Nuclear NF-κB Levels Research Articles

Acute ethanol administration inhibits Toll-like receptor 4 signaling pathway in rat intestinal epithelia

Excess alcohol intake, as in binge drinking, increases susceptibility to microbial pathogens. Alcohol impairs macrophage function by suppression of the Toll-like receptor 4 (TLR4) pathway. This study investigated the effects of acute ethanol intake on the TLR4 pathway in rat intestinal epithelia, which usually encounters luminal antigens at first and participates in the development of intestinal immunity. Twenty Wistar rats were randomly assigned to an ethanol group given ethanol as a 25% (v/v) solution in water at 7.5 g/kg, or a control group given saline, by oral gavage daily for 3 days. The epithelial histology and ultrastructure, the intestinal microflora, peripheral and portal venous plasma lipopolysaccharide (LPS) levels, and somatostatin (SST) levels in the peripheral plasma and small intestine were evaluated. Somatostatin receptor 2 (SSTR2), TLR4, TANK binding kinase-1 (TBK1), activated nuclear factor-κB (NF-κB), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the intestinal mucosa were assayed. LPS responsiveness with or without SST pretreatment was assayed in vitro by quantification of TLR4, TBK1, activated NF-κB, IFN-γ and TNF-α in isolated intestinal epithelia. Mucosal damage was observed in the ethanol group by light and electron microscopy. Escherichia coli cultures were unchanged in rat intestine of the ethanol group compared with controls, but lactobacilli cultures were reduced (p < 0.05). LPS levels increased in peripheral and portal venous plasma (p < 0.05), but mucosal TLR4, TBK1, nuclear NF-κB, IFN-γ and TNF-α were unchanged in the ethanol group. LPS treatment in vitro up-regulated the level of TLR4, TBK1 and nuclear NF-κB as well as the production of IFN-γ and TNF-α in isolated intestinal epithelia in the control (p < 0.05), but not the ethanol group. The stimulatory effects of LPS on intestinal epithelia isolated from the control group were significantly inhibited by SST pretreatment (p < 0.05). The peripheral plasma and intestinal levels of SST and the mucosal expression of SSTR2 in the ethanol group were significantly higher than in the control group (p < 0.05). These findings suggest the hyposensitivity of intestinal epithelial TLR4 to LPS induced by acute alcohol abuse probably through ethanol per se and ethanol-enhanced intestinal mucosal SST pathway may be a novel mechanism for increased susceptibility to intestinal pathogens.

Cocoa polyphenols prevent inflammation in the colon of azoxymethane-treated rats and in TNF-α-stimulated Caco-2 cells

Numerous lines of evidence support a relationship between intestinal inflammation and cancer. Therefore, much attention has recently been focused on the identification of natural compounds with anti-inflammatory activities as a strategy to suppress the early stages of colorectal cancer. Because cocoa is a rich source of bioactive compounds, the present study investigated its anti-inflammatory properties in a rat model of azoxymethane (AOM)-induced colon carcinogenesis and in TNF-α-stimulated Caco-2 cells. A total of forty male rats were fed with control or cocoa-enriched diets (12 %) during 8 weeks and injected with saline or AOM (20 mg/kg body weight) during the third and fourth week (n 10 rats/group). At the end of the experiment, colon samples were evaluated for markers of inflammation. The anti-inflammatory activity of a cocoa polyphenolic extract (10 μg/ml) was examined in TNF-α-stimulated Caco-2 cells, an in vitro model of experimentally induced intestinal inflammation. The signalling pathways involved, including NF-κB and the mitogen-activated protein kinase family such as c-Jun NH₂-terminal kinases (JNK), extracellular signal-regulated kinases and p38, were also evaluated. The results show that the cocoa-rich diet decreases the nuclear levels of NF-κB and the expression of pro-inflammatory enzymes such as cyclo-oxygenase-2 and inducible NO synthase induced by AOM in the colon. Additionally, the experiments in Caco-2 cells confirm that cocoa polyphenols effectively down-regulate the levels of inflammatory markers induced by TNF-α by inhibiting NF-κB translocation and JNK phosphorylation. We conclude that cocoa polyphenols suppress inflammation-related colon carcinogenesis and could be promising in the dietary prevention of intestinal inflammation and related cancer development.

