• 대한한방종양학회지
• /
• 제6권1호
• /
• pp.67-79
• /
• 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.

• Preventive Nutrition and Food Science
• /
• 제4권3호
• /
• pp.209-214
• /
• 1999

In this study, the effectof ethanol and acetaldehyde on DNA binding activities of NF-$textsc{k}$B and AP-1 were evaluated in C6 rat glial cells. Both NF-$textsc{k}$B and AP-1 are important transcription factors for the expression of various cytokines in glial cells. Our data showed that neither ethanol nor acetaldehyde induced conspicuous cell death of C6 cells at clinically realistic concentrations. When the DNA binding activities of nuclear NF-$textsc{k}$B and AP-1 were estimated using electrophoretic mobility shift assay (EMSA), ethanol(0.3%) or acetaldehyde(1mM) induced transient activation of these transcription factors, which attained peak levels at 4~8 hours and declined to basal levels at 12 hours after treatement . The supershift analysis showed that the increased activities of NF-$textsc{k}$B in ethanol/acetaldehyde-treated C6 cells were due to the preferential induction of p65/p50 heterodimer complex. The DNA binding activities of these transcriptional factors decreased below basal levels when cells were cultured with either ethanol or acetaldehyde for 24 hours, and showed the inhibitory effect of chronic ehtanol /acetaldehyde treatment on the activities of these transsriptional factors. Our data indicate that either ethanol or acetaldehyde can induce functional changes of glial cells throught bi-directional modulation of NF-$textsc{k}$B and AP-1 DNA binding activities.

• Food Science and Biotechnology
• /
• 제15권2호
• /
• pp.303-307
• /
• 2006

Effect of Oenanthe javanica ethanol extract (OJE) on nuclear factor-${\kappa}B$ (NF-${\kappa}B$)-mediated inflammatory reaction in RAW 264.7 macrophage cells was investigated. The OJE dose-dependently inhibited secretions of tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) and prostaglandins $E_2\;(PGE_2)$ from lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and blocked LPS-induced expression of cyclooxygenase-2. To clarify mechanistic basis for its inhibitions of NF-${\kappa}B$ and activator protein-1 (AP-1) activations, effects of OJE on activations of NF-${\kappa}B$ and AP-1 genes by luciferase reporter activity were examined. The LPS-stimulated activations of NF-${\kappa}B$ and AP-1 were significantly blocked by 400 and $600\;{\mu$}g/mL of OJE, implicating that OJE might regulate gene expression through more than one signaling pathway. Cytosolic degradation of I-${\kappa}B{\alpha}$ was inhibited by OJE dose-dependently, indicating that the nuclear translocation of p65 was inhibited by OJE. These findings suggest that the inhibition of LPS-stimulated COX-2 expression by OJE is due to its inhibition of NF-${\kappa}B$ activation by blocking I-${\kappa}B{\alpha}$ degradation, which may be mechanistic basis of anti-inflammatory effects of OJE.

• 생약학회지
• /
• 제35권3호통권138호
• /
• pp.244-249
• /
• 2004

The effects of hesperetin and naringenin on $NF-{\kappa}B$ activation were investigated in normal rat kidney cells transformed by temperature sensitive Rous Sarcoma Virus (tsNRK). The flavonoids, naringenin and hesperetin, significantly reduced v-Src-induced $NF-{\kappa}B$ activation as well as phosphorylation of Akt and GSK-3 in tsNRK cells, whereas these compounds did not effect on platelet-derived growth factor (PDGF)-induced $NF-{\kappa}B$ activation in $NIH3T3{\gamma}l$ cells. In addition, the DNA binding activity of SP-I was also reduced but that of AP-1 was not affected by the compounds. Our study suggests that Src-induced $NF-{\kappa}B$ activation could occur via Akt-GSK-3 pathway without $IkB{\alpha}$ degradation and that naringenin and hesperetin could be used in the treatment of cancer through the inhibition of $NF-{\kappa}B$ activation.

