NF-kappaB DNA Binding Activity Research Articles

Suppression of inducible nitric oxide synthase and cyclooxygenase-2 by cell-permeable superoxide dismutase in lipopolysaccharide-stimulated BV-2 microglial cells.

Oxidative stress plays a pivotal role in uncontrolled neuro-inflammation leading to many neurological diseases including Alzheimer's. One of the major antioxidant enzymes known to prevent deleterious effects due to oxidative stress is Cu,Zn-superoxide dismutase (SOD). In this study, we examined the regulatory function of SOD on the LPS-induced signaling pathways leading to NF-kappaB activation, expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in BV-2 cells using cell-permeable SOD. Treatment of BV-2 cells with cell-permeable SOD led to a decrease in LPS-induced reactive oxygen species (ROS) generation and significantly inhibited protein and mRNA levels of iNOS and COX-2 upregulated by LPS. Production of NO and PGE2 in LPS stimulated BV-2 cells was significantly abrogated by pretreatment with a cell-permeable SOD fusion protein. Furthermore, cell-permeable SOD inhibited LPS-induced NF-kappaB DNA-binding activity and activation of MAP kinases including ERK, JNK, and p38 in BV-2 cells. These data indicate that SOD has a regulatory function for LPS-induced NF-kappaB activation leading to expression of iNOS and COX-2 in BV-2 cells and suggest that cell-permeable SOD is a feasible therapeutic agent for regulation of ROS-related neurological diseases.

Glabridin inhibits lipopolysaccharide‐induced activation of a microglial cell line, BV‐2, by blocking NF‐κB and AP‐1

Glabridin, a flavonoid present in licorice root, is known to have antiinflammatory and cardiovascular protective activities. The present study reports an inhibitory effect of glabridin on microglial activation. Glabridin dose-dependently attenuated lipopolysaccharide (LPS)-induced production of inflammatory mediators, including nitric oxide, tumor necrosis factor-alpha and interleukin-1beta, in BV-2 cells, a murine microglia cell line. Moreover, mRNA expression of these inflammatory mediators was also suppressed by glabridin in LPS-stimulated BV-2 cells. Further study demonstrated that glabridin inhibited LPS-induced DNA binding activity of NF-kappaB and AP-1 in BV-2 cells. Collectively, the results presented in this report demonstrate that glabridin inhibits the production of inflammatory mediators in BV-2 cells and this is mediated, at least in part, by blocking NF-kappaB and AP-1 activation. The results suggest that glabridin might be a potential therapeutic agent for the treatment of neuroinflammatory and neurodegenerative diseases.

Beta-lapachone suppresses radiation-induced activation of nuclear factor-kappaB.

Anticancer effects of beta-lapachone (beta-lap) are due to generation of ROS and metabolic catastrophes as a result of NAD(P)H:quinone oxidoreductase (NQO1)-mediated futile cycling between the oxidized and reduced forms of beta-lap. It has been shown that NQO1 is also essential for the TNF-induced activation of NF-kappaB and that beta-lap suppresses the TNF-induced NF-kappaB activation. We investigated whether or not NQO1 is involved and beta-lap suppresses the radiation-induced NF-kappaB activation using A549 human lung cancer cells and NQO1-knock down A549 cells (shNQO1 A549 cells). Irradiation with 4 Gy markedly increased the DNA binding activity of NF-kappaB in A549 cells, but not in the shNQO1 A549 cells, thus demonstrating that NQO1 plays a pivotal role in irradiation-induced NF-kappaB activation. Treatment with 10 micronM beta-lap for 4 h almost completely abrogated the radiation-induced increase in NF-kappaB activation and the transcription of NF-kappaB target genes such as bcl2, gadd45beta and cyclinD1. Moreover, beta-lap markedly suppressed the activation of IkappaB kinase gamma (IKKgamma) and the subsequent phosphorylation of IkappaBalpha, thereby inhibiting NF-kappaB activation. It is concluded that beta-lap suppresses the radiation-induced activation of NF-kappaB by interrupting the involvement of NQO1 in the activation of NF-kappaB, thereby inhibiting the transcription of survival signals. The radiosensitization caused by beta-lap may, in part, be attributed to beta-lap-induced suppression of NF-kappaB activation.

