• Tuberculosis and Respiratory Diseases
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• 제52권5호
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• pp.485-496
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• 2002

연구배경 : Nuclear factor ${\kappa}B$ (NF-${\kappa}B$)는 면역기능, 급성기 반응, 세포주기 조절 등 다양한 세포활동을 조절하는 전사인자로서 외부 자극에 의해 세포질에 존재하던 NF-${\kappa}B$가 핵 속으로 이동되어 여러 유전자의 ${\kappa}B$ element에 결합하여 그 유전자의 전사를 가져온다. 최근 들어 암의 발생과 증식 및 전이에 있어 NF-${\kappa}B$의 역할이 주목받고 있다. 즉, 여러 종류의 암세포에서 NF-${\kappa}B$의 과발현 및 지속적인 활성화가 알려져 NF-${\kappa}B$와 암의 발생 및 증식과의 관련성이 제시되고 있고, NF-${\kappa}B$의 항 아포프토시스 기능은 암세포의 생존에서 중요한 역할을 하는 것으로 이해되고 있다. 또한 ICAM-l, VCAM-l 등 세포 부착물질의 발현에 영향을 끼쳐 암 전이와의 관련성도 제시되고 있다. 폐암에서 NF-${\kappa}B$의 역할에 관한 연구는 많지 않은 상태로 폐암 조직 및 폐암 세포주에서 p50과 c-Rel의 과발현이 보고된 바 있다. 그러나 NF-${\kappa}B$의 과발현이 NF-${\kappa}B$의 활성화를 의미하는 것은 아니며 현재까지 폐암 세포에서 NF-${\kappa}B$의 활성화 유무에 관한 연구는 없는 실정이다. 방 법 : 본 연구에서는 정상 기관지 세포주와 폐암 세포주에서 기저 상태와 외부 자극에 의한 NF-${\kappa}B$의 활성화를 비교하여 폐암 세포에서 NF-${\kappa}B$의 활성도를 평가하였다. 정상 기관지 상피세포로는 BEAS-2B 세포주를 사용하였고 폐암 세포주로는 A549, NCI-H358, NCI-H441, NCI-H522, NCI-H2009, NCI-H460, NCI-H1229, NCI-H1703, NCI-H157, NCI-H187, NCI-H417, NCI-H526 등 12종을 실험에 사용하였다. NF-${\kappa}B$의 활성화는 p65와 p50의 핵내 발현과 electrophoretic mobility shift assay (EMSA)를 이용한 NF-${\kappa}B$ DNA binding activity로 평가하였다. 결 과 : NCI-H358과 NCI-H460 세포를 제외한 모든 폐암 세포주와 BEAS-2B 세포의 기저상태에서 핵 단백질내에 p65와 p50의 발현이 관찰되었다. TNF-$\alpha$로 자극하고 30분이 경과한 후에는 핵 내 p65와 p50의 발현이 증가하였다. NCI-H358과 NCI-H460 세포에서는 기저 상태와 TNF-${\alpha}$ 자극 시 핵 단백질 내의 p65의 발현이 관찰되지 않았고 TNF-${\alpha}$ 자극했을 때에도 p65의 발현은 증가하지 않았다. 그러나 이 두 세포주에서는 TNF-${\alpha}$로 자극 시 p50보다 분자량이 작은 두 종의 단백질의 발현이 증가되어 p50의 변형된 형태로 생각되었다. 기저 상태에서의 NF-${\kappa}B$의 DNA 결합능은 실험에 사용한 모든 세포주에서 거의 관찰되지 않았고 TNF-${\alpha}$ 자극 시 유의하게 증가하였다. TNF-${\alpha}$ 자극으로 활성화된 NF-${\kappa}B$ complex는 NCI-H358 과 NCI-H460을 제외한 모든 세포주에서는 p50/p65 heterodimer로 확인되었고 NCI-H358과 NCI-H460에서는 변형된 p50/p50 homodimer가 활성화되었다. 결 론 : 이상의 결과로 일부 폐암 세포주에서 외부 자극으로 활성화된 NF-${\kappa}B$ complex의 구성에 차이를 보였지만 전체적으로는 정상 기관지 세포주와 비교해 폐암 세포해서 NF-${\kappa}B$ 활성화에 있어 큰 차이가 없었다.

