Repression Of NF-κB Research Articles

The anti-inflammatory and autophagy-induced effects of baicalin in ankylosing spondylitis hip ligament fibroblasts

IntroductionAnkylosing spondylitis (AS) is characterised by chronic inflammation that affects the axial skeleton and peripheral joints. Hip involvement occurs in nearly one-third of patients with AS and is considered a poor prognostic sign. Inflammation is also associated with AS progression. Baicalin is a bioactive flavonoid possessing both anti-inflammatory and antioxidant properties, although its activity in AS has not been investigated. The aim of our study was to evaluate the effects of baicalin on AS pathogenesis-related cells and investigate the potential mechanisms. MethodsFibroblasts were isolated from AS patients who met the modified New York criteria and underwent total hip arthroplasty. Flow cytometry was used to identify the fibroblasts. Cytokine expression was evaluated by enzyme-linked immunosorbent assay and real-time polymerase chain reaction, and protein expression was detected using western blotting. Immunofluorescence and transmission electron microscopy were used to assess autophagy. ResultsBaicalin effectively inhibited the expression of inflammatory cytokines upregulated by lipopolysaccharides in AS hip ligament fibroblasts. Moreover, the lipopolysaccharide-triggered nuclear factor kappa-B (NF-κB) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathways were also suppressed by baicalin. Furthermore, baicalin markedly induced autophagy, which suppressed inflammation dose-dependently. ConclusionsThe anti-inflammatory activity of baicalin was mediated by the repression of NF-κB and JAK/STAT signalling pathways. Furthermore, the induction of autophagy may contribute to inhibiting inflammation.

Elevation of hsa-miR-7-5p level mediated by CtBP1-p300-AP1 complex targets ATXN1 to trigger NF-κB-dependent inflammation response.

Nuclear factor-κB (NF-κB)-mediated inflammation is a major cause of acute respiratory distress syndrome (ARDS). However, the regulatory mechanisms by which NF-κB transactivates proinflammatory cytokines remain unclear in the pathogenesis of ARDS. Herein, we report that the activating protein 1 (AP1) transcription factor recruits a histone acetyltransferase p300 and a transcriptional regulator C-terminal binding protein 1 (CtBP1) to assemble the CtBP1-p300-AP1 complex, which transactivates the expression of hsa-miR-7-5p in ARDS biopsies. Overexpressed hsa-miR-7-5p binds to the three prime untranslated regions (3'-UTRs) of ataxin 1 (ATXN1), suppressing its expression. Decreased ATXN1 expression relieves its repression of NF-κB, causing the induction of proinflammatory cytokine genes and triggering an inflammatory response. Depletion of CtBP1 or treatments with two CtBP1 inhibitors (NSC95397 and 4-methylthio-2-oxobutanoate (MTOB)) in human macrophages impairs the assembly of the CtBP2-p300-AP1 complex, resulting in decreased hsa-miR-7-5p levels, upregulation of ATXN1, and attenuation of proinflammatory cytokines. A similar regulatory mechanism was observed in lipopolysaccharide-treated mice. Our results reveal that increased hsa-miR-7-5p level mediated by the CtBP1-p300-AP1 complex targets ATXN1 to trigger an NF-κB-dependent inflammatory response. Interfering with this signaling pathway to block the inflammatory response may be a strategy for treating ARDS. KEY MESSAGES : The transcription factor AP1 recruits p300 and CtBP1 to form a transcriptional complex, which transactivates the expression of hsa-miR-7-5p in ARDS biopsies. Overexpressed hsa-miR-7-5p binds to the 3'-UTR of ATXN1, suppressing its expression. The decreased ATXN1 impaired its suppression of NF-κB, causing the induction of proinflammatory cytokine genes and triggering inflammation response. Disruption of the assembly of CtBP2-p300-AP1 complex upregulates ATXN1 and attenuates inflammation.

GB1a Ameliorates Ulcerative Colitis via Regulation of the NF-κB and Nrf2 Signaling Pathways in an Experimental Model.