6b,11b-Dihydroxy-6b,11b-dihydro-7H-indeno[1,2-b]naphtho[2,1-d]furan-7-one (DHFO), a small molecule targeting NF-κB, demonstrates therapeutic potential in immunopathogenic chronic inflammatory conditions

6b,11b-Dihydroxy-6b,11b-dihydro-7H-indeno[1,2-b]naphtho[2,1-d]furan-7-one (DHFO), an easily synthesisable, orally bioavailable and relatively non-toxic small molecule synthesised in our lab, was previously reported to possess anti-oxidant, 5-lipoxygenase inhibitory, anti-inflammatory and peripheral analgesic activities. The present work deals with exploration of DHFO's efficacy in immunopathogenic chronic inflammatory conditions — arthritis and allergy. In carrageenan-induced inflammatory air pouch, which resembles the arthritic synovium, DHFO effectively reduced inflammatory redness and swelling and neutrophil infiltration. In complete Freund's adjuvant-induced arthritis, DHFO significantly decreased paw oedema and nitrite levels with efficacy comparable to diclofenac. DHFO inhibited neutrophil activation (observed as decreased myeloperoxidase levels), in both the in vivo models of inflammation. Interestingly, DHFO did not ulcerate the gastrointestinal tract, while diclofenac was observed to be extremely ulcerogenic. In antigen-induced active and passive anaphylaxis (allergy) models, DHFO dose-dependently prevented mesenteric mast cell (MC) degranulation with efficacy comparable to ketotifen. DHFO also inhibited compound 48/80 (C48/80)-induced paw oedema and peritoneal MC degranulation. DHFO stabilised p815 murine MCs stimulated by C48/80 and calcium ionophore—A23187, indicating an action downstream of calcium mobilisation. DHFO's anti-allergic mechanism could be two-pronged involving (1) inhibition of IgE production and/or (2) MC stabilisation. DHFO inhibited lipopolysaccharide (LPS)-induced pro-inflammatory mediator release (ROS, NO, IL-6 levels) and COX2 expression in RAW264.7 murine macrophages. Protein expression studies confirmed DHFO's ability to reduce nuclear levels of NF-κB in LPS-stimulated macrophages. Thus, DHFO is a promising non-ulcerogenic synthetic small molecule lead for immunopathogenic chronic inflammatory conditions.

Lipoteichoic acid induces surfactant protein-A biosynthesis in human alveolar type II epithelial cells through activating the MEK1/2-ERK1/2-NF-κB pathway

BackgroundLipoteichoic acid (LTA), a gram-positive bacterial outer membrane component, can cause septic shock. Our previous studies showed that the gram-negative endotoxin, lipopolysaccharide (LPS), could induce surfactant protein-A (SP-A) production in human alveolar epithelial (A549) cells.ObjectivesIn this study, we further evaluated the effect of LTA on SP-A biosynthesis and its possible signal-transducing mechanisms.MethodsA549 cells were exposed to LTA. Levels of SP-A, nuclear factor (NF)-κB, extracellular signal-regulated kinase 1/2 (ERK1/2), and mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1 were determined.ResultsExposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability. Meanwhile, when exposed to 30 μg/ml LTA for 1, 6, and 24 h, the biosynthesis of SP-A mRNA and protein in A549 cells significantly increased. As to the mechanism, LTA enhanced cytosolic and nuclear NF-κB levels in time-dependent manners. Pretreatment with BAY 11–7082, an inhibitor of NF-κB activation, significantly inhibited LTA-induced SP-A mRNA expression. Sequentially, LTA time-dependently augmented phosphorylation of ERK1/2. In addition, levels of phosphorylated MEK1 were augmented following treatment with LTA.ConclusionsTherefore, this study showed that LTA can increase SP-A synthesis in human alveolar type II epithelial cells through sequentially activating the MEK1-ERK1/2-NF-κB-dependent pathway.