• 한국환경성돌연변이발암원학회:학술대회논문집
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• 한국환경성돌연변이발암원학회 2002년도 Current Trends in Toxicological Sciences
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• pp.100-100
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• 2002

• Toxicological Research
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• 제22권3호
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• pp.293-299
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• 2006

We demonstrate that magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, inhibits LPS-induced expression of iNOS gene in RAW 264.7 cells(murine macrophage cell line). Treatment of RAW 264.7 cells with magnolol inhibited LPS-stimulated nitric oxide production in a dose-related manner. RT-PCR analysis showed that the decrease of NO was due to the inhibition of iNOS gene expression. Western immunoblot analysis of phosphorylate p38 kinase showed magnolol significantly inhibited the phosphorylation of p38 kinase which is important in the regulation of iNOS gene expression. The specific p38 inhibiter SB203580 abrogated the LPS-induced NO generation and iNOS expression, whereas the selective MEK-1 inhibitor PD98059 did not affect the NO induction. Immunostaining of p65 and reporter gene assay showed that magnolol inhibited NF-${\kappa}/Rel$ nuclear translocation and transcriptional activation, respectively. Collectively, this series of experiments indicates that magnolol inhibits iNOS gene expression by blocking NF-k/Rel and p38 kinase signaling. Due to the critical role that NO release plays in mediating inflammatory responses, the inhibitory effects of magnolol or iNOS suggest that magnolol may represent a useful anti-inflammatory agent.

• 대한한방종양학회지
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• 제6권1호
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• pp.67-79
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• 2000

Objective : Hedyotis diffusa has been used as an anticancer agent for several decades in oriental medicine. We test whether the methanol extract of the herb affects transcriptional activation factors including $NF-{\kappa}B$ and AP-1. Methods : 1. HL-60 cells were treated with various concentrations(from 200 to $50{\mu}g/ml$) of methanol extract and $H_2O$ extract($200{\mu}g/ml$)of hedyotis diffusa, After 48h later, the cells were tested for viability by MTT assay. 2. The HL-60 cells were treated with $200{\mu}g/ml$ of methanol extract for the indicated periods. First. Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$ and AP-1. Second. Nuclear extracts were isolated and reacted with p50, p65. c-rel pan-Jun, c-Jun, JunB. JunD antibody on ice for 30min. Finally The cell lysates were prepared and analyzed by western blotting using anti-Fas, anti-FasL and anti-p53 antibody. Results : 1. The methanol extract decreases the viability of human lymphoid origin leukemia HL-60 cells in a dose-dependent manner. 2. $NF-{\kappa}B$ is rapidly activated by the addition of the methanol extract, reaches a peak at 30min and gradually returns to resting level. We confirm that $NF-{\kappa}B$ is a heterodimer mainly composed of p65 subunit with c-Rel. 3. Transcriptional activation of AP-1 is detected at 30min and reaches a maximum at 1hr after stimulation of the cells with the methanol extract. AP-1 is mainly composed with Jur-D and partially Jug-B proteins. 4. the methanol extract of Hedyotis diffusa induces the expression of Fas, Fas ligand and p53 proteins of HL-60 cells in a time dependent fashion. Conclusions : These results suggest that the methanol extract of Hedyotis diffusa exerts anticancer effects to induce the death of human leukomic HL-60 cells via activation of trascriptional factors such as $NF-{\kappa}B$ and AP-1, increase in expression of Fas mediated signalling proteins, and induction of tumor suppressor gene. p53.

• 대한한의학회지
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• 제29권5호
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• pp.111-117
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• 2008