Ulcerative colitis (UC) is an inflammatory bowel disease. The intake of African Garcinia Kola nuts has been reported as a therapy for diarrhea and dysentery in the African population. However, the mechanism of action through which Garcinia Kola nuts act to ameliorates UC remains unknown. GB1a is the main active component of Garcinia Kola nuts. In this study, we explored the therapeutic effects and underlying mechanism of GB1a on dextran sodium sulfate (DSS)-induced UC. Human Colonic Epithelial Cells (HCoEpic) were challenged with TNF-α to test the effects of GB1a in protecting against oxidative stress and inflammation in vitro. Our data showed that GB1a significantly attenuated DSS-induced colonic inflammatory injury manifested as reversed loss of body weight and disease activity index (DAI) scores in UC mice. We also showed that GB1a improved the permeability of the intestinal epithelium by modulating the expression of tight junction proteins (ZO-1, Occludin). Mechanistically, GB1a may activate the Nrf2 antioxidant signaling pathway and suppress the nuclear translocation of NF-κB in reduced oxidative stress and expression of inflammatory genes induced by TNF-α in HCoEpic cells. Our study suggests that GB1a alleviates inflammation, oxidative stress and the permeability of the colonic epithelial mucosa in UC mice via the repression of NF-κB and activation of Nrf2 signaling pathway.

The Transcription Factor NF-κB in Stem Cells and Development.

NF-κB (nuclear factor kappa B) belongs to a family of transcription factors known to regulate a broad range of processes such as immune cell function, proliferation and cancer, neuroprotection, and long-term memory. Upcoming fields of NF-κB research include its role in stem cells and developmental processes. In the present review, we discuss one role of NF-κB in development in Drosophila, Xenopus, mice, and humans in accordance with the concept of evo-devo (evolutionary developmental biology). REL domain-containing proteins of the NF-κB family are evolutionarily conserved among these species. In addition, we summarize cellular phenotypes such as defective B- and T-cell compartments related to genetic NF-κB defects detected among different species. While NF-κB proteins are present in nearly all differentiated cell types, mouse and human embryonic stem cells do not contain NF-κB proteins, potentially due to miRNA-dependent inhibition. However, the mesodermal and neuroectodermal differentiation of mouse and human embryonic stem cells is hampered upon the repression of NF-κB. We further discuss NF-κB as a crucial regulator of differentiation in adult stem cells such as neural crest-derived and mesenchymal stem cells. In particular, c-REL seems to be important for neuronal differentiation and the neuroprotection of human adult stem cells, while RELA plays a crucial role in osteogenic and mesodermal differentiation.

Genomics Analysis of Metabolic Pathways of Human Stem Cell-Derived Microglia-Like Cells and the Integrated Cortical Spheroids.

Brain spheroids or organoids derived from human pluripotent stem cells (hiPSCs) are still not capable of completely recapitulating in vivo human brain tissue, and one of the limitations is lack of microglia. To add built-in immune function, coculture of the dorsal forebrain spheroids with isogenic microglia-like cells (D-MG) was performed in our study. The three-dimensional D-MG spheroids were analyzed for their transcriptome and compared with isogenic microglia-like cells (MG). Cortical spheroids containing microglia-like cells displayed different metabolic programming, which may affect the associated phenotype. The expression of genes related to glycolysis and hypoxia signaling was increased in cocultured D-MG spheroids, indicating the metabolic shift to aerobic glycolysis, which is in favor of M1 polarization of microglia-like cells. In addition, the metabolic pathways and the signaling pathways involved in cell proliferation, cell death, PIK3/AKT/mTOR signaling, eukaryotic initiation factor 2 pathway, and Wnt and Notch pathways were analyzed. The results demonstrate the activation of mTOR and p53 signaling, increased expression of Notch ligands, and the repression of NF-κB and canonical Wnt pathways, as well as the lower expression of cell cycle genes in the cocultured D-MG spheroids. This analysis indicates that physiological 3-D microenvironment may reshape the immunity of in vitro cortical spheroids and better recapitulate in vivo brain tissue function for disease modeling and drug screening.