The p50 Subunit of NF-κB Orchestrates Dendritic Cell Lifespan and Activation of Adaptive Immunity

Dendritic cells play a central role in keeping the balance between immunity and immune tolerance. A key factor in this equilibrium is the lifespan of DC, as its reduction restrains antigen availability leading to termination of immune responses. Here we show that lipopolysaccharide-driven DC maturation is paralleled by increased nuclear levels of p50 NF-κB, an event associated with DC apoptosis. Lack of p50 in murine DC promoted increased lifespan, enhanced level of maturation associated with increased expression of the proinflammatory cytokines IL-1, IL-18 and IFN-β, enhanced capacity of activating and expanding CD4+ and CD8+ T cells in vivo and decreased ability to induce differentiation of FoxP3+ regulatory T cells. In agreement, vaccination of melanoma-bearing mice with antigen-pulsed LPS-treated p50−/− BM-DC boosted antitumor immunity and inhibition of tumor growth. We propose that nuclear accumulation of the p50 NF-κB subunit in DC, as occurring during lipopolysaccharide-driven maturation, is a homeostatic mechanism tuning the balance between uncontrolled activation of adaptive immunity and immune tolerance.

Anti-Inflammatory Effects of Lutein in Retinal Ischemic/Hypoxic Injury: In Vivo and In Vitro Studies

Lutein protects retinal neurons by its anti-oxidative and anti-apoptotic properties in ischemia/reperfusion (I/R) injury while its anti-inflammatory effects remain unknown. As Müller cells play a critical role in retinal inflammation, the effect of lutein on Müller cells was investigated in a murine model of I/R injury and a culture model of hypoxic damage. Unilateral retinal I/R was induced by a blockade of internal carotid artery using the intraluminal method in mice. Ischemia was maintained for 2 hours followed by 22 hours of reperfusion, during which either lutein (0.2 mg/kg) or vehicle was administered. Flash electroretinogram (flash ERG) and glial fibrillary acidic protein (GFAP) activation were assessed. Lutein's effect on Müller cells was further evaluated in immortalized rat Müller cells (rMC-1) challenged with cobalt chloride-induced hypoxia. Levels of IL-1β, cyclooxygenase-2 (Cox-2), TNFα, and nuclear factor-NF-kappa-B (NF-κB) were examined by Western blot analysis. Lutein treatment minimized deterioration of b-wave/a-wave ratio and oscillatory potentials as well as inhibited up-regulation of GFAP in retinal I/R injury. In cultured Müller cells, lutein treatment increased cell viability and reduced level of nuclear NF-κB, IL-1β, and Cox-2, but not TNFα after hypoxic injury. Reduced gliosis in I/R retina was observed with lutein treatment, which may contribute to preserved retinal function. Less production of pro-inflammatory factors from Müller cells suggested an anti-inflammatory role of lutein in retinal ischemic/hypoxic injury. Together with our previous studies, our results suggest that lutein protected the retina from ischemic/hypoxic damage by its anti-oxidative, anti-apoptotic, and anti-inflammatory properties.

Antitumor effects of the novel quinazolinone MJ-33: Inhibition of metastasis through the MAPK, AKT, NF-κB and AP-1 signaling pathways in DU145 human prostate cancer cells

Quinazolinone compounds have been shown to have antitumor activity in many human cancer cell lines. In the present study, we investigated the anti-metastatic activity of MJ-33 (2-(3-ethoxyphenyl)-6-pyrrolidinylquinazolinone), a novel quinazolinone derivate, and the signaling pathway of MJ-33 in human prostate cells. MJ-33 exhibited a growth inhibitory effect on DU145, LNCaP and PC-3 cells by MTT assay. DU145 cells showed greater sensitivity to the growth inhibition of MJ-33 than that of LNCaP and PC-3 cells. MJ-33 also had an inhibitory effect on the invasion, migration and adhesion of DU145 cells using Boyden chamber transwell assays, wound-healing and adhesion assay. In addition, MJ-33 inhibited cell metastasis through the reduction of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator (u-PA) enzyme activities and protein levels by gelatin zymography assay and western blot analysis, respectively. MJ-33 reduced the protein levels of p-JNK, p-p38, p-ERK, p-AKT and nuclear NF-κB (p65), c-fos and c-Jun protein levels by western blotting. Using electrophoretic mobility-shift assay (EMSA), we demonstrated that MJ-33 blocked the activation of transcription factor AP-1 (activator protein-1) and NF-κB, which led to the inhibition of MMP-2 and MMP-9 expression. Collectively, our data showed that MJ-33 decreased protein levels of MAPKs (mitogen-activated protein kinases), AKT, AP-1 and NF-κB, resulting in the inhibition of matrix metalloproteinases. Downregulation of MMP-2 and MMP-9 reduces the invasion, migration and adhesion activities of DU145 cells. MJ-33 may be a promising agent against prostate cancer metastasis.

