• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.33 no.6
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• pp.636-642
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• 2007

Background: CpG DNA plays an important role in immune cell function. This study examined whether the temporal control of toll-like receptor (TLR)9 by CpG DNA can regulate the expression of matrix metalloproteinase-9(MMP-9). Methods and materials: Macrophages were cultured in the presence of 10% FBS. For the various MMP genes analysis, RT-PCR and real-time PCR were performed. In addition, zymography assay performed for the MMP activity. The phosphorylation assay did for the ERK1/2 and NF${\kappa}B$ activation, and luciferase promoter assay was for the NF${\kappa}B$ activity. Results: CpG DNA induced the mRNA expression of MMP-2, MMP-9, and MMP-13, but not of MMP-7, MMP-8, and MMP-12, in a time-dependent manner. Especially, the mRNA expression of MMP-9 was strongly induced by CpG DNA using real-time RT-PCR. The TLR9 inhibitor, chloroquine, suppressed CpG DNA-induced MMP-9 expression and its activity. Moreover, CpG DNA induced the phosphorylation of ERK and the inhibition of ERK by U0126 suppressed CpG DNA-induced MMP-9 expression and its activity. CpG DNA stimulated $I{\kappa}B-{\alpha}$ degradation and luciferase activity. In addition, pretreatment of SN-50, the inhibitor of NF${\kappa}B$, strongly blocked the CpG DNA-induced MMP-9 expression and activity. Conclusion: These observations suggest that CpG DNA may play important roles in the activation of macrophages by regulating the production of MMP-9 via the sequential TLR9-ERK-NF${\kappa}B$ signaling pathway.

• Biomolecules & Therapeutics
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• v.23 no.2
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• pp.110-118
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• 2015

According to the expansion of lifespan, neuronal disorder based on inflammation has been social problem. Therefore, we isolated shikonin from Lithospermum erythrorhizon and evaluated anti-inflammatory effects of shikonin in lipopolysaccharide (LSP)-stimulated BV2 microglial cells. Shikonin dose-dependently inhibits the expression of the proinflammatory mediators, nitric oxide (NO), prostaglandin $E_2$ ($PGE_2$), and tumor necrosis factor-${\kappa}B$ (TNF-${\alpha}$) as well as their main regulatory genes and products such as inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-${\alpha}$ in LPS-stimulated BV2 microglial cells. Additionally, shikonin suppressed the LPS-induced DNA-binding activity of nuclear factor-${\kappa}B$ (NF-${\kappa}B$) to regulate the key regulatory genes of the proinflammatory mediators, such as iNOS, COX-2, and TNF-${\alpha}$, accompanied with downregulation of reactive oxygen species (ROS) generation. The results indicate that shikonin may downregulate the expression of proinflammatory genes involved in the synthesis of NO, $PGE_2$, and TNF-${\alpha}$ in LPS-treated BV2 microglial cells by suppressing ROS and NF-${\kappa}B$. Taken together, our results revealed that shikonin exerts downregulation of proinflammatory mediators by interference the ROS and NF-${\kappa}B$ signaling pathway.

• Journal of Microbiology and Biotechnology
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• v.18 no.10
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• pp.1683-1688
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• 2008

Lactobacillus casei 3260 (L. casei 3260) was evaluated in relation to the inflammatory response mediated by lipopolysaccharide (LPS)-induced nuclear factor-${\kappa}B$ (NF-${\kappa}B$) and cyclooxygenase-2 (COX-2) expression in Raw264.7 macrophage cells. The treatment of Raw264.7 cells with L. casei 3260 significantly inhibited the secretion of tumor necrosis factor-$\alpha$ (TNF-$\alpha$) and prostaglandins $E_{2}\;(PGE_{2})$, followed by suppression of COX-2. To clarify the molecular mechanism, the inhibitory effect of L. casei 3260 on the NF-${\kappa}B$ signaling pathway was examined based on the luciferase reporter activity. Although the treatment of Raw264.7 cells with L. casei 3260 did not affect the transcriptional activity of NF-${\kappa}B$, it did inhibit NF-${\kappa}B$ activation, as determined by the cytosolic p65 release and degradation of I-${\kappa}B{\alpha}$. Therefore, these findings suggest that the suppression of COX-2 through inhibiting the NF-${\kappa}B$ activation by LPS may be associated with the antiinflammatory effects of L. casei 3260 on Raw264.7 cells.

