre-treating cells with proteasome inhibitor followed by IL-$1{\beta}$ or TNF-$\alpha$ stimulation. The basal level of IKK $\alpha$ expression was evaluated by Western blot analysis. Results: $I{\kappa}B{\alpha}$ and $I{\kappa}B{\alpha}$ was rapidly degraded after 5 minutes of incubation with IL-$1{\beta}$ or TNF-$\alpha$ in BEAS-2B, A549, and NCI-H157 cells. The activation of NF-${\kappa}B{\alpha}$ and the induction of IL-8 and TNF-$\alpha$ mRNA expression were observed by IL-$1{\beta}$ or TNF-$\alpha$ stimulation in these cells. In contrast, neither the changes in NF-${\kappa}B/I{\kappa}B$ pathway nor IL-8 and TNF-$\alpha$mRNA expression was induced by IL-$1{\beta}$ or TNF-$\alpha$ stimulation in NCI-H719 cells. IL-$1{\beta}$ and TNF-$\alpha$-induced $I{\kappa}B$ phosphorylation was observed in BEAS-2B, A549, and NCI-H157 cells, but not in NCI-H719 cells. The basal level of IKK$\alpha$ expression was not different between cell. Conclusion : NF-${\kappa}B/I{\kappa}B$ pathway plays an important role in the expression of pro-inflammatory cytokine in most lung epithelial cells. The absence of the effect on NF-${\kappa}B/I{\kappa}B$ pathway in NCI-H719 cells sæms to be due to the defect in the intracellular signal transduction pathway upstream to IKK.

• BMB Reports
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• v.43 no.12
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• pp.807-812
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• 2010

$NF{\kappa}B$ and ZAS3 are transcription factors that control important cellular processes including immunity, cell survival and apoptosis. Although both proteins bind the ${\kappa}B$-motif, they produce opposite physiological consequences; $NF{\kappa}B$ activates transcription, promotes cell growth and is often found to be constitutively expressed in cancer cells, while ZAS3 generally represses transcription, inhibits cell proliferation and is downregulated in some cancers. Here, we show that ZAS3 inhibits $NF{\kappa}B$-dependent transcription by competing with $NF{\kappa}B$ for the ${\kappa}B$-motif. Transient transfection studies show that N-terminal 645 amino acids is sufficient to repress transcription activated by $NF{\kappa}B$, and that the identical region also possesses intrinsic repression activity to inhibit basal transcription from a promoter. Finally, in vitro DNA-protein interaction analysis shows that ZAS3 is able to displace $NF{\kappa}B$ by competing with $NF{\kappa}B$ for the ${\kappa}B$-motif. It is conceivable that ZAS3 has therapeutic potential for controlling aberrant activation of $NF{\kappa}B$ in various diseases.

• 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.

• Journal of Acupuncture Research
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• v.27 no.3
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• pp.1-13
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• 2010

목적 : 이 연구는 봉약침의 봉독과 그 주요성분인 멜리틴이 NF-${\kappa}B$의 활성억제와 세포자멸사 관련 단백질의 발현 조절을 통하여 세포자멸사를 유도함으로써 전립선 암세포주인 PC-3 세포의 성장을 억제하는지를 확인하고 해당 기전을 살펴보고자 하였다. 방법 : 봉독이나 멜리틴을 처리한 후 PC-3의 성장억제를 관찰하기 위해 WST-1 assay, CCK-8 assay를 시행하였고, 세포자멸사 조절단백질의 변동 관찰에는 western blot analysis를 시행하였고, 세포자멸사와 연관된 NF-${\kappa}B$의 활성 변화를 관찰하기 위해 EMSA를 시행하였으며, PC-3에서 봉독이나 멜리틴과 NF-${\kappa}B$의 상호작용을 관찰하기 위해 transient transfection assay를 시행하여 세포생존율과 NF-${\kappa}B$의 활성 변동을 측정하였다. 결과 : PC-3 세포에 봉독이나 멜리틴을 처리한 후, 전립선암세포의 성장, 세포자멸사의 유발, 세포자멸사 관련 단백질의 발현, NF-${\kappa}B$의 활성, NF-${\kappa}B$의 p50, $IKK{\alpha}$, $IKK{\beta}$ 치환 후 NF-${\kappa}B$의 활성과 PC-3 세포 증식에 미치는 영향을 관찰하여 다음과 같은 결과를 얻었다. 1. PC-3 세포에서 봉독이나 멜리틴을 처리한 후 세포자멸사가 유도되어 세포성장이 억제되었고, 세포자멸사 관련 단백질 중 분리된 PARP, caspase-3, -9는 유의한 증가를, Bcl-2, XIAP, cXIAP2는 유의한 감소를 나타내었다. 2. PC-3 세포에서 봉독이나 멜리틴을 처리한 후 NF-${\kappa}B$의 활성은 유의한 감소를 나타내었다. 3. PC-3 세포에서 NF-${\kappa}B$의 p50, $IKK{\alpha}$, $IKK{\beta}$를 치환하여 작용기를 없애고 봉독이나 멜리틴을 처리하였을 경우에도 NF-${\kappa}B$의 활성이 유의한 감소를 나타내었다. 결론 : 이상의 결과는 봉독이나 멜리틴이 NF-${\kappa}B$의 활성 억제를 통하여 인간 전립선암세포주인 PC-3의 세포자멸사를 유발함으로써 증식억제 효과가 있음을 입증한 것으로, 전립선암의 예방과 치료에 대한 효과적인 치료제 개발에 도움이 될 것으로 기대된다.