Calcium signal-initiated early activation of NF-κB in neurons is a neuroprotective event in response to kainic acid-induced excitotoxicity

We demonstrate that activation of nuclear factor kappaB (NF-kappaB) in neurons is neuroprotective in response to kainic acid (KA)-induced excitotoxicity. Combination of Western blotting, immunocytochemistry, and electrophoresis mobility shift assay showed that KA exposure induced a fast but transient nuclear translocation of the NF-kappaB p65 subunit and increased DNA-binding activity of NF-kappaB in primary cultured cortical neurons. The transient NF-kappaB activity was associated with upregulation of antiapoptotic Bcl-xL and XIAP gene products revealed by real-time PCR. Knockdown of p65 decreased neuronal viability and antiapoptotic gene expression. In addition, we showed that KA-stimulated DNA-binding activity of NF-kappaB was associated with reactive oxygen species and calcium signals, using AMPA/KA receptor antagonist, calcium chelator, and antioxidant. These results suggest that the fast and transient activation of NF-kappaB initiated by calcium signals is one of the important proximal events in response to KA-induced excitotoxicity, which has neuroprotective effect against KA-induced apoptosis.

Commensal microflora induce host defense and decrease bacterial translocation in burn mice through toll-like receptor 4

BackgroundMajor burn is associated with decreased gut barrier function and increased bacterial translocation (BT). This study is to investigate whether commensal microflora induce host defense and decrease BT in burn mice.MethodsFirst, we treated Wild type (WT) mice with antibiotics in drinking water for 4 weeks to deplete gut commensal microflora. At week 3, drinking water was supplemented with lipopolysaccharide (LPS); a ligand for TLR4, to trigger TLRs in gut. The intestinal permeability, glutathione level, NF-κB DNA-binding activity, TLR4 expression of intestinal mucosa, BT to mesenteric lymph nodes (MLNs), and bacterial killing activity of peritoneal cells were measured after thermal injury. Second, lung of animals were harvested for MPO activity and TNFα mRNA expression assay. Third, WT animals were treated with oral antibiotics with or without LPS supplement after burn. At 48 hr after burn, TLR4 expression of intestinal mucosa and bacterial killing activity of cells were examined. Finally, bacterial killing activity and BT to MLNs after thermal injury in C3H/HeJ (TLR4 mutant) mice were measured.ResultsBurn induced BT to MLNs in WT mice. Commensal depletion decreased TLR4 expression as well as NF-κB activation of intestine, myeloperoxidase (MPO) activity as well as TNFα expression of lung, and bacterial killing activity of peritoneal cells. Oral LPS supplement markedly reduced 81% of burn-induced BT and increased TLR4 expression, MPO activity of lung, as well as bacterial killing activity of peritoneal cells. LPS supplement did not change BT or bacterial killing activity in C3H/HeJ mice.ConclusionsCollectively, commensal microflora induce TLR4 expression of intestine and bacterial killing activity of inflammatory cells in burn. TLR4 ligand increases bacterial killing activity and decreases burn-induced BT. Taken together with the abolition of LPS effect in TLR4 mutant mice, we conclude that commensal microflora induce host defense and decrease bacterial translocation in burn mice through toll-like receptor 4.

Growth inhibitory effects of dihydroartemisinin on pancreatic cancer cells: involvement of cell cycle arrest and inactivation of nuclear factor-κB