• The Korean journal of internal medicine
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• v.34 no.1
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• pp.146-155
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• 2019

Background/Aims: Indoxyl sulfate (IS) is a uremic toxin and an important causative factor in the progression of chronic kidney disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to exhibit protective effects in kidney injury. Here, we investigated the effects of paricalcitol treatment on IS-induced renal tubular injury. Methods: The fluorescent dye 2',7'-dichlorofluorescein diacetate was used to measure intracellular reactive oxygen species (ROS) following IS administration in human renal proximal tubular epithelial (HK-2) cells. The effects of IS on cell viability were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and levels of apoptosis-related proteins (Bcl-2-associated protein X [Bax] and B-cell lymphoma 2 [Bcl-2]), nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) p65, and phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) were determined by semiquantitative immunoblotting. The promoter activity of $NF-{\kappa}B$ was measured by luciferase assays and apoptosis was determined by f low cytometry of cells stained with f luorescein isothiocyanate-conjugated Annexin V protein. Results: IS treatment increased ROS production, decreased cell viability and induced apoptosis in HK-2 cells. IS treatment increased the expression of apoptosis-related protein Bax, decreased Bcl-2 expression, and activated phosphorylation of MAPK, $NF-{\kappa}B$ p65, and Akt. In contrast, paricalcitol treatment decreased Bax expression, increased Bcl-2 expression, and inhibited phosphorylation of MAPK, $NF-{\kappa}B$ p65, and Akt in HK-2 cells. $NF-{\kappa}B$ promoter activity was increased following IS, administration and was counteracted by pretreatment with paricalcitol. Additionally, flow cytometry analysis revealed that IS-induced apoptosis was attenuated by paricalcitol treatment, which resulted in decreased numbers of fluorescein isothiocyanate-conjugated Annexin V positive cells. Conclusions: Treatment with paricalcitol inhibited IS-induced apoptosis by regulating MAPK, $NF-{\kappa}B$, and Akt signaling pathway in HK-2 cells.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.4
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• pp.2325-2331
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• 2013

Objective: To detect effects of plumbagin on proliferation and apoptosis in non-small cell lung cancer cell lines, and investigate the underlying mechanisms. Materials and Methods: Human non-small cell lung cancer cell lines A549, H292 and H460 were treated with various concentrations of plumbagin. Cell proliferation rates was determined using both cell counting kit-8 (CCK-8) and clonogenic assays. Apoptosis was detected by annexin V/propidium iodide double-labeled flow cytometry and TUNEL assay. The levels of reactive oxygen species (ROS) were detected by flow cytometry. Activity of NF-${\kappa}B$ was examined by electrophoretic mobility shift assay (EMSA) and luciferase reporter assay. Western blotting was used to assess the expression of both NF-${\kappa}B$ regulated apoptotic-related gene and activation of p65 and $I{\kappa}B{\kappa}$. Results: Plumbagin dose-dependently inhibited proliferation of the lung cancer cells. The IC50 values of plumbagin in A549, H292, and H460 cells were 10.3 ${\mu}mol/L$, 7.3 ${\mu}mol/L$, and 6.1 ${\mu}mol/L$ for 12 hours, respectively. The compound concentration-dependently induced apoptosis of the three cell lines. Treatment with plumbagin increased the intracellular level of ROS, and inhibited the activation of NK-${\kappa}B$. In addition to inhibition of NF-${\kappa}B$/p65 nuclear translocation, the compound also suppressed the degradation of $I{\kappa}B{\kappa}$. ROS scavenger NAC highly reversed the effect of plumbagin on apoptosis and inactivation of NK-${\kappa}B$ in H460 cell line. Treatment with plumbagin also increased the activity of caspase-9 and caspase-3, downregulated the expression of Bcl-2, upregulated the expression of Bax, Bak, and CytC. Conclusions: Plumbagin inhibits cell growth and induces apoptosis in human lung cancer cells through an NF-${\kappa}B$-regulated mitochondrial-mediated pathway, involving activation of ROS.

• Biomolecules & Therapeutics
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• v.31 no.3
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• pp.306-311
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• 2023

The current study aimed to reveal the potential effect of meclofenamate, a nonsteroidal anti-inflammatory drug, on the gene expression of airway MUC5AC mucin. Human pulmonary mucoepidermoid NCI-H292 cells were pretreated with meclofenamate for 30 min and stimulated with phorbol 12-myristate 13-acetate (PMA) for 24 h. Thereafter, the effect of meclofenamate on the PMA-induced nuclear factor kappa B (NF-kB) signaling pathway was assessed. Meclofenamate inhibited glycoprotein production and mRNA expression of MUC5AC mucins induced by PMA by inhibiting the degradation of inhibitory kappa Bα (IkBα) and NF-kB p65 nuclear translocation. These results suggest meclofenamate suppresses mucin gene expression by regulating NF-kB signaling pathway in human pulmonary epithelial cells.