• Journal of Acupuncture Research
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• v.26 no.3
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• pp.39-48
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• 2009

목적 : 이 연구는 봉약침의 주요성분인 멜리틴이 NF-${\kappa}$B의 활성억제를 통하여 세포자멸사를 유도하고, 전립선 암세포주인 DU-145 세포의 성장을 억제하는지를 확인하고 멜리틴의 NF-${\kappa}$B 활성억제기전을 살펴보고자 하였다. 방법 : 멜리틴을 처리한 후 DU-145의 성장억제를 관찰하기 위해 WST-1 assay를 시행하였고, 세포자멸 사의 관찰에는 DAPI stairung assay를 통한 세포형태관찰을 시행하였으며, 염증관련유전자 발현 관찰에는 western blot analysis를 시행하였고, 세포자멸사와 연관된 NF-${\kappa}$B의 활성 변화를 관찰하기 위해 EMSA와 luciferase assay를 시행하였으며, DU-145에서 멜리틴과 NF-${\kappa}$B의 상호작용을 관찰하기 위해 transient transfection assay를 시행 시 세포생존율과 NF-${\kappa}$B의 활성 변동을 측정하였다. 결과 : DU-145 세포에 멜리틴을 처리한 후, 전립선암세포의 성장, 세포자멸사의 유발, 염중관련유전자 발현 및 NF-${\kappa}$B의 활성, NF-${\kappa}$B의 p50 치환 후 NF-${\kappa}$B의 활성과 DU-145 세포 증식에 미치는 영향을 관찰하여 다음과 같은 결과를 얻었다. 1. DU-145 세포에서 멜리틴을 처리한 후 세포자멸사가 유도되어 세포성장이 억제되었다. 2. DU-145 세포에서 멜리틴을 처리한 후 염증관련유전자 발현 및 NF-${\kappa}$B의 활성에 유의한 감소를 나타내었다. 3. DU-145 세포에서 NF-${\kappa}$B의 p50와 IKK들을 치환하여 작용기를 없애고 멜리틴을 처리하였을 경우에도 세포활성 및 NF-${\kappa}$B의 활성의 유의한 감소를 나타내었다.

• Tuberculosis and Respiratory Diseases
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• v.54 no.4
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• pp.449-458
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• 2003

Background : Cyclosporin A(CsA) and tacrolimus(FK506) have been widely used as immunosuppressants. The effects of CsA, or FK506, on the $I{\kappa}B/NF-{\kappa}B$ pathway have been shown to vary according to the cell type. However, their effects on the $I{\kappa}B/NF-{\kappa}B$ pathway have not been reported in bronchial epithelial cells. In this study, the effects of CsA and FK506 on the $I{\kappa}B/NF-{\kappa}B$ pathway in bronchial epithelial cells, monocytes, lymphocytes and alveolar macrophages were evaluated. The relationship between their effects on the $I{\kappa}B/NF-{\kappa}B$ pathway and $I{\kappa}B$ kinase(IKK) activity was also investigated. Methods : BEAS-2B and A549 cells, pulmonary alveolar macrophages, peripheral blood monocytes and lymphocytes were used. The cells were pre-treated with CsA, or FK506, for various time periods, followed by stimulation with TNF-${\alpha}$, LPS or IL-$1{\beta}$. The $I{\kappa}B{\alpha}$ expressions were assayed by Western blot analyses. The IKK activity was evaluated by an in vitro immune complex kinase assay, using GST-$I{\kappa}B{\alpha}$ as the substrate. Results : Neither CsA nor FK506 affected the level of $I{\kappa}B{\alpha}$ expression in any of the cell types used in this study. CsA pre-treatment inhibited the TNF ${\alpha}$-induced $I{\kappa}B{\alpha}$ degradation in bronchial epithelial cells. In contrast, the TNF ${\alpha}$-induced $I{\kappa}B{\alpha}$ degradation was not affected by FK506 pre-treatment. However, FK506 suppressed the cytokine-induced $I{\kappa}B{\alpha}$ degradation in the pulmonary alveolar macrophages, peripheral blood monocytes and lymphocytes. The inhibitory effect of CsA, or FK506, on $I{\kappa}B{\alpha}$ degradation was not related to IKK. Conclusions : CsA and FK506 suppressed the $I{\kappa}B{\alpha}$ degradation in bronchial epithelial cells, monocytes, lymphocytes and alveolar macrophages, so this may not be mediated through IKK.