NF-κB contributes to the detrimental effects of social isolation after experimental stroke

Social isolation (SI) is increasingly recognized as a risk factor for stroke. Individuals with lack of social support systems have an increased incidence of stroke, poorer recovery, and greater functional decline after injury compared to individuals with social support. Attesting to the importance of social factors in stroke outcome is that these same effects can be reproducibly demonstrated in animals; social interaction improves behavioral deficits and reduces damage after experimental stroke, whereas SI enhances injury. The mechanism by which SI exacerbates injury is unclear. We investigated the role of nuclear factor-kappaB (NF-κB) signaling in male mice that were pair housed (PH) with an ovariectomized female prior to random assignment into continued PH or SI for 7 days prior to middle cerebral artery occlusion. The effects of SI on infarct volume and functional recovery were assessed at 72 h post-stroke. Nuclear NF-κB levels and activity were assessed by Western blot and transcriptional assays. SI significantly exacerbated infarct size in both male and female mice compared to PH mice. SI mice had delayed functional recovery compared to PH mice. An elevation of systemic IL-6 levels, increased nuclear NF-κB transcriptional activity, and enhanced nuclear translocation of NF-κB was seen in SI stroke animals. Interference with NF-κB signaling using either a pharmacological inhibitor or genetically engineered NF-κB p50 knockout mice abolished the detrimental effects of SI on both infarct size and functional recovery. This suggests that NF-κB mediates the detrimental effects of SI.

Butein impairs the protumorigenic activity of malignant pleural mesothelioma cells

Chronic inflammation appears to be a driving force behind cancer progression. NFκB and STAT3 activation plays a pertinent role in this process by mediating chemoresistance and the acquisition of mesenchymal features of protumorigenic cells. Epidemiological data and experimental observations suggest that Malignant Pleural Mesothelioma (MPM) is a prototype of chronic inflammation-driven cancer. Chemoresistance is a major feature of MPM. Thus, this paper explores the effect of butein (3,4,2',4'-tetrahydroxychalcone), a naturally occurring NFκB and STAT3 inhibitor, on the tumorigenic properties of MPM cells. MPM cells harbor high nuclear levels of NFκB and pSTAT3(Y705). Butein inhibits pSTAT3(Y705) phosphorylation, nuclear localization of NFκB and the physical interaction of NFκB and pSTAT3. This correlates with a downregulation of several genes involved in cancer progression (such as ICAM1, Vimentin, MMP9, Twist) of proangiogenic cytokines (VEGF) and of IL-6 and IL-8, key growth factors for MPM. Hence, butein inhibits the migration of MPM cells and strongly affects the clonogenicity of MPM cells in vitro. Finally, we show that the in vitro actions of butein translate into anticancer effects in vivo. In fact, butein treatment severely affects tumor engraftment and potentiates the anticancer effects of pemetrexed in mouse xenograft models. Butein does not significantly affect the viability of human, untransformed mesothelial cells in vitro, nor does it affect survival of tumor-free mice in vivo. The possibility of using butein as an additional treatment to current MPM therapies is discussed here.

Bojesodok-eum, a Herbal Prescription, Ameliorates Acute Inflammation in Association with the Inhibition of NF-κB-Mediated Nitric Oxide and ProInflammatory Cytokine Production.

Bojesodok-eum (BSE) is a herbal prescription consisting of Coptidis Rhizoma and Scutellariae Radix as main components. This paper investigated the effects of BSE on the induction of nitric oxide (NO), prostaglandin E2 (PGE2), and proinflammatory cytokines that are caused by lipopolysaccharide (LPS) in murine macrophage cell line and on the paw edema formation in animals. Administration of BSE (0.3 g/kg and 1 g/kg) in rats significantly inhibited carrageenan-induced paw edema formation, as did dexamethasone, an anti-inflammatory positive control drug. In cell model, treatment of BSE decreased the production of NO and PGE2 in RAW264.7 cells stimulated by LPS. BSE also inhibited the expression of iNOS and COX-2 protein as well as COX activity in a concentration-dependent manner. Consistently, BSE suppressed the ability of LPS to produce TNF-α, interleukin-1β, and interleukin-6. LPS treatment induced nuclear NF-κB level and I-κBα phosphorylation, which were inhibited subsequent treatment of BSE, suggesting its repression of LPS-inducible NF-κB activation. BSE abrogated the induction of NO, PGE2, and proinflammatory cytokines, as well as iNOS and COX-2 protein expression in RAW264.7 cells stimulated by LPS as mediated with NF-κB inhibition.