Angelica polysaccharide mitigates lipopolysaccharide-evoked inflammatory injury by regulating microRNA-10a in neuronal cell line HT22

Background Angelica polysaccharide (AP) is disengaged from the roots of Angelica sinensis. The extensive pathological activities of AP have been discovered in disparate diseases. Nevertheless, the impression of AP in epilepsy (EP) remains unaware. The research attempted to probe the impact of AP on lipopolysaccharide (LPS)-evoked inflammatory injury in HT22 cells. Methods AP were exploited to stimulate HT22 cells, cell cytotoxicity was monitored by CCK-8 assay. LPS was utilized to administrate HT22 cells to evoke inflammatory injury, meanwhile the involvements of AP in cell proliferation, apoptosis and inflammatory cytokines productions were examined. MicroRNA-10a (miR-10a) inhibitor and its negative control were one by one transfected into HT22 cells, the effect of miR-10a inhibition on LPS- and AP-treated cells was determined. NF-κB and JAK2/STAT3 pathways were ultimately detected. Results AP promoted cell proliferation, inhibited apoptosis and suppressed IL-1β, TNF-α and IL-6 productions in LPS-stimulated HT22 cells. Additionally, AP raised miR-10a expression in HT22 cells administration with LPS. These functions of AP in LPS-disposed cells were conversed by miR-10a suppression. Further, AP interdicted NF-κB and JAK2/STAT3 pathways via enhancing miR-10a. Conclusions Data corroboarted that AP mitigated LPS-evoked inflammatory injury through repression of NF-κB and JAK2/STAT3 pathways by regulating miR-10a in HT22 cells.

Fabrication of resveratrol coated gold nanoparticles and investigation of their effect on diabetic retinopathy in streptozotocin induced diabetic rats

This study includes the fabrication of gold nanoparticles (AuNPs) with the help of a plant polyphenol called Resveratrol through an ecofriendly synthetic process without any use of harmful reductants. In the fabrication of AuNPs, Resveratrol acts as both stabilizing and reducing agent. The prepared AuNPs is tested on streptozotocin (STZ) induced diabetic rats for their amelioration consequence. The images of TEM displayed the development of spherical nanoparticles (NPs) with a median of 20 nm particle size. The STZ injected diabetic rats were administrated orally with calcium dobesilate (CD; 500 mg/kg/day) or AuNPs (200, 300 mg/kg/day) for a period of 3 months. The characteristics displayed by AuNPs were found to be similar with CD in decreasing permeability of blood–retinal barrier in STZ injected diabetic rats. The retinal vessels in the AuNPs administrated diabetic rats were observed to be decreased through the retinal histopathological examination. In the AuNPs administrated diabetic rats, the retinal expression of renal Pigment Epithelium-Derived Factor (PEDF) was observed to be increased and the Vascular Endothelial Growth Factor (VEGF-1), which was increased in diabetic rats was declined on treating with AuNPs. On treating the STZ injected diabetic rats with AuNPs, all the retinal mRNA expressions of VEGF-1, Tumor Necrosis Factor (TNFα), Monocyte Chemotactic Proteins-1 (MCP-1), Intercellular Adhesion Molecule-1 (ICAM-1), and Interleukin (IL)-6, IL-1β were observed to be reduced. Furthermore, AuNPs can reduce phosphorylation of Nuclear Factor Kappa B (NF-κB) p65 and Extracellular signal Regulated Kinase (ERK) 1/2 along with a growth in nuclear translocation of pNF-κB p65 produced by STZ. To conclude, the protective effect of AuNPs on STZ injected diabetic rats could help in redeveloping the balance among the inhibitors and stimulators of angiogenesis. Furthermore, on treating with AuNPs results in inhibiting the signaling pathway of ERK1/2 as well as with amelioration of retinal inflammation through trans repression of NF-κB.

Nitric Oxide Modulates Macrophage Responses to Mycobacterium tuberculosis Infection through Activation of HIF-1α and Repression of NF-κB.