In a recent publication, we have shown that dihydroartemisinin (DHA), a derivative of antimalaria drug artemisinin, inhibits growth of pancreatic cancer cells in vitro and in vivo mediated by its anti-proliferative and pro-apoptotic effects. As it has been shown that the apoptosis might be induced due to cell cycle arrest, and that transcriptional factor nuclear factor-kappa B (NF-kappaB) plays vital roles in the apoptosis of pancreatic cancer cells, we extend our study to investigate the effects of DHA on cell cycle progression and NF-kappaB activity in pancreatic cancer cells to further reveal the anticancer effects of DHA on pancreatic cancer. Cell cycle progression was determined by propidium iodide staining and flow cytometry. Changes in the expression of cell cycle-associated proteins were detected using Western blot analysis. Measurement of NF-kappaB activity was performed with immunoblot analyzing the nuclear protein expression of NF-kappaB/p65 and ELISA detecting the NF-kappaB DNA-binding activity. The treatment with DHA resulted in a dose-dependent G(0)/G(1) cell cycle arrest and regulated the expression of some cyclins, cdks and cdk inhibitors that involved in the G(0)/G(1) cell cycle progression such as cyclin E, cdk2, cdk4 and p27(Kip1) in pancreatic cancer BxPC-3 and AsPC-1 cells. The translocation and DNA-binding activity of NF-kappaB were inhibited in DHA-treated cells in a dose-dependent manner, indicated the inactivation effects of DHA in pancreatic cancer cells. Together with our previous observations, our data show that DHA induces cell cycle arrest and apoptosis in pancreatic cancer cells, and this effect might be due to inhibition of NF-kappaB signaling. We suggest that DHA could be developed as a novel agent against pancreatic cancer.

Heat stress triggers apoptosis by impairing NF-κB survival signaling in malignant B cells

Nuclear factor-kappaB (NF-kappaB) is involved in multiple aspects of oncogenesis and controls cancer cell survival by promoting anti-apoptotic gene expression. The constitutive activation of NF-kappaB in several types of cancers, including hematological malignancies, has been implicated in the resistance to chemo- and radiation therapy. We have previously reported that cytokine- or virus-induced NF-kappaB activation is inhibited by chemical and physical inducers of the heat shock response (HSR). In this study we show that heat stress inhibits constitutive NF-kappaB DNA-binding activity in different types of B-cell malignancies, including multiple myeloma, activated B-cell-like (ABC) type of diffuse large B-cell lymphoma (DLBCL) and Burkitt's lymphoma presenting aberrant NF-kappaB regulation. Heat-induced NF-kappaB inhibition leads to rapid downregulation of the anti-apoptotic protein cellular inhibitor-of-apoptosis protein 2 (cIAP-2), followed by activation of caspase-3 and cleavage of the caspase-3 substrate poly(adenosine diphosphate ribose)polymerase (PARP), causing massive apoptosis under conditions that do not affect viability in cells not presenting NF-kappaB aberrations. NF-kappaB inhibition by the proteasome inhibitor bortezomib and by short-hairpin RNA (shRNA) interference results in increased sensitivity of HS-Sultan B-cell lymphoma to hyperthermic stress. Altogether, the results indicate that aggressive B-cell malignancies presenting constitutive NF-kappaB activity are sensitive to heat-induced apoptosis, and suggest that aberrant NF-kappaB regulation may be a marker of heat stress sensitivity in cancer cells.

Tetrandrine prevents acquired drug resistance of K562 cells through inhibition of mdr1 gene transcription

There have been no reports describing successful drug resistance reversal in tumors in the clinic until now. Conversion of drug resistance reversal studies to drug resistance prevention studies may assist in the development of more efficient anti-tumor strategies. The present study demonstrates the prevention of doxorubicin-induced drug resistance by tetrandrine on leukemia cells. K562 cells were either solely treated with doxorubicin (0.6 mg/ml) or pretreated with tetrandrine of different concentrations (0.5, 1.0 and 2.0 microg/ml) for 24 h followed by combined-treatment with doxorubicin (0.6 microg/ml). The results showed that doxorubicin treatment only resulted in elevated levels of mdr1 mRNA/P-gp expression. Doxorubicin also induced up-regulation of P-gp functional activities, as intracellular retention of rhodamine was decreased; however, 2.0 microg/ml tetrandrine significantly inhibited the overexpression of doxorubicin-induced mdr1 mRNA/P-gp. Consistently, the functional activity of P-gp was also inhibited, which led to increased intracellular drug retention and the recovery of cell sensitivity to chemotherapeutic drugs in combined treatment groups. Both mRNA and protein levels of NF-kappaB were up-regulated in the cells treated with doxorubicin only. Results from an electrophoretic mobility shift assay and a chromatin immunoprecipitation assay demonstrated the enhanced binding to the promoter region of mdr1 gene compared to the control group. However, tetrandrine could markedly inhibit the doxorubicin-induced expression of NF-kappaB mRNA and protein. In addition, it also attenuated the NF-kappaB DNA-binding activity. In summary, tetrandrine can prevent doxorubicin-induced mdr1 mRNA/P-gp expression and P-gp functions in a dose-dependent manner through a mechanism that may involve inhibition of doxorubicin-induced NF-kappaB mRNA expression and protein activity.