Abstract 3350: Inhibition of breast cancer and cancer stem cells via a novel micheliolide analogue CY-9

Abstract Natural products that target cancer stem cells (CSCs) have the potential to treat cancers and reduce the occurrence of relapse. The sesquiterpene lactone parthenolide has received much attention for its ability to preferentially target CSCs over their normal equivalents. However, poor pharmacokinetics have severely limited the clinical use of parthenolide, and prompted synthesis of a water soluble analogue, LC-1. Although currently in Phase I trials for disseminated cancers, LC-1 suffers from a short half-life, which could potentially limit its efficacy. In order to identify new sesquiterpene lactones which function similarly to parthenolide and LC-1 but with improved pharmacokinetics, we tested a new micheliolide analogue CY-9 for activity against both breast cancer and CSCs. Using the MTS assay, SUM159 breast cancer cells displayed sensitivity to CY-9 with an IC50 of 5 μM, which is similar to reported inhibitory concentrations of parthenolide. SUM159 cells assayed for Aldefluor expression after a three day treatment with CY-9 (1, 2.5, 5 μM) showed a 27%, 64%, and 83% reduction in their CSC populations, respectively. This CSC depletion was largely reversed in SUM159 cells that were pre-treated with the antioxidant N-Acetyl-Cysteine (NAC) before exposure to CY-9. Inhibition of SUM159 CSC self-renewal after CY-9 treatment was assayed using tumorsphere formation of single SUM159 Aldefluor-positive cells. A seven day treatment of 5 μM CY-9 reduced SUM159 Aldefluor-positive cell tumorsphere formation by 74%. Under drug-free conditions, tumorsphere formation was reduced by 26% in the second passage, but returned to control levels by the third passage. Western blotting also showed that 5 μM CY-9 reduced the levels of nuclear NF-κB. These results demonstrate the effectiveness of our new micheliolide analogue CY-9 at targeting populations of both bulk breast tumor cells and CSCs. The slight reduction in tumorsphere formation under drug-free conditions suggests that inhibition of self-renewal plays a minor role in CY-9's activity against CSCs. Loss of CY-9 activity with cells pre-treated with NAC suggests that the inhibitory effect of CY-9 is partially mediated by its interaction with biologically relevant cysteines and/or ROS generation. Additionally, as NF-κB is vital for defense against cellular stressors such as ROS, depletion of nuclear NF-κB might also explain the observed inhibition with CY-9. Together, these results warrant further study of CY-9 as a potential therapy for breast cancer and CSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3350. doi:10.1158/1538-7445.AM2011-3350

MTOR complex 2 mediates Notch signaling in thymocyte and T-cell leukemia development (64.8)

Abstract Mammalian target of rapamycin (mTOR) is an important component of the PI3-kinase (PI3K) pathway that regulates cell growth and proliferation. PI3K signaling cascades can be initiated by activation of Notch, a critical regulator for T lineage commitment and leukemia development. Here we show that mTOR complex 2 (mTORC2) is functionally vital as a downstream target of Notch in thymocytes and T-cell acute lymphoblastic leukemia (T-ALL). Conditional deletion of rictor, an essential component of mTORC2, results in impaired proliferation and differentiation of pre-T cells driven by Notch ligation. Moreover, mTORC2 is a primary kinase for Akt S473 and enhances Akt activity in thymocytes. Furthermore, mTORC2 regulates NF-κB activity, such that constitutively active Akt can restore nuclear NF-κB levels in the rictor-depleted pre-T cells, as well as normalizing Notch-initiated proliferation and differentiation. Strikingly, interruption of mTORC2 significantly delays T-ALL induced by Notch1 activating mutation and suppresses infiltration of leukemic cells into the non-lymphoid organs. Our study provides evidence of a key role of mTORC2 in relaying Notch signaling in thymocyte development, and reveals that mTORC2 promotes Notch-driven leukemia progression.