IFN-γ is essential for control of Mycobacterium tuberculosis infection in vitro and in vivo. However, the mechanisms by which IFN-γ controls infection remain only partially understood. One of the crucial IFN-γ target genes required for control of M. tuberculosis is inducible NO synthase (iNOS). Although NO produced by iNOS is thought to have direct bactericidal activity against M. tuberculosis, the role of NO as a signaling molecule has been poorly characterized in the context M. tuberculosis infection. In this study, we found that iNOS broadly regulates the macrophage transcriptome during M. tuberculosis infection, activating antimicrobial pathways while also limiting inflammatory cytokine production. The transcription factor hypoxia inducible factor-1α (HIF-1α) was recently shown to be critical for IFN-γ-mediated control of M. tuberculosis infection. We found that HIF-1α function requires NO production, and that HIF-1α and iNOS are linked by a positive feedback loop that amplifies macrophage activation. Furthermore, we found that NO inhibits NF-κB activity to prevent hyperinflammatory responses. Thus, NO activates robust microbicidal programs while also limiting damaging inflammation. IFN-γ signaling must carefully calibrate an effective immune response that does not cause excessive tissue damage, and this study identifies NO as a key player in establishing this balance during M. tuberculosis infection.

A unique tolerizing dendritic cell phenotype induced by the synthetic triterpenoid CDDO-DFPA (RTA-408) is protective against EAE

Tolerogenic dendritic cells (DCs) have emerged as relevant clinical targets for the treatment of multiple sclerosis and other autoimmune disorders. However, the pathways essential for conferring the tolerizing DC phenotype and optimal methods for their induction remain an intense area of research. Triterpenoids are a class of small molecules with potent immunomodulatory activity linked to activation of Nrf2 target genes, and can also suppress the manifestations of experimental autoimmune encephalomyelitis (EAE). Here we demonstrate that DCs are a principal target of the immune modulating activity of triterpenoids in the context of EAE. Exposure of DCs to the new class of triterpenoid CDDO-DFPA (RTA-408) results in the induction of HO-1, TGF-β, and IL-10, as well as the repression of NF-κB, EDN-1 and pro-inflammatory cytokines IL-6, IL-12, and TNFα. CDDO-DFPA exposed DCs retained expression of surface ligands and capacity for antigen uptake but were impaired to induce Th1 and Th17 cells. TGF-β was identified as the factor mediating suppression of T cell proliferation by CDDO-DFPA pretreated DCs, which failed to passively induce EAE. These findings demonstrate the potential therapeutic utility of CDDO-DFPA in the treatment and prevention of autoimmune disorders, and its capacity to induce tolerance via modulation of the DC phenotype.

Abstract 2332: Integral modulation of nuclear factor-kappa B activation by C/EBPβ and the endoplasmic reticulum stress sensor PERK to mediate estrogen-induced apoptosis in estrogen-deprived breast cancer cells

Abstract Stress responses are critical for estrogen (E2) to induce apoptosis in E2-deprived breast cancer cells. Nuclear factor-kappa B (NF-κB) is well known as a therapeutic target to prevent stress responses in chronic inflammatory diseases including cancer. However, whether E2 activates NF-κB to participate in stress-associated apoptosis in E2-deprived breast cancer cells is unclear. We demonstrated that E2 differentially modulates NF-κB activity in E2-deprived breast cancer cells according to the treatment time. Because E2 initially has significant potential to down modulate the NF-κB activation, it completely suppresses the tumor necrosis factor alpha (TNFα)-induced NF-κB activation. We found that E2 preferentially and constantly enhances the expression of transcription factor CCAAT/enhancer binding protein beta (C/EBPβ) which is responsible for suppression of NF-κB activation by E2 in MCF-7:5C cells. The mTOR signaling pathway promotes repression of NF-κB by C/EBPβ which is confirmed by the evidence that inhibition of mTOR is synergistic with E2 to upregulate NF-κB-dependent genes, such as TNFα. Interestingly, NF-κB p65 activity is upregulated when E2-treatment is administered for 48 hours, leading to induction of TNFα. Blocking the nuclear translocation of NF-κB completely prevents E2 from induction of TNFα and apoptosis. Importantly, protein kinase RNA-like endoplasmic reticulum kinase (PERK), a stress sensor of unfolded protein response, is activated by E2 and plays an essential role in increasing NF-κB p65 DNA binding through the activation of STAT3, independently of canonical IκBα signal pathway. Thus, inhibition of PERK kinase activity completely blocks nuclear activation of NF-κB and NF-κB-dependent induction of TNFα, thereby preventing E2-induced apoptosis. All of these findings illustrate a crucial role for the novel PERK/NF-κB/TNFα axis in E2-induced apoptosis which is integrally modulated by the stress responsive transcription factor C/EBPβ and endoplasmic reticulum stress. This study provides an important rationale for exercising caution in clinical trials when considering targeting PERK or NF-κB following the development of acquired resistance to aromatase inhibitors whereas mTOR may be a target to enhance the therapeutic effects of E2 in antihormone resistant breast cancer. Citation Format: Ping Fan, Amit K. Tyagi, Fadeke A. Agboke, Niranjana Pokharel, V. Craig Jordan. Integral modulation of nuclear factor-kappa B activation by C/EBPβ and the endoplasmic reticulum stress sensor PERK to mediate estrogen-induced apoptosis in estrogen-deprived breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2332. doi:10.1158/1538-7445.AM2017-2332