• Tuberculosis and Respiratory Diseases
• /
• 제55권5호
• /
• pp.488-498
• /
• 2003

연구배경 : 세포에 방사선을 조사하면 세포사멸과 함께 AP-l, NF-${\kappa}B$와 같은 여러 전사인자가 활성화 되는 것으로 알려져 있다. 이중 염증과 면역반응의 중요한 전사인자인 NF-${\kappa}B$는 항아포프토시스의 기능이 있으며 NF-${\kappa}B$ 활성화를 차단함으로써 TNF-${\alpha}$나 daunorubicine 등에 의 한 항암효과를 상승시킬 수 있음이 밝혀져 있다. NF-${\kappa}B$의 항아포프토시스 기전은 NF-${\kappa}B$ 의존성 단백질인 cIAPI, 2의 전사발현 유도에 의한 것으로 cIAPl, 2는 caspase 3, 7, pro-caspase-9의 활성을 차단함으로써 아포프토시스를 억제하는 것으로 알려져 있다. 이에 저자들은 방사선 유도성 세포사멸에 비교적 내성을 보이는 A549 세포주에서 방사선에 의한 NF-${\kappa}B$ 활성화와 그에 따른 cIAP 발현유도를 조사하고 NF-${\kappa}B$ 활성화를 차단하여 방사선 유도성 세포사멸의 감작효과를 확인하기 위하여 본 연구를 시행하였다. 방 법 : 세포주는 A549 폐암세포주를 이용하였고, 방사선 조사는 Varian사의 Clinac 1800C 선형가속기를 이용하였으며 조사량은 10GY를 사용하였다. 세포독성 검사는 MTT Assay를 이용하였고, NF-${\kappa}B$ 활성화 검사는 luciferase reporter gene assay, electromobility shift assay, $I{\kappa}B-{\alpha}$ degradation에 대한 westem blot을 이용하였다. NF-${\kappa}B$활성을 차단하기 위하여 proteosome inhibitor인 MGI32와 $I{\kappa}B{\alpha}$-superrepressor plasmid를 transfection한 안정적 세포주 A549-$I{\kappa}B{\alpha}$-superrepressor를 이용하였다. cIAP의 발현은 RT-PCR을 이용하였고, cIAP2 promoter 활성은 NF-${\kappa}B$ site를 포함한 cIAP2 유전자 5' f1anking region(1.4kb)을 pGL2-Basic luciferase vector에 cloning 한 construct를 사용하여 transfection 후 luciferase assay를 시행하였다. 결 과 : A549 cell에서 10Gy 방사선 조사에 의한 세포독성은 24hr, 48hr에 각각 $10.82{\pm}.3%$, $17.7{\pm}6.4%$로 비교적 내성이 있음을 확인하였다. 방사선에 의한 NF-${\kappa}B$의 활성은 $I{\kappa}B-{\alpha}$ 분해에 대한 western blot과 EMSA로 확인하였으며 luciferase assay에서도 약 1.6 배 정도의 NF-${\kappa}B$ 활성화가 있었다. NF-${\kappa}B$활성을 차단하기 위해 사용한 MG132는 방사선 유도성 세포사멸에 영향을 주지 않았으며 A549-$I{\kappa}B{\alpha}$-superrepressor 세포주에서도 세포사멸의 감작효과는 없었다. 또한 RT-PCR 결과 방사선에 의한 cIAP1,2 mRNA 발현유도는 관찰되지 않았고 cIAP2 promoter luciferase assay에서도 cIAP2 전사활성 유도는 없었다. 결 론 : A549 폐암세포주에서 방사선에 의해 NF-${\kappa}B$의 활성화는 확인하였으나 활성화 정도가 미약하였고 NF-${\kappa}B$ 의존성 항아포프토시스 유전자인 cIAP가 방사선에 의해 발현유도 되지 않았으며, NF-${\kappa}B$ 활성을 차단함에도 세포독성에 감작효과가 없었으므로 A549 폐암세포주에서 방사선 유도성 세포사멸에 내성을 보이는 기전에는 NF-${\kappa}B$의 역할이 미미하리라고 사료된다.