The Anti-Inflammatory Effects of Dimethyl Fumarate in Astrocytes Involve Glutathione and Haem Oxygenase-1

DMF (dimethyl fumarate) exerts anti-inflammatory and pro-metabolic effects in a variety of cell types, and a formulation (BG-12) is being evaluated for monotherapy in multiple sclerosis patients. DMF modifies glutathione (GSH) levels that can induce expression of the anti-inflammatory protein HO-1 (haem oxygenase-1). In primary astrocytes and C6 glioma cells, BG-12 dose-dependently suppressed nitrite production induced by either LI [LPS (lipopolysaccharide) at 1 μg/ml plus IFNγ (interferon γ) at 20 units/ml] or a mixture of pro-inflammatory cytokines, with greater efficacy in C6 cells. BG-12 reduced NOS2 (nitric oxide synthase 2) mRNA levels and activation of a NOS2 promoter, reduced nuclear levels of NF-κB (nuclear factor κB) p65 subunit and attenuated loss of IκBα (inhibitory κBα) in both cell types, although with greater effects in astrocytes. In astrocytes, LI decreased mRNA levels for GSHr (GSH reductase) and GCL (c-glutamylcysteine synthetase), and slightly suppressed GSHs (GSH synthetase) mRNAs. Co-treatment with BG-12 prevented those decreased and increased levels above control values. In contrast, LI reduced GSHp (GSH peroxidase) and GCL in C6 cells, and BG-12 had no effect on those levels. BG-12 increased nuclear levels of Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2), an inducer of GSH-related enzymes, in astrocytes but not C6 cells. In astrocytes, GSH was decreased by BG-12 at 2 h and increased at 24 h. Prior depletion of GSH using buthionine-sulfoximine increased the ability of BG-12 to reduce nitrites. In astrocytes, BG-12 increased HO-1 mRNA levels and effects on nitrite levels were blocked by an HO-1 inhibitor. These results demonstrate that BG-12 suppresses inflammatory activation in astrocytes and C6 glioma cells, but with distinct mechanisms, different dependence on GSH and different effects on transcription factor activation.

Attenuation of LPS-induced apoptosis in NGF-differentiated PC12 cells via NF-κB pathway and regulation of cellular redox status by an oxazine derivative

Neuronal cell death due to apoptosis is a common characteristic of neurodegenerative diseases. In this study, we report protective effect of 2-ethoxy-4,5-diphenyl-1,3-oxazine-6-one (EDPOO) against lipopolysaccharide (LPS)-induced cell death in rat pheochromocytoma (PC12) cells, as assessed by MTT test, acridine orange/ethidium bromide staining, determination of Bax, Bcl-2 and caspase-3 levels. We further show that this compound could increase heat shock proteins Hsp-70 and Hsp-32 levels. EDPOO also modulates nuclear levels of Nrf2 and NF-κB, transcription factors that are activated by intracellular reactive oxygen species and/or mediators generated due to chemical exposure of cells. Pretreatment of the cells with this oxazine derivative also increases γ-GCS level, as well as antioxidant enzyme activities, in a dose-dependent manner. Protective effect of this compound could represent a promising approach for treatment of neurodegenerative diseases.

Low Levels of NF-κB/p65 Mark Anergic CD4+T Cells and Correlate with Disease Severity in Sarcoidosis

T lymphocytes from patients with sarcoidosis respond weakly when stimulated with mitogen or antigen. However, the mechanisms responsible for this anergy are not fully understood. Here, we investigated the protein levels of nuclear transcription factor NF-κB (p50, p65, and p105), IκBα (inhibitor of NF-κB), T-cell receptor (TCR) CD3ζ-chain, tyrosine kinase p56(LCK), and nuclear factor of activated T cells c2 (NF-ATc2) in peripheral blood CD4(+) T cells from patients with sarcoidosis. Baseline expression of p65 in these lymphocytes was reduced in 50% of patients. The reduced levels of p65 in sarcoid CD4(+) T cells concurred with decreased levels of p50, p105, CD3ζ, p56(LCK), IκBα, and NF-ATc2. Polyclonal stimulation of NF-κB-deficient sarcoid T cells resulted in reduced expression of CD69 and CD154, decreased proliferation, and cytokine (i.e., interleukin 2 [IL-2] and gamma interferon [IFN-γ]) production. The clinical significance of these findings is suggested by the association between low p65 levels and the development of more severe and active sarcoidosis. Although correlative, our results support a model in which multiple intrinsic signaling defects contribute to peripheral T-cell anergy and the persistence of chronic inflammation in sarcoidosis.