Genistein inhibited ammonia induced astrocyte swelling by inhibiting NF-κB activation-mediated nitric oxide formation.

Our previous study has indicated the involvement of epidermal growth factor receptor (EGFR) transactivation in ammonia-induced astrocyte swelling, which represents a major pathogenesis of brain edema in hepatic encephalopathy. In this study, we examined the effect of genistein, a naturally occurred broad-spectrum protein tyrosine kinase (PTK) inhibitor, on ammonia-induced cell swelling. We found that genistein pretreatment significantly prevented ammonia-induced astrocyte swelling. Mechanistically, ammonia triggered EGFR/extracellular signal-regulated kinase (ERK) association and subsequent ERK phosphorylation were alleviated by genistein pretreatment. Moreover, ammonia-induced NF-κB nuclear location, iNOS expression, and consequent NO production were all prevented by AG1478 and genistein pretreatment. This study suggested that genistein could alleviate ammonia-induced astrocyte swelling, which may be, at least partly, related to its PTK-inhibiting activity and repression of NF-κB mediated iNOS-derived NO accumulation.

Long-acting β2-agonists promote glucocorticoid-mediated repression of NF-κB by enhancing expression of the feedback regulator TNFAIP3.

Glucocorticoids, or corticosteroids, are effective treatments for many chronic inflammatory diseases, and in mild/moderate asthma, long-acting β2-adrenoceptor agonists (LABAs) enhance the efficacy of inhaled corticosteroids (ICSs) more than increasing the ICS dose. In human bronchial epithelial, BEAS-2B, cells, expression of TNFα-induced protein-3 (TNFAIP3), or A20, a dual-ubiquitin ligase that provides feedback inhibition of NF-κB, was induced by budesonide, an ICS, and formoterol, a LABA, and was further enhanced by budesonide-formoterol combination. The proinflammatory cytokine TNF induced TNFAIP3 and TNF expression. Whereas subsequent budesonide treatment enhanced TNF-induced TNFAIP3 and reduced TNF expression, formoterol amplified these differential effects. In primary human airway smooth muscle cells, TNFAIP3 expression was induced by TNF. This was largely unaffected by budesonide but was acutely enhanced by budesonide-formoterol combination. In BEAS-2B cells, TNF recruited RELA, the main NF-κB transactivating subunit, to a 3' region of the TNF gene. RELA binding was reduced by budesonide, was further reduced by formoterol cotreatment, and was associated with reduced RNA polymerase II recruitment to the TNF gene. This is consistent with reduced TNF expression. TNFAIP3 knockdown enhanced TNF expression in the presence of TNF, TNF plus budesonide, and TNF plus budesonide-formoterol combination and confirms feedback inhibition. A luciferase reporter containing the TNF 3' RELA binding region recapitulated TNF inducibility and was inhibited by an IκB kinase inhibitor and TNFAIP3 overexpression. Repression of reporter activity by budesonide was increased by formoterol and involved TNFAIP3. Thus LABAs may improve the anti-inflammatory properties of ICSs by augmenting TNFAIP3 expression to negatively regulate NF-κB.