• 생명과학회지
• /
• 제20권2호
• /
• pp.275-280
• /
• 2010

Sulforaphane은 십자가화 채소에 존재하는 화합물로 항염증, 항암 및 신생혈관 생성의 억제 효과가 알려짐으로써 최근 많은 연구가 활발히 이루어지고 있으나, LPS에 의한 MMP-9 활성 조절에 대한 연구는 매우 미흡한 편이다. 따라서 본 연구에서 sulforaphane이 LPS 유도에 의한 MMP-9 활성에 미치는 영향에 대해서 조사해 보았다. Raw 264.7 세포에 sulforaphane을 전처리 한 후 LPS를 처리하여 gelatin zymography를 실시해 본 결과, LPS에 의해 유도된 MMP-9 활성 증가가 sulforaphane 농도 의존적으로 감소됨을 확인 하였다. 또한 RT-PCR과 MMP-9의 luciferase assay를 통한 실험에서 sulforaphane의 MMP-9 억제효과가 전사단계에서 조절됨을 추측 할 수 있었다. MMP-9 promoter 부위에 여러 가지의 전사조절인자 결합부위가 존재한다. 특히 AP-1과 NF-${\kappa}B$가 중요 전사조절인자로 작용하여 MMP-9 발현조절에 관여한다. 본 실험에서 sulforaphane에 의한 MMP-9 억제효과 기전에 이들 전사조절인자들의 중요한 역할을 조사하였다. AP-1과 NF-${\kappa}B$ 결합부위를 변형 시킨 vector를 transfection하여 MMP-9의 promoter 활성을 측정한 결과, 정상 vector에 비해 그 활성도가 현저히 떨어짐을 확인하였고, LPS에 의해 증가되는 AP-1과 NF-${\kappa}B$의 basal promoter 활성 또한 sulforaphane에 의해 감소됨을 관찰 할 수 있었다. 이상의 결과에서 sulforaphane의 MMP-9 활성억제효과는 AP-1과 NF-${\kappa}B$와 같은 전사인자들이 MMP-9의 전사를 조절함으로써 일어나는 것임을 알 수 있었다. 그리고 sulforaphane은 세포의 invasion능력 또한 효과적으로 억제시킴을 관찰 할 수 있었는데 이는 MMP-9 활성억제효과와 밀접한 관련이 있음을 추측 할 수 있었다.

• Toxicological Research
• /
• 제24권1호
• /
• pp.17-22
• /
• 2008

Sopungsan (SS) is a traditional Korean decoction used for the treatment of dermatitis. The aim of this study is to confirm whether or not SS has a preventive effect on the development of atopic dermatitis in dinitrochlorobenzene-applied Nc/Nga mice. SS was administered orally to Nc/Nga mice, which led to the remarkable suppression of the development of dermatitis, as determined by a histological examination and the serum IgE levels. Moreover, SS inhibited the production of thymus- and activation-regulated chemokine (TARC) and its mRNA expression in a keratinocyte cell line, HaCaT, which had been stimulated with tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) and interferon-${\gamma}$ (IFN-${\gamma}$). Activation of the nuclear factor-${\kappa}B$ (NF-${\kappa}B$) or activator protein-1 (AP-1) is one of key steps in the signaling pathways mediating induction of TARC. In this study, SS selectively suppressed NF-${\kappa}B$ activation which may be essential for TARC expression in $TNF-{\alpha}/IFN-{\gamma}$ treated keratinocytes. The inhibitory effect of SS on NF-${\kappa}B$ activation and TARC production might be associated with the anti-dermatitic effects of SS.