Nobiletin, a citrus flavonoid, suppresses invasion and migration involving FAK/PI3K/Akt and small GTPase signals in human gastric adenocarcinoma AGS cells

Nobiletin, a compound isolated from citrus fruits, is a polymethoxylated flavone derivative shown to have anti-inflammatory, antitumor, and neuroprotective properties. This study has investigated that nobiletin exerted inhibitory effects on the cell adhesion, invasion, and migration abilities of a highly metastatic AGS cells under non-cytotoxic concentrations. Data also showed nobiletin could inhibit the activation of focal adhesion kinase (FAK) and phosphoinositide-3-kinase/Akt (PI3K/Akt) involved in the downregulation of the enzyme activities, protein expressions, messenger RNA levels of matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-2 (MMP-9). Also, our data revealed that nobiletin inhibited FAK/PI3K/Akt with concurrent reduction in the protein expressions of Ras, c-Raf, Rac-1, Cdc42, and RhoA by western blotting, whereas the protein level of RhoB increased progressively. Otherwise, nobiletin-treated AGS cells showed tremendously decreased in the phosphorylation and degradation of inhibitor of kappaBα (IκBα), the nuclear level of NF-κB, and the binding ability of NF-κB to NF-κB response element. Furthermore, nobiletin significantly decreased the levels of phospho-Akt and MMP-2/9 in Akt1-cDNA-transfected cells concomitantly with a marked reduction in cell invasion and migration. These results suggest that nobiletin can reduce invasion and migration of AGS cells, and such a characteristic may be of great value in the development of a potential cancer therapy.

Inhibition of the IKK/NF-κB pathway by AAV gene transfer improves muscle regeneration in older mdx mice

The IκB kinase (IKKα, β and the regulatory subunit IKKγ) complex regulates nuclear factor of κB (NF-κB) transcriptional activity, which is upregulated in many chronic inflammatory diseases. NF-κB signaling promotes inflammation and limits muscle regeneration in Duchenne muscular dystrophy (DMD), resulting in fibrotic and fatty tissue replacement of muscle that exacerbates the wasting process in dystrophic muscles. Here, we examined whether dominant-negative forms of IKKα (IKKα-dn) and IKKβ (IKKβ-dn) delivered by adeno-associated viral (AAV) vectors to the gastrocnemius (GAS) and tibialis anterior (TA) muscles of 1, 2 and 11-month-old mdx mice, a murine DMD model, block NF-κB activation and increase muscle regeneration. At 1 month post-treatment, the levels of nuclear NF-κB in locally treated muscle were decreased by gene transfer with either AAV-CMV-IKKα-dn or AAV-CMV-IKKβ-dn, but not by IKK wild-type controls (IKKα and β) or phosphate-buffered saline (PBS). Although treatment with AAV-IKKα-dn or AAV-IKKβ-dn vectors had no significant effect on muscle regeneration in young mdx mice treated at 1 and 2 months of age and collected 1 month later, treatment of old (11 months) mdx with AAV-CMV-IKKα-dn or AAV-CMV-IKKβ-dn significantly increased levels of muscle regeneration. In addition, there was a significant decrease in myofiber necrosis in the AAV-IKKα-dn- and AAV-IKKβ-dn-treated mdx muscle in both young and old mice. These results demonstrate that inhibition of IKKα or IKKβ in dystrophic muscle reduces the adverse effects of NF-κB signaling, resulting in a therapeutic effect. Moreover, these results clearly demonstrate the therapeutic benefits of inhibiting NF-κB activation by AAV gene transfer in dystrophic muscle to promote regeneration, particularly in older mdx mice, and block necrosis.