α‐Fetoprotein as a modulator of the pro‐inflammatory response of human keratinocytes

The immunomodulatory effects of alpha-fetoprotein (AFP) on lymphocytes and macrophages have been described in vitro and in vivo. Recombinant forms of human AFP have been proposed as potential therapeutic entities for the treatment of autoimmune diseases. We examined the effects of embryonic and recombinant human AFP on the spontaneous, UVA- and cytokine-induced pro-inflammatory responses of human keratinocytes. Cultures of primary and immortalized human keratinocytes (HaCaT) and human blood T lymphocytes were used. The effects of AFP on cytokine expression were studied by bioplexed elisa and quantitative reverse transcriptase polymerase chain reaction assay. Kinase and nuclear factor kappa B (NFkappaB) phosphorylation were quantified by intracellular elisa. Nuclear activator protein 1 and NFkappaB DNA binding activity was measured by specific assays. Nitric oxide and H(2)O(2) production and redox status were assessed by fluorescent probe and biochemical methods. All forms of AFP enhanced baseline expression of cytokines, chemokines and growth factors. AFP dose-dependently increased tumour necrosis factor alpha-stimulated granulocyte macrophage colony stimulating factor and interleukin 8 expression and decreased tumour necrosis factor alpha-induced monocyte chemotactic protein 1 and IP-10 (interferon gamma-produced protein of 10 kDa) expression. AFP induced a marked activator protein 1 activation in human keratinocytes. AFP also increased H(2)O(2) and modulated nitrite/nitrate levels in non-stimulated keratinocytes whereas it did not affect these parameters or cytokine release from UVA-stimulated cells. Phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Akt1 but not NFkappaB was activated by AFP alone or by its combination with UVA. Exogenous AFP induces activation of human keratinocytes, with de novo expression of a number of pro-inflammatory mediators and modulation of their pro-inflammatory response to cytokines or UVA. AFP may modulate inflammatory events in human skin.

Oxidized low density lipoprotein decreases Rankl‐induced differentiation of osteoclasts by inhibition of Rankl signaling

The role of OxLDL in the generation and progression of atherosclerosis is well admitted. In addition, it is well known that atherosclerosis is often accompanied by perturbations in bone remodeling, resulting in osteoporosis. In the current studies, the effect of Cu(2+)-oxidized LDL (OxLDL) on RANKL-induced RAW264.7 mouse monocytes-macrophages differentiation to osteoclasts and on RANKL signaling pathway was investigated. OxLDL, within the range of 10-50 microg protein/ml, prevented RANKL-induced generation of multinucleated osteoclast-like cells and RANKL-induced tartrate resistant acid phosphatase (TRAP) activity. OxLDL also prevented the RANKL-induced phosphorylation of ERK, p38 and JNK kinases, together with the RANKL-induced DNA binding activities of NFkappaB and NFAT transcription factors. Concomitantly, OxLDL enhanced RANKL-induced generation of reactive oxygen species in a dose-dependent manner. The antioxidant glutathione (GSH) prevented whereas the prooxidant compound buthionine-sulfoximine (BSO) enhanced the effect of OxLDL on RANKL-induced oxidative stress and RANKL-induced differentiation. Finally, OxLDL also prevented RANKL-induced TRAP activity and RANKL-induced bone resorbing activity of human peripheral blood mononuclear cells. These results demonstrate that OxLDL, by generation of an intracellular oxidative stress, prevents the differentiation of osteoclasts by inhibition of RANKL signaling pathway. This might be related to the fact that atherosclerosis is accompanied by perturbations in bone and vascular remodeling, leading to osteoporosis and vascular calcification.