Asymmetric arginine dimethylation of RelA provides a repressive mark to modulate TNFα/NF-κB response

Nuclear factor kappa B (NF-κB) is an inducible transcription factor that plays critical roles in immune and stress responses and is often implicated in pathologies, including chronic inflammation and cancer. Although much has been learned about NF-κB-activating pathways, the specific repression of NF-κB is far less well understood. Here we identified the type I protein arginine methyltransferase 1 (PRMT1) as a restrictive factor controlling TNFα-induced activation of NF-κB. PRMT1 forms a cellular complex with NF-κB through direct interaction with the Rel homology domain of RelA. We demonstrate that PRMT1 methylates RelA at evolutionary conserved R30, located in the DNA-binding L1 loop, which is a critical residue required for DNA binding. Asymmetric R30 dimethylation inhibits the binding of RelA to DNA and represses NF-κB target genes in response to TNFα. Molecular dynamics simulations of the DNA-bound RelA:p50 predicted structural changes in RelA caused by R30 methylation or a mutation that interferes with the stability of the DNA-NF-κB complex. Our findings provide evidence for the asymmetric arginine dimethylation of RelA and unveil a unique mechanism controlling TNFα/NF-κB signaling.

Anatomy of a negative feedback loop: the case of IκBα.

The magnitude, duration and oscillation of cellular signalling pathway responses are often limited by negative feedback loops, defined as an 'activator-induced inhibitor' regulatory motif. Within the NFκB signalling pathway, a key negative feedback regulator is IκBα. We show here that, contrary to current understanding, NFκB-inducible expression is not sufficient for providing effective negative feedback. We then employ computational simulations of NFκB signalling to identify IκBα molecular properties that are critical for proper negative feedback control and test the resulting predictions in biochemical and single-cell live-imaging studies. We identified nuclear import and nuclear export of IκBα and the IκBα-NFκB complex, as well as the free IκBα half-life, as key determinants of post-induction repression of NFκB and the potential for subsequent reactivation. Our work emphasizes that negative feedback is an emergent systems property determined by multiple molecular and biophysical properties in addition to the required 'activator-induced inhibitor' relationship.

Anti-inflammatory function of ginsenoside Rg1 on alcoholic hepatitis through glucocorticoid receptor related nuclear factor-kappa B pathway

Ethnopharmacological relevanceGinseng is the dried root of Panax ginseng C.A. Mayer. Since ancient times, ginseng has been used as one kind of treatment drug or tonic in China and even other eastern countries like Korea and Japan. Pharmacological active chemical ingredients and its extract of ginseng are a mixture of triterpenoid saponins, collectively called ginsenosides. Among them, ginsenoside Rg1 is the most pharmacological active one. Aim of the studyBased on prior experimental results and the understanding of alcoholic hepatitis, the major aim of this study is to investigate whether Rg1 is beneficial in a rodent model mimic alcoholic hepatic injury associated with binge drinking and explore the underlying possible mechanisms. Materials and methodsC57BL/6 mice were given oral consumption of 6g/kg alcohol 1h after treated with Rg1 (10, 20 and 40mg/kg) or dexamethasone (1mg/kg) for 9 consecutive days. Biochemical analyses were performed and liver fragments were processed for microscopy, immunohistochemistry and western blot analysis. ResultsAccording to our data, Rg1 treatment significantly reversed the high mortality rate induced by alcohol consumption and also alleviated liver impairment as evidenced by the decrease of serum parameters. Meanwhile, histological and ultrastructural analysis of alcoholic groups showed hepatocellular impairment but restored in Rg1-treated groups. Overproductive inflammatory cytokines were also suppressed by Rg1 in alcohol-intoxicated mouse livers. In addition, changes of GR related NF-κB pathway, including phospho-IκB-α, were also modulated to normal levels. ConclusionThis study demonstrates that Rg1 might promote GR mediating the repression of NF-κB and inhibit the inflammatory reactions in alcoholic hepatitis.