Sesamin Inhibits Macrophage-Induced Vascular Endothelial Growth Factor and Matrix Metalloproteinase-9 Expression and Proangiogenic Activity in Breast Cancer Cells

Sesamin is a sesame component with antihypertensive and antioxidative activities and has recently aroused much interest in studying its potential anticancer application. Macrophage is one of the infiltrating inflammatory cells in solid tumor and may promote tumor progression via enhancement of tumor angiogenesis. In this study, we investigated whether sesamin inhibited macrophage-enhanced proangiogenic activity of breast cancer cell lines MCF-7 and MDA-MB-231. Using vascular endothelial cell capillary tube and network formation assays, both breast cancer cell lines exhibited elevated proangiogenic activities after coculture with macrophages or pretreatment with macrophage-conditioned medium. This elevation of proangiogenic activity was drastically suppressed by sesamin. Vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) induced by macrophages in both cell lines were also inhibited by sesamin. Nuclear levels of HIF-1α and NF-κB, important transcription factors for VEGF and MMP-9 expression, respectively, were obviously reduced by sesamin. VEGF induction by macrophage in MCF-7 cells was shown to be via ERK, JNK, phosphatidylinositol 3-kinase, and NF-κB-mediated pathways. These signaling molecules and additional p38(MAPK) were also involved in macrophage-induced MMP-9 expression. Despite such diverse pathways were induced by macrophage, only Akt and p38(MAPK) activities were potently inhibited by sesamin. Expression of interleukin (IL)-6, IL-8, and tumor necrosis factor-α were substantially increased and involved in macrophage-induced VEGF and MMP-9 mRNA expression in MCF-7 cells. Sesamin effectively inhibited the expression of these cytokines to avoid the reinforced induction of VEGF and MMP-9. In conclusion, sesamin potently inhibited macrophage-enhanced proangiogenic activity of breast cancer cells via inhibition of VEGF and MMP-9 induction.

Effects of sodium houttuyfonate on expression of NF-kappaB and MCP-1 in membranous glomerulonephritis

Aims of the study Sodium houttuyfonate (SH) is an addition compound of sodium bisulfite and houttuynin. Houttuynin is one of the main ingredients in the volatile oil of Houttuynia cordata Thunb, which has been widely used in traditional Chinese medicines. In this study, we investigated the effect of SH in membranous glomerulonephritis (MGN) induced by cationic Bovine Serum Albumin (C-BSA) in BALB/c mice. Materials and methods Mice were divided into four groups, including normal vehicle-treated controls (N group), model (M group), low SH of 60 mg/kg body weight (L group), or high SH of 120 mg/kg body weight (H group). Urine protein quantification was detected by the urine protein strip test. Morphological assessment in kidneys was observed by light microscope and electron microscopy. The level of nuclear factor-kappaB (NF-κB) in the nuclear was evaluated by Western blot. Immunohistochemical was used to analyze the expression of MCP-1. Results SH was shown to reverse C-BSA induced increases in urinary protein, and changes in morphology. Treatment with SH at 60–120 mg/kg (L and H groups, respectively) dose-dependently decreased the level of nuclear NF-κB and MCP-1 expression compared to that of the M group. Conclusions This study reveals that SH could treat C-BSA induced MGN in BALB/c mice by suppressing NF-κB activation and MCP-1 expression. Therefore, the most likely mechanism underlying the biological effects of SH is inhibition of an NF-κB mediated-cytokine pathway.

Circulating nucleic acids in type 1 diabetes may modulate the thymocyte turnover rate

The autoimmunity of type 1 diabetes is associated with T-cell hyperactivity. Current study was designed to examine the effect of circulating ribonucleic acids (RNAs), isolated from type 1 diabetic patients on proliferative, apoptotic and inflammatory potential of rat thymocytes. Rat thymocytes were assayed for proliferating nuclear cell antigen (PCNA), Bcl-2, Bax and NF-κB level, using the flow cytometric and fluorometric assays. Cells were allocated into groups, treated with RNAs purified from plasma of juvenile diabetics, adult type 1 diabetic patients, control healthy children, healthy adult persons, nucleic acids and polynucleotide standards (RNA, polyC, PolyA, PolyIC, and CpG). The upregulation of PCNA and Bcl-2 protein and downregulation of Bax protein and NF-κB was shown when the thymocytes where incubated with RNA purified from plasma of juvenile type 1 diabetic patients. The dysregulation of inflammatory cascade and central tolerance may be a defect in autoimmune diseases related to innate immunity leading to corresponding alteration in adaptive immune response.