Objectives and methods : Previous mechanistic studies suggest the cyclooxygenase-2 (COX-2) inhibitors represent the good candidates against tumor progression. MeOH extract of the stem barks of Euonymus alatus induced the strong inhibition of COX-2. A phenolic compound responsible for the anti- COX-2 known to involve in tumor adhesion and invasion has been studied through the methanol extracts. The compound, rosmarinic acid (ROS-A) was an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. ROS-A showed a strong inhibitory effect of COX-2 activity in a concentration-dependent manner. Then we have measured the IL-1${\beta}$, IL-6 and TNF-${\alpha}$ production related the immune regulation, induction of inflammatory related genes. Results and Conclusions :Hep3B cells produce proinflammatory cytokines of IL-1${\beta}$, IL-6 and TNF-${\alpha}$ while ROS A inhibited the cytokines production. Since IL-1${\beta}$, IL-6 and TNF-${\alpha}$ need the transcription factors such as nuclear factor- ${\kappa}$B (NF-${\kappa}$B) and activated protein-1 (AP-1), we measured the transcription factors. ROS-A inhibited the activation of p65, p50, c-Rel subunits of NF-${\kappa}$B and AP-1 transcription factors. These findings indicate that ROS A from the stem bark of E. alatus inhibits proliferation in metastatic cancer cells. It was suggested that stem barks of E. alatus could be suitable for anti-cancer drugs.

• 생명과학회지
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• 제20권9호
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• pp.1332-1338
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• 2010

사람 신경모세포종 세포주 SH-SY5Y에서 Fenretinide (FenR)에 의한 GD3합성효소(hST8Sia I)의 발현증가기작을 규명하게 위하여 hST8Sia I의 프로모터 활성을 조사해 본 결과 -1146에서 -646영역에서 FenR에 의한 활성증가를 나타내었다. 또한 부위특이적 변이의 분석은 -731에서 -722영역에 위치한 전사인자 NF-kB 결합부위가 hST8Sia I의 FenR에 의한 활성증가에 중요하게 관여하고 있음을 나타내었다. FenR에 의한 hST8Sia I 유전자의 발현유도에 포함된 신호전달기작을 전사인자 단백질의 항체를 이용하여 조사해 본 결과 FenR처리에 의해 세포질에서는 인산화된 AKT단백질 수준의 증가가 관찰되었고 핵내에서는 NF-kB의 p65단백질의 증가가 관찰되었다. 이러한 결과들은 FenR에 의한 hST8Sia I 유전자의 발현증가는 AKT신호전달경로에 의해 활성화된 NF-kB의 핵내로 이동하여 hST8Sia I 유전자의 프로모터에 결합함으로서 전사가 촉진되어 일어난다는 것을 나타낸다.

• 생명과학회지
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• 제32권11호
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• pp.833-840
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• 2022

만성 염증은 종양의 발생 및 진행과 밀접하게 연관되어 있다. 핵인자 kappa B (NF-κB)는 5개의 전사인자로 구성되며 염증 반응에 필수적인 역할을 한다. 다양한 암에서 NF-κB의 조절장애가 보고되고 있으며 NF-κB 조절이 암 치료에 있어 핵심 표적이 된다. 본 연구에서는 CDC Like Kinase 3 (CLK3)를 NF-κB 신호전달 경로를 조절하는 새로운 키네이스임을 확인하였다. 우리는 CLK3가 정규 및 비정규 NF-κB 신호전달경로를 억제하는 것을 밝혔다. CLK3 과발현 또는 knock-down 세포주를 이용한 루시퍼레이즈 분석 결과, 이 키네이스는 TNFα와 PMA가 유도하는 정규 NF-κB 신호전달경로 활성을 억제하였다. 또한 CLK3 과발현은 잘 알려진 비정규 NF-κB 신호경로 유도제인 NF-κB-inducing kinase (NIK) 또는 CD40에 의한 NF-κB 활성을 저해하였다. 추가적으로 CLK3의 NF-κB 신호전달 저해기전을 설명하고자 TNFα 처리 후 웨스턴 블롯 분석으로 이 키네이스 영향권 내에 있는 NF-κB 신호경로 분자들을 식별하였다. 그 결과 CLK3가 TAK1, IKKα/α, p65, IκBα 및 ERK1/2-MAPK의 인산화/활성화를 저해하여 TNFα 처리로 유도된 NF-κB 및 MAPK 신호경로를 모두 억제함을 밝혔다. 앞으로의 연구는 CLK3가 정규 및 비정규 NF-κB 경로를 억제하는 기작을 밝히는데 초점을 맞출 것이다. 위 연구 결과들을 토대로 CLK3가 NF-κB 신호전달경로의 새로운 음성 조절자로써 기능함을 제시하였다.