Fisetin, a natural flavonoid, targets chemoresistant human pancreatic cancer AsPC‐1 cells through DR3‐mediated inhibition of NF‐κB

Death receptors of the tumor necrosis factor (TNF) receptor super family have been implicated in constitutive activation of nuclear factor-kappa B (NF-kappaB) in pancreatic cancer (PaC) cells. In this study, we demonstrate that fisetin, a natural flavonoid, induces apoptosis and inhibits invasion of chemoresistant PaC AsPC-1 cells through suppression of DR3-mediated NF-kappaB activation. Fisetin treatment resulted in dose-dependent inhibition of PaC cell growth and cell proliferation with concomitant induction of apoptosis. A cDNA array analysis revealed that fisetin modulates expression of more than 20 genes at transcription level with maximum decrease observed in DR3 expression and a parallel increase observed in the expression levels of IkappaBalpha, an NF-kappaB inhibitor. Down-regulation of DR3 in PaC cells was found to down regulate activated pNF-kappaB/p65, pIkBalpha/beta kinases (pIKK's), MMP9 and XIAP that mostly impart chemoresistance in PaC. Immunoblotting and EMSA analysis showed a marked decrease in pNF-kappaB and NF-kappaB DNA binding activity, respectively, with modest decrease in NF-kappaB promoter activity and significant decrease in MMP9 promoter activity with fisetin treatment. Importantly, consistent with these findings, we further found that transient down-regulation of DR3 by RNA interference significantly augmented fisetin induced changes in cell proliferation, cell invasion and apoptosis paralleled with decrease in pNF-kappaB, pIKKalpha/beta, MMP9, XIAP and NF-kappaB DNA binding activity. Blocking of DR3 receptor with an extra cellular domain blocking antibody demonstrated similar effects. These data provide evidence that fisetin could provide a biological rationale for treatment of pancreatic cancer or as an adjuvant with conventional therapeutic regimens.

Benzyl isothiocyanate exhibits anti-inflammatory effects in murine macrophages and in mouse skin

Benzyl isothiocyanate (BITC) is detected in abundance in Brassica vegetables, and some previous studies have demonstrated that BITC may potentially function as a chemopreventive agent in humans. This study examined whether BITC inhibits lipopolysaccharide (LPS)-induced inflammatory responses in Raw 264.7 macrophages and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema formation. The treatment of macrophages with various concentrations of BITC resulted in a dose-dependent reduction in the LPS-induced secretion of interleukin (IL)-1beta, TNF-alpha, and IL-6 and their corresponding mRNA levels, as well as in the production of nitric oxide and PGE(2). Consistent with these findings, BITC inhibited the LPS-induced expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 proteins and their corresponding mRNAs. BITC inhibited LPS-induced phosphorylation and the degradation of the inhibitor of kappaBalpha, translocation of p65 into the nucleus, and the DNA binding activity and transcriptional activity of NFkappaB. Moreover, the LPS-stimulated phosphorylation of extracellular signal regulated kinase (ERK)1/2 and Akt was suppressed by BITC. BITC also inhibited ear edema formation and the protein expression of iNOS and COX-2 in mouse skin treated with TPA. We demonstrate that BITC is a potent anti-inflammatory agent, and the anti-inflammatory properties of BITC may result from the downregulation of NFkappaB signaling.

Nod2 Mutation Enhances NF-kappaB Activity and Bacterial Killing Activity of Macrophages

NOD2, an intracellular sensor of bacteria, are linked to increased susceptibility to bacteria in Crohn's disease (CD). The NOD2 protein is expressed mainly by macrophages and dendritic cells. This study is to examine the role of NOD2 in the innate response of macrophages to bacterial challenge. First, peritoneal macrophages and alveolar macrophages were harvested from WT, Nod2(2939iC), as well as TLR4(-/-) mice and incubated with E. coli or P. aeruginosa. Bacterial killing activity; IL-1beta and TLR4 protein expression; NF-kappaB DNA binding activity assay; as well as IL-1beta, TNFalpha, TLR2, TLR4 and TLR9 mRNA expression of macrophages were examined. We found that alveolar macrophages and peritoneal macrophages of Nod2(2939iC) mice but not WT mice or TLR4(-/-) mice demonstrated a significant increase of E. coli killing activity. Bacterial challenge also induced a significant increase of pro-IL-1beta protein expression; NF-kappaB DNA binding activity; as well as IL-1beta and TNFalpha mRNA expression of the peritoneal macrophages in Nod2(2939iC) mice. Collectively, the increase of bacterial killing activity, IL-1beta expression, and NF-kappaB DNA binding activity of macrophages in Nod2(2939iC) mice suggests that NOD2 is a positive regulator of NF-kappaB/IL-1beta-mediated innate response to bacteria challenge in Crohn's disease.