• Journal of Food Hygiene and Safety
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• v.21 no.3
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• pp.181-188
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• 2006

Tumor necrosis factor-alpha $(TNF-{\alpha})$ plays a variety of biological functions such as apoptosis, inflammation and immunity. PTEN also has various cellular function including cell growth, proliferation, migration and differentiation. Thus, possible relationships between two molecules are suggested. $(TNF-{\alpha})$has been known to downregulate PTEN via nuclear factor-kappa $B(NF-{\kappa}B)$ pathway in the human colon cell line, HT-29. However, here we show the opposite finding that $(TNF-{\alpha})$ upregulates PTEN via activation of $NF-{\kappa}B$ in HL-60 cells. $TNF-{\alpha}$ increased PTEN expression at HL-60 cells in a time- and dose-dependent manner, but the response was abolished by disruption of $NF-{\kappa}B$ with p65 anisense oligonucleotide or pyrrolidine dithiocarbamate (PDTC). We found that $TNF-{\alpha}$ activated the $NF-{\kappa}B$ pathways, evidenced by the translocation of p65 to the nucleus in $TNF-{\alpha}-treated$ cells. We conclude that $TNF-{\alpha}$ induces upregulation of PTEN expression through $NF-{\kappa}B$ activation in HL-60 cells.

• Biomolecules & Therapeutics
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• v.22 no.4
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• pp.288-294
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• 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.

• The Korean Journal of Physiology and Pharmacology
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• v.27 no.1
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• pp.61-73
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• 2023

Esophageal squamous cell carcinoma (ESCC) is a kind of malignant tumor with high incidence and mortality in the digestive system. The aim of this study is to explore the function of lnc-ABCA12-3 in the development of ESCC and its unique mechanisms. RT-PCR was applied to detect gene transcription levels in tissues or cell lines like TE-1, EC9706, and HEEC cells. Western blot was conducted to identify protein expression levels of mitochondrial apoptosis and toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling pathway. CCK-8 and EdU assays were carried out to measure cell proliferation, and cell apoptosis was examined by flow cytometry. ELISA was used for checking the changes in glycolysis-related indicators. Lnc-ABCA12-3 was highly expressed in ESCC tissues and cells, which preferred it to be a candidate target. The TE-1 and EC9706 cells proliferation and glycolysis were obviously inhibited with the downregulation of lnc-ABCA12-3, while apoptosis was promoted. TLR4 activator could largely reverse the apoptosis acceleration and relieved the proliferation and glycolysis suppression caused by lnc-ABCA12-3 downregulation. Moreover, the effect of lnc-ABCA12-3 on ESCC cells was actualized by activating the TLR4/NF-κB signaling pathway under the mediation of exosome. Taken together, the lnc-ABCA12-3 could promote the proliferation and glycolysis of ESCC, while repressing its apoptosis probably by regulating the TLR4/NF-κB signaling pathway under the mediation of exosome.

• Journal of Life Science
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• v.32 no.11
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• pp.833-840
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• 2022

Chronic inflammation has been shown to be closely associated with tumor development and progression. Nuclear factor kappa B (NF-κB) is composed of a family of five transcription factors. NF-κB signaling plays a crucial role in the inflammatory response and is often found to be dysregulated in various types of cancer, making it an attractive target in cancer therapeutics. In this study, CDC-like kinase 3 (CLK3) was identified as a novel kinase that regulates the NF-κB signaling pathway. Our data demonstrate that CLK3 inhibits the canonical and non-canonical NF-κB pathways. Luciferase assays following the transient or stable expression of CLK3 indicated that this kinase inhibited NF-κB activation mediated by Tumor necrosis factor-alpha (TNFα) and Phorbol 12-myristate 13-acetate (PMA), which are known to activate NF-κB signaling via the canonical pathway. Consistent with data on the ectopic expression of CLK3, CLK3 knockdown using shRNA constructs increased NF-κB activity 1.5-fold upon stimulation with TNFα in HEK293 cells compared with the control cells. Additionally, overexpression of CLK3 suppressed the activation of this signaling pathway induced by NF-κB-inducing kinase (NIK) or CD40, which are well-established activators of the non-canonical pathway. To further examine the negative impact of CLK3 on NF-κB signaling, we performed Western blotting following the TNFα treatment to directly identify the molecular components of the NF-κB pathway that are affected by this kinase. Our results revealed that CLK3 mitigated the phosphorylation/activation of transforming growth factor-α-activated kinase 1 (TAK1), inhibitor of NF-κB kinase alpha/beta (IKKα/α), NF-κB p65 (RelA), NF-κB inhibitor alpha (IκBα), and Extracellular signal-regulated kinase 1/2-Mitogen-activated protein kinase (ERK1/2-MAPK), suggesting that CLK3 inhibits both the NF-κB and MAPK signaling activated by TNFα exposure. Further studies are required to elucidate the mechanism by which CLK3 inhibits the canonical and non-canonical NF-κB pathways. Collectively, these findings reveal CLK3 as a novel negative regulator of NF-κB signaling.