• Molecules and Cells
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• v.37 no.3
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• pp.189-195
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• 2014

The NF-${\kappa}B$ pathway transcriptionally controls a large set of target genes that play important roles in cell survival, inflammation, and immune responses. While many studies showed anti-tumorigenic and pro-survival role of NF-${\kappa}B$ in cancer cells, recent findings postulate that NF-${\kappa}B$ participates in a senescence-associated cytokine response, thereby suggesting a tumor restraining role of NF-${\kappa}B$. In this review, we discuss implications of the NF-${\kappa}B$ signaling pathway in cancer. Particularly, we emphasize the connection of NF-${\kappa}B$ with cellular senescence as a response to chemotherapy, and furthermore, present examples how distinct oncogenic network contexts surrounding NF-${\kappa}B$ produce fundamentally different treatment outcomes in aggressive B-cell lymphomas as an example.

• Journal of Acupuncture Research
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• v.23 no.3
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• pp.91-102
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• 2006

목적 : 이 연구에서는 FBS에 의하여 유도되는 혈관 평활근 세포 증식에 대한 봉약침액과 Melittin의 세포 고사효과의 영향 및 작용 기전을 살펴보고자 하였다. 방법 : $I{\kappa}Ba$, p-$I{\kappa}Ba$, p-ERK1/2, p-Akt, p53, Bcl-2, Bax 및 active caspase-3는 Western blotting을, $NF-{\kappa}B$는 EMSA와 immunofluorescence staining을 이용하여 측정하였다. 결과 : 1. Melittin은 $NF-{\kappa}B$ 활성에 대하여 $I{\kappa}Ba$의 인산화를 유의하게 익제하고 $I{\kappa}Ba$를 증가시켰으며, $NF-{\kappa}B$의 DNA 결합과 $NF-{\kappa}B$ p50의 핵 내 유입을 유의하게 감소시켰다. 2. Melittin은 $NF-{\kappa}B$ 활성을 증가시키는 물질인 Akt의 인산화를 유의하게 억제하였고, ERK1/2의 인산화도 억제하였다. 3. Melittin은 세포사멸 전구 단백질인 p53, Bax 및 caspase-3의 발현을 유의하게 증가시켰고, 세포사멸억제 단백질인 Bcl-2의 발현은 감소시켰다. 결론 : 이상의 결과는 $NF-{\kappa}B$ 와 Akt 활성을 억제함으로써 혈관평활근세포 증식을 억제하는 효과가 있음을 입증한 것이며, 향후 안전성 연구를 바탕으로 혈관성형술 후 재발성협착증과 동맥경화증의 치료제로 사용될 수 있을 것으로 기대된다.

• Tuberculosis and Respiratory Diseases
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• v.68 no.3
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• pp.168-174
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• 2010

Background: Sepsis still has a high mortality rate despite adequate supportive care. Newer therapeutic modalities have been developed but they have generally ended in failure. Recently, insulin was reported to have an anti-inflammatory effect by inhibiting the $I{\kappa}B/NF-{\kappa}B$ pathway, and may have therapeutic potential in sepsis. However, the precise mechanism of the anti-inflammatory effect of insulin is unclear. This study examined the role of insulin in activating $I{\kappa}B/NF-{\kappa}B$ in macrophage. Methods: Raw 264.7 cells, a murine macrophage cell line, were used in this experiment. Western blotting using $I{\kappa}B$ Ab and phosphor-specific $I{\kappa}B$ Ab was performed to evaluate the degradation and phosphorylation of $I{\kappa}B$ cells. For the $I{\kappa}B$ Kinase (IKK) activity, an immune complex kinase assay was performed. The level of interleukin-6 (IL-6) was measured by ELISA to determine the level of proinflammatory cytokine. Results: $I{\kappa}B{\alpha}$ degradation began 30 min after lipopolysaccharide (LPS) treatment. However, an insulin pretreatment suppressed the $I{\kappa}B{\alpha}$ degradation caused by the LPS treatment. The phosphorylation of $I{\kappa}B{\alpha}$ and IKK activity was also inhibited by the insulin pretreatment. Finally, the insulin pretreatment showed a tendency to suppress the induction of IL-6 by LPS. Conclusion: Insulin might have an anti-inflammatory effect though partial inhibition of the $I{\kappa}B/NF{\kappa}B$ pathway in macrophage cell lines.