• Biomolecules & Therapeutics
• /
• 제22권4호
• /
• pp.288-294
• /
• 2014

Mangostenone F (MF) is a natural xanthone isolated from Garcinia mangostana. However, little is known about the biological activities of MF. This study was designed to investigate the anti-inflammatory effect and underlying molecular mechanisms of MF in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. MF dose-dependently inhibited the production of NO, iNOS, and pro-inflammatory cytokines (TNF-${\alpha}$, IL-6, and IL-$1{\beta}$) in LPS-stimulated RAW264.7 macrophages. Moreover, MF decreased the NF-${\kappa}B$ luciferase activity and NF-${\kappa}B$ DNA binding capacity in LPS-stimulated RAW264.7 macrophages. Furthermore, MF suppressed the NF-${\kappa}B$ activation by inhibiting the degradation of $I{\kappa}B{\alpha}$ and nuclear translocation of p65 subunit of NF-${\kappa}B$. In addition, MF attenuated the AP-1 luciferase activity and phosphorylation of ERK, JNK, and p38 MAP kinases. Taken together, these results suggest that the anti-inflammatory effect of MF is associated with the suppression of NO production and iNOS expression through the down-regulation of NF-${\kappa}B$ activation and MAPK signaling pathway in LPS-stimulated RAW264.7 macrophages.

• 대한한의학회지
• /
• 제23권1호
• /
• pp.50-60
• /
• 2002

Objectives: Sunghyangjunggisan (SHJS) is a commonly prescribed drug with a wide neuropharmacological spectrum. The water extracts of SHJS were found to be protective against neurotoxicity elicited by deprivation of serum and glucose. Methods: The morphological examination and Hoechst staining of nucleus also clearly showed that the extracts attenuated the cell shrinkage, membrane blebbing, representing typical neuronal apoptotic phenomena and nucleosome-sized fragmentation under the microscope in PC 12 rat pheochromocytoma cells. Results: Activation of protein kinase A (PKA) with dibutyl-cAMP and forskolin also protected during glucose deprivation, although it was not additive with the effect provided by phorbol ester. Interestingly, treatment with the protein kinase A inhibitor, KT5720, was not neuroprotective in the presence of SHJS. Electrophoretic mobility shift assays were used to characterize the neuroprotective binding of nuclear proteins to consensus sequences for AP-l, nuclear factor kappa B ($NF-{\kappa}B$) after glucose deprivation. When PC 12 cells are induced to undergo apoptosis by serum deprivation, AP-l and $NF-{\kappa}B$ DNA binding activity transiently increases to a slight degree. This stimulation is blocked by the water extracts of SHJS. The site of action of the drugs appeared to involve specific inhibition of AP-1 and nuclear factor kB binding activity. Conclusions: Taken together, these results suggested the possibility that the extracts of SHJS might provide a neurotrophic-like activity in PC 12 cells.

• BMB Reports
• /
• 제45권3호
• /
• pp.177-182
• /
• 2012

Camellia japonica oil (CJ oil) has been used traditionally in East Asia to nourish and soothe the skin as well as help restore the elasticity of skin. CJ oil has also been used on all types of bleeding instances. However, little is known about its anti-inflammatory effects. Therefore, the anti-inflammatory effects of CJ oil and its mechanisms of action were investigated. CJ oil inhibited LPS-induced production of NO, $PGE_2$, and TNF-${\alpha}$ in RAW264.7 cells. In addition, expression of COX-2 and iNOS genes was reduced. To evaluate the mechanism of the anti-inflammatory activity of CJ oil, LPS-induced activation of AP-1 and NF-${\kappa}B$ promoters was found to be significantly reduced by CJ oil. LPS-induced phosphorylation of $I{\kappa}B{\alpha}$, ERK, p38, and JNK was also attenuated. Our results indicate that CJ oil exerts anti-inflammatory effects by downregulating the expression of iNOS and COX-2 genes through inhibition of NF-${\kappa}B$ and AP-1 signaling.