Epidermal growth factor increases prostaglandin E2 production via ERK1/2 MAPK and NF-κB pathway in fibroblast like synoviocytes from patients with rheumatoid arthritis

High concentration of epidermal growth factor (EGF) is found in the synovial fluid of rheumatoid arthritis (RA) that might imply the involvement of EGF in the pathogenesis of arthritic diseases. In order to investigate if EGF is involved in the regulation of cyclooxygenase-2 (COX-2) and the prostaglandin E(2) (PGE(2)) production in fibroblast like synoviocytes (FLS) from patients with RA. The levels of COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) were evaluated using RT-PCR and Western blot analysis. Electrophoretic mobility shift assay (EMSA) was performed to investigate EGF mediated DNA binding activity of nuclear factor-kappaB (NF-kappaB). PGE(2) levels were analyzed by ELISA. EGF enhanced both COX-2 protein and mRNA expressions. mPGES-1 mRNA level was also increased by EGF treatment. EGF also stimulated ERK1/2 MAPK activity and the inhibition of ERK1/2 by PD098059 (ERK1/2 specific inhibitor) resulted in the suppression of EGF-induced COX-2 expression. The DNA binding activity of NF-kappaB was remarkably increased by EGF treatment and the pretreatment of PD098059 abolished EGF-stimulated NF-kappaB activity. We also observed that the level of PGE(2) was significantly elevated with the treatment of EGF in FLS, and the pretreatment of PD098059 abolished this stimulating effect. These results suggest that EGF is involved in the inflammatory process of RA by stimulating COX-2 expression and PGE(2) production. And EGF enhanced PGE(2) production appears to be mediated via ERK1/2 MAPK and NF-kappaB pathway in FLS.

Effects of Exercise on Cyclooxygenase‐2 Expression and Nuclear Factor‐κB DNA Binding in Human Peripheral Blood Mononuclear Cells

There are multiple lines of compelling evidence supporting the beneficial effect of exercise on the prevention and/or improvement of certain chronic diseases. However, exhaustive or intense exercise causes oxygen free radical generation and oxidative stress, which can lead to injuries and chronic fatigue as well as inflammation. Abnormal upregulation of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostaglandin biosynthesis, has been implicated in many inflammation-associated chronic disorders. Nuclear factor-kappaB (NF-kappaB) is a major transcription factor involved in regulation of COX-2 gene expression. To determine whether inflammation induction is dependent on intensity of exercise, COX-2 expression and NF-kappaB activation were adopted as the main targets. Thirteen volunteers who participated in the exercise program were subject to four exercise intensities [40, 60, 80, and 100% of heart rate reserve (HRR)] on a treadmill and to resting conditions. Isolated human peripheral blood mononuclear cells (PBMCs) were collected during the resting state and immediately after exercise and subjected to the electrophoretic mobility gel shift assay and Western blot analysis. As exercise intensity increased, both COX-2 expression and NF-kappaB DNA-binding activity were enhanced. The expression of IkappaB kinase alpha (IKKalpha) and IkappaBalpha were not significantly altered. However, exhaustive/vigorous exercise (100% HRR) could induce the phosphorylation of both IKKalpha and IkappaBalpha. In conclusion, a single bout of exercise induced COX-2 expression and DNA-binding activity of NF-kappaB in human PBMCs, and both COX-2 expression and DNA-binding activity of NF-kappaB were dependent on exercise intensity.