Hepatitis C virus-mediated Aurora B kinase inhibition modulates inflammatory pathway and viral infectivity

Chronic hepatitis C is a leading cause of chronic liver disease, cirrhosis and hepatocellular carcinoma. DNA methylation and histone covalent modifications constitute crucial mechanisms of genomic instability in human disease, including liver fibrosis and hepatocellular carcinoma. The present work studies the consequences of HCV-induced histone modifications in early stages of infection. Human primary hepatocytes and HuH7.5 cells were transiently transfected with the core protein of hepatitis C virus (HCV) genotypes 1a, 1b, and 2a. Infectious genotype 2a HCV in culture was also used. We show that HCV and core protein inhibit the phosphorylation of Serine 10 in histone 3. The inhibition is due to the direct interaction between HCV core and Aurora B kinase (AURKB) that results in a decrease of AURKB activity. HCV and core significantly downregulate NF-κB and COX-2 transcription, two proteins with anti-apoptotic and proliferative effects implicated in the control of the inflammatory response. AURKB depletion reduced HCV and core repression of NF-κB and COX-2 gene transcription and AURKB overexpression reversed the viral effect. AURKB abrogation increased HCV specific infectivity which was decreased when AURKB was overexpressed. The core-mediated decrease of AURKB activity may play a role in the inflammatory pathway during the initial steps of viral infection, while ensuring HCV infectivity.

O9.06 * PROGNOSTIC RELEVANCE AND ONCOGENIC CORRELATES OF EPILEPSY IN GLIOBLASTOMA PATIENTS

BACKGROUND: In 30-40% of glioblastoma patients, the disease present with an epileptic seizure. Some studies suggest that tumor-associated epilepsy influences the survival of glioblastoma patients, possibly due to early diagnosis or the tumors' biology. However, the in vivo mechanisms underlying the suggested prognostic effect of epilepsy in glioblastoma patients are still unclear. METHODS: In a retrospective cohort study, we investigated the effect of epilepsy on survival of 230 glioblastoma patients using multivariate Weibull analysis. Gene set enrichment analyses defined the transcriptional effects of epilepsy and anti-epileptic drug treatments using gene expression microarrays of 50 fresh frozen surgical specimens. Protein expression was analyzed using immunohistochemistry on tissue microarrays containing surgical specimens of 230 patients. In vitro reporter gene assays, western blotting, kinase assays and immunocytochemistry experiments were performed to confirm our findings. RESULTS: Epilepsy is an independent prognostic factor in glioblastoma patients (HR 0.71, 95%CI: 0.53 - 0.96, p = 0.024), irrespective of other clinical characteristics. The epilepsy subgroup displayed a distinct gene expression pattern, with a significant downregulation of, notably, the oncologically relevant NF-κB, C/EBPβ and STAT3, -5A and -5B transcription factor associated genes. At a protein level, a significant downregulation of phosphorylated STAT3 and C/EBPβ expression, but not of phosphorylated STAT5b or NF-κB p65, was confirmed in the surgical samples. In addition, a significant repression of NF-κB was observed in the patients who were treated with the anti-epileptic drug valproic acid (VPA) prior to their surgery. In vitro, VPA inhibited NF-κB transcriptional activity and was shown to increase acetylated histone H3 and H4 expression. CONCLUSION: Glioblastoma patients with tumor-associated epilepsy form a distinct subgroup with a favorable prognosis and a distinct gene expression pattern involving downregulation of oncologically relevant pathways. The anti-epileptic drug VPA downregulates NF-κB signaling, which might, as suggested in recent literature, contribute to a longer survival of glioblastoma patients.

Metallothionein 1G and Zinc Sensitize Human Colorectal Cancer Cells to Chemotherapy

Metallothioneins (MT) are a family of low molecular weight proteins that are silenced during colorectal cancer progression, mainly through epigenetic mechanisms, and this loss is associated with poor survival. In this article, we show that overexpression of the MT1G isoform sensitizes colorectal cell lines to the chemotherapeutic agents oxaliplatin (OXA) and 5-fluorouracil (5-FU), in part through enhancing p53 and repressing NF-κB activity. Despite being silenced, MTs can be reinduced by histone deacetylase inhibitors such as trichostatin A and sodium butyrate. In fact, this induction contributes to the cytotoxicity of these agents, given that silencing of MTs by siRNAs reduces their growth-inhibitory activities. Zinc ions also potently enhance MT expression and are cytotoxic to cancer cells. We show for the first time that OXA and 5-FU induce higher levels of intracellular labile zinc, as measured using the fluorescent probe FLUOZIN-3, and that such zinc contributes to the activation of p53 and repression of NF-κB. Addition of zinc enhanced growth inhibition by OXA and 5-FU, and was also capable of resensitizing 5-FU-resistant cell lines to levels comparable with sensitive cell lines. This effect was MT independent because silencing MTs did not affect zinc cytotoxicity. In conclusion, we show that MT induction and zinc administration are novel strategies to sensitize colorectal cancer cells to presently utilized chemotherapeutic agents.