• Asian Pacific Journal of Cancer Prevention
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• 제15권23호
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• pp.10483-10487
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• 2015

Heptaphylline derivatives are carbazoles in Clausena harmandiana, a medicinal plant that is utilized for headache, stomach ache, and other treatments of illness. The present study examined the effects of heptaphylline and 7-methoxyheptaphylline on apoptosis of human colon adenocarcinoma cells (HT-29 cell line). Quantification of cell viability was performed using cell proliferation assay (MTT assay) and of protein expression through immunoblotting. The results showed that only heptaphylline, but not 7-methoxyheptaphylline, significantly significantly activated cleaved of caspase-3 and poly (ADP-ribose) polymerase (PARP-1) which resulted in HT-29 cell death. We found that heptaphylline activated BH3 interacting-domain death agonist (Bid) and Bak, proapoptotic proteins. In contrast, it suppressed X-linked inhibitor-of-apoptosis protein (XIAP), Bcl-xL and survivin, inhibitors of apoptosis. In addition, heptaphylline inhibited activation of NF-${\kappa}B$/p65 (rel), a regulator of apoptotic regulating proteins by suppressing the activation of Akt and $IKK{\alpha}$, upstream regulators of p65. The findings suggested that heptaphylline induces apoptosis in human colon adenocarcinoma cells.

• Preventive Nutrition and Food Science
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• 제8권4호
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• pp.390-394
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• 2003

In the post-genome period, the technique for identifying gene expression has been progressed to high throughput screening. In the field of molecular nutrition, the use of screening techniques to clarify molecular function of specific nutrients would be very advantageous. In this study, we have evaluated Zn-regulated gene expression in Zn-deficient, homocystein-treated EA.hy926 cells, using cDNA microarray, which can be used to screen the expression of many genes simultaneously. The information obtained can be used for preliminary assessment of molecular and signaling events modulated by Zn under pro-atherogenic conditions. EA.hy926 cells derived from human umbilical vein endothelial cells were cultured in Zn-adequate (control, 15 $\mu$M Zn) or Zn-deficient (experimental, 0 $\mu$M Zn) Dulbecco's MEM media under high homocysteine level (100 $\mu$M) for 3 days of post-confluency. Cells were harvested and RNA was extracted. Total RNA was reverse-transcribed and the synthesized cDNA was labeled with Cy3 or Cy5. Fluorescent labeled cDNA probe was applied to microarray slides for hybridization, and the slide was then scanned using a fluorescence scanner. The expression of seven genes was found to be significantly decreased, and one significantly increased, in response to treatment of EA.hy926 cells with Zn-deficient medium, compared with Zn-supplemented medium. The upregulated genes were oncogenes and tumor suppressor genes, cell cycle-related genes and transporter genes. The down-regulated gene was RelB, a component of the NF-kappaB complex of transcription factors. The results of this study imply the effectiveness of cDNA microarray for expression profiling of a singly nutrient deficiency, namely Zn. Furthur study, using tailored-cDNA array and vascular endothelial cell lines, would be beneficial to clarify the molecular function of Zn in atherosclerosis, more in detail.

• The Korean Journal of Physiology and Pharmacology
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• 제17권4호
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• pp.315-320
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• 2013

Here, we show that radicicol, a fungal antibiotic, resulted in marked inhibition of inducible nitric oxide synthase (iNOS) transcription by the pancreatic beta cell line MIN6N8a in response to cytokine mixture (CM: TNF-${\alpha}$, IFN-${\gamma}$, and IL-$1{\beta}$). Treatment of MIN6N8a cells with radicicol inhibited CM-stimulated activation of NF-${\kappa}B$/Rel, which plays a critical role in iNOS transcription, in a dose-related manner. Nitrite production in the presence of PD98059, a specific inhibitor of the extracellular signal-regulated protein kinase-1 and 2 (ERK1/2) pathway, was dramatically diminished, suggesting that the ERK1/2 pathway is involved in CM-induced iNOS expression. In contrast, SB203580, a specific inhibitor of p38, had no effect on nitrite generation. Collectively, this series of experiments indicates that radicicol inhibits iNOS gene expression by blocking ERK1/2 signaling. Due to the critical role that NO release plays in mediating destruction of pancreatic beta cells, the inhibitory effects of radicicol on iNOS expression suggest that radicicol may represent a useful anti-diabetic activity.