Plant Polyphenols Effectively Protect HaCaT Cells from Ultraviolet C–Triggered Necrosis and Suppress Inflammatory Chemokine Expression

Oxidative stress is a common response of epidermal cells to a variety of noxious stimuli such as ultraviolet (UV) radiation from solar light and proinflammatory cytokines from skin-infiltrating leukocytes. Here, we report that two types of plant-derived antioxidants, the phenylpropanoid glycoside verbascoside as well as the flavonoids rutin and quercetin possess protective effects against UVC-induced cell damage and proinflammatory activation. The molecules under investigation were effective against the loss of cell integrity associated with necrosis in doses consistent with their antioxidant activity, whereas they did not significantly oppose UVC-induced proliferation arrest and apoptosis. By contrast, only verbascoside effectively inhibited cytokine-induced release of proinflammatory mediators in a dose-dependent fashion. Verbascoside and its homologue teupolioside dramatically impaired NF-kappaB and AP-1 DNA binding activity. These results suggest that plant polyphenols with antioxidant properties have distinct mechanisms in the suppression of oxidative stress induced in keratinocytes by different stimuli. Verbascoside and teupolioside are hence of potential interest in the protection of the skin from both environmental and inflammatory insults.

Role for nuclear factor-κB in augmented lung injury because of interaction between hyperoxia and high stretch ventilation

High-tidal-volume mechanical ventilation and hyperoxia used in patients with acute lung injury (ALI) can induce alveolar coagulopathy and fibrin depositions within the airways. Hyperoxia has been shown to increase ventilator-induced lung injury (VILI), but the mechanisms that regulate interaction between high-tidal-volume mechanical ventilation and hyperoxia are unclear. We hypothesized that mechanical stretch with hyperoxia synergistically augmented neutrophil infiltration and production of plasminogen activator inhibitor-1 (PAI-1) via the nuclear factor-kappaB (NF-kappaB) pathway. C57BL/6 mice (n=5 per group) were exposed to high-tidal-volume (30 mL/kg) or low-tidal-volume (6 mL/kg) mechanical ventilation with room air or hyperoxia for 1 to 5h after 2-microg/g NF-kappaB inhibitor (SN-50) administration. Nonventilated mice with room air or hyperoxia served as control groups. Evans blue dye, myeloperoxidase, electrophoretic mobility shifting of nuclear protein, and inflammatory cytokine were measured. The expression of tumor necrosis factor-alpha (TNF-alpha) and PAI-1 were studied by immunohistochemistry. The addition of hyperoxia to high-tidal-volume ventilation-augmented lung injury, as demonstrated by increased microvascular leak, neutrophil migration into the lung, TNF-alpha and active PAI-1 production, DNA binding activity of NF-kappaB, and NF-kappaB activation. No statistically significant increase of neutrophil infiltration and inflammatory cytokine production was found in the mice ventilated at 6 mL/kg using hyperoxia. Hyperoxia-induced augmentation of VILI was attenuated in mice with pharmacologic inhibition of NF-kappaB activity by SN-50. We conclude that hyperoxia increased high-tidal-volume-induced cytokine production and neutrophil influx through activation of the NF-kappaB pathway.

Oxidative Stress Enhances Toll-Like Receptor 3 Response to Double-Stranded RNA in Airway Epithelial Cells

Virus infections are a major cause of chronic obstructive pulmonary disease (COPD) exacerbations. Recently, Toll-like receptor 3 (TLR3) has been demonstrated to react to double-stranded RNA (dsRNA) and to be involved in the immune responses after viral infections. In the present study, we examined whether oxidative stress, which is involved in the pathogenesis of COPD, enhances the responses of TLR3 in airway epithelial cells. The effect of hydrogen peroxide (H(2)O(2)) on the release of IL-8 from BEAS-2B cells and primary human bronchial epithelial cells after stimulation with polyinosine-polycytidylic acid [poly(I:C)], a synthetic analog of viral dsRNA and a ligand for TLR3, and the signal transduction were examined. One hundred to 150 muM H(2)O(2) significantly potentiated the release of IL-8 from the epithelial cells after stimulation with 10 microg/ml poly(I:C). The H(2)O(2)-augmented IL-8 release was inhibited by treatment with N-acetylcysteine. One hundred micromoles of H(2)O(2) enhanced the translocation of nuclear factor (NF)-kappaB p65, but not that of interferon regulatory factor-3 (IRF-3), into the nucleus and the NF-kappaB DNA binding activity after poly(I:C) stimulation, which effect was inhibited not by the silencing of IRF-3 but by MG132, a proteasome inhibitor, or dexamethasone. One hundred micromoles of H(2)O(2) potentiated the TLR3 expression on the airway epithelial cells treated with poly(I:C). These data suggest that oxidative stress augments the response of TLR3 in airway epithelial cells via NF-kappaB and that this effect might be partly mediated by the enhancement of TLR3 expression. Modulation of this pathway may be a therapeutic target for viral-induced exacerbations of COPD.