Nucleo-Cytoplasmic Trafficking of TRIM8, a Novel Oncogene, Is Involved in Positive Regulation of TNF Induced NF-κB Pathway

TNF induced nuclear factor kappa B (NF-κB) is one of the central signaling pathways that plays a critical role in carcinogenesis and inflammatory diseases. Post-translational modification through ubiquitin plays important role in the regulation of this pathway. In the current study, we investigated the role of TRIM8, member of RING family ubiquitin ligase in regulation of NF-κB pathway. We observed that TRIM8 positively regulates TNF induced NF-κB pathway. Different domains of TRIM8 showed discrete functions at the different steps in regulation of TNF induced NF-κB pathway. Ubiquitin ligase activity of TRIM8 is essential for regulation of NF-κB activation in both cytoplasm as well as nucleus. TRIM8 negates PIAS3 mediated negative repression of NF-κB at p65 by inducing translocation of PIAS3 from nucleus to cytoplasm as well as its turnover. TNF induces translocation of TRIM8 from nucleus to cytoplasm, which positively regulates NF-κB. The cytoplasmic translocation of TRIM8 is essential for TNF induced NF-κB but not for p65 mediated NF-κB regulation. TRIM8 also enhanced the clonogenic and migration ability of cells by modulating NF-κB. The further study will help to understand the role of TRIM8 in inflammation and cancer.

Abstract A84: ω3 rich diet protects against Kras-induced metaplasia via repression of NF-κB

Abstract Expression of mutant Kras is observed and often implicated in both pancreatitis and pancreatic cancer. Yet, the means by which inflammation occurs is relatively unexplored, although a previous report supports that induction of inflammation in the presence of oncogenic Kras initiates an NF-κB–mediated positive feedback loop that incorporates Cox-2 and production of PGE2 which further amplify mutant Kras. The involvement of PGE2 is interesting, as recent data suggests diets rich in ω6 fatty acids promote Kras induced pancreatic inflammation and neoplastic lesions. However, the precise mechanism and role of epigenetic events derived from diets rich in fatty acids have yet to be completely resolved. In this work, we demonstrate that a key step in this process is the induction and accumulation of NF-κB in pancreatic epithelia, a factor sufficient to promote chronic inflammation and progression to pancreatic adenocarcinoma. In HPDE cells, inhibition of NF-κB shows promise, as treatment with the IKK inhibitor Bay11-7085 noticeably reduced levels of the pro-proliferative factor pAkt. In EL-Kras mice fed a diet rich in ω6 fatty acids, NF-kB is upregulated in the pancreas, predominantly in metaplastic lesions. Conversely, these transgenic mice fed an ω3 fatty acid diet show little, if any, NF-kB expression, which is similar to the response in mice fed an intermediate, isocaloric high fat diet. pAkt is known to act upstream of NF-κB, implicating that NF-κB may be involved in a positive feedback loop with a variety of Kras effectors culminating in pancreatitis and eventually pancreatic neoplasia/adenocarcinoma. It has also been demonstrated that the ω6 Cox2-metabolite PGE2 is mediating this response, though in cultured human pancreatic epithelia, Cox-2 inhibition showed no significant change on levels of NF-κB. Yet, Cox2 inhibition in the stroma may be synergistic with epithelial inhibition of NF-κB, offering protection against the progression from inflammation to cancer. This data provides support to multiple targets of chemoprevention and therapy, including Ras and IKK/NF-κB, for pancreatic neoplasia and cancer. Citation Format: Daniel R. Principe, Windel Emman T. Mascarinas, Chintan Chheda, Kevin Adrian, Riley Mangan, Lindsay C. Boven, Paul J. Grippo. ω3 rich diet protects against Kras-induced metaplasia via repression of NF-κB. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A84.