Loss of NADPH Quinone Oxidoreductase in the Prostate and Enhanced Serum Levels of Cytokine-Induced Neutrophil Chemoattractant 2α in Hormone-Stimulated Noble Rats: Potential Role in Prostatic Intraepithelial Neoplasia Development

The Noble rat is an established model for studying hormone-induced development of prostatic intraepithelial neoplasia and prostatic adenocarcinoma. It is known that for a period, hormones in the prostate generate reactive molecules that have the capacity to overwhelm intracellular defenses, damage macromolecules, and modulate redox-regulated signaling pathways leading to increased oxidative stress. Such hormone-induced imbalance in the oxidative stress/antioxidant defense enzymes may lead to neoplastic transformation of the prostate. We investigated alteration in the expression of critical antioxidant defense enzymes, a redox-regulated transcription factor nuclear factor kappaB (NFkappaB) and its downstream target inflammation-associated cyclooxygenase 2 (Cox-2) in the prostate from hormone-stimulated Noble rats using immunohistochemistry. Further, we also analyzed serum levels of cytokines and chemokines associated with inflammation using multiplex immunoassay. Our results show that there was no significant change in the expression of glutathione peroxidase, glutathione S-transferase pi, superoxide dismutase, or catalase. However, the level of NADPH quinone oxidoreductase decreased in hormone-stimulated animals compared with their unstimulated counterparts. Further, the prostate from hormone-stimulated rats showed very strong expressions of p65, Cox-2, and NFkappaB DNA binding activity. In addition, the cytokine-induced neutrophil chemoattractant 2alpha was significantly upregulated by more than 10-fold (P = .001) in serum from animals stimulated with hormones. Although further studies are required, we speculate that activation of NFkappaB/cytokine-induced neutrophil chemoattractant 2alpha/Cox-2 along with modulation of antioxidant defense mechanisms may create a proinflammatory environment suitable for tumor growth and survival.

The canonical NF-κB pathway is required for formation of luminal mammary neoplasias and is activated in the mammary progenitor population

The role of the canonical NF-kappaB pathway in mammary tumorigenesis was investigated using a transgenic (TG) mouse expressing a dominant-negative inhibitor of kappaB (IkappaBalpha(SR (S32A/S36A))) in the mammary gland under the control of the mouse mammary tumor virus promoter (MMTV). TG and control mice were subjected to a chemical carcinogenesis protocol. Hyperkeratinized squamous metaplasias (cytokeratin-6+/p63+) sometimes with a basaloid island component, were found in both TG and control mice whereas luminal (cytokeratin-19+/MUC1+) ErbB2+ papillary and adenomatous lesions developed almost exclusively in control mice. p65/RelA- and NF-kappaB DNA-binding activity were detected in mammary luminal lesions, but rarely in squamous metaplasias. Analysis of NF-kappaB family proteins and target genes using microarray data from a cohort of human mammary tumors revealed the expression of a canonical NF-kappaB pathway, but not non-canonical pathway proteins in HER2+ luminal cancers. HER2+ tumors also showed differential regulation of specific NF-kappaB target genes relative to basal and ER+ luminal cancers. Isolation of mammary cell populations enriched for stem and progenitor cell characteristics from an NF-kappaB-EGFP reporter mouse by fluorescence-activated cell sorting demonstrated that luminal progenitors contain activated NF-kappaB whereas the mammary stem cell-enriched population, does not. Together these data suggest that the canonical NF-kappaB pathway is active in normal luminal progenitor cells before transformation and is required for the formation of mammary luminal-type epithelial neoplasias.