bay-11-7082 and prolinedithiocarbamate

Long-standing untreated hyperuricemia could lead to gout. Several recent studies have demonstrated a significant decrease of serum urate during acute gout attack, which is an aseptic inflammation process focusing on IL-1β. However, how IL-1β, by itself, alters the expression and the functional activity of urate transporters in renal tubular epithelial cells is still unclear. Herein, we revealed that IL-1β could attenuate the mRNA and protein levels of ABCG2, a major urate efflux pump, in HK-2 cells by real-time PCR and Western-blot assays. Moreover, using an ABCG2 specific inhibitor and a new sensitive and specific detection system, it was found that IL-1β also reduced the ABCG2 transporter activities. Incubation with specific inhibitors of the NF-κB pathway partly dampened the inhibitory effect of IL-1β on ABCG2, indicating that IL-1β reduced the ABCG2 expression partially through the NF-ĸB pathway. Furthermore, the decreased expression of PDZK1 induced by IL-1β, which is dependent on the NF-κB pathway, could account for the imbalance between the functions and expressions of ABCG2 on this status. These findings demonstrated a new role for IL-1β, whereby it leads to the inhibition of ABCG2 in renal tubular epithelial cells; this new role probably does not encompass its involvement in the process of renal urate excretion mediated by inflammation. Therefore, other regulation mechanisms of urate reabsorption in renal tubular epithelial cells deserve to be examined in further studies.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; Base Sequence; Biological Transport; Carrier Proteins; Cell Line; Humans; Interleukin-1beta; Membrane Proteins; Neoplasm Proteins; NF-kappa B; Nitriles; Polymorphism, Single Nucleotide; Proline; RNA, Messenger; Sulfones; Thiocarbamates; Time Factors; Uric Acid

The present study was designed to determine whether sulfur dioxide (SO2) could be endogenously produced in adipocyte and served as a novel adipocyte-derived inflammatory inhibitor. SO2 was detected in adipose tissue using high-performance liquid chromatography with fluorescence detection. SO2 synthase aspartate aminotransferase (AAT1 and AAT2) mRNA and protein expressions in adipose tissues were measured. For in vitro study, 3T3-L1 adipocytes were cultured, infected with adenovirus carrying AAT1 gene or lentivirus carrying shRNA to AAT1, and then treated with tumor necrosis factor-α (TNF-α). We found that endogenous SO2/AAT pathway existed in adipose tissues including perivascular, perirenal, epididymal, subcutaneous and brown adipose tissue. AAT1 overexpression significantly increased SO2 production and inhibited TNF-α-induced inflammatory factors, monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) secretion from 3T3-L1 adipocytes. By contrast, AAT1 knockdown decreased SO2 production and exacerbated TNF-α-stimulated MCP-1 and IL-8 secretion. Mechanistically, AAT1 overexpression attenuated TNF-α-induced IκBα phosphorylation and degradation, and nuclear factor-κB (NF-κB) p65 phosphorylation, while AAT1 knockdown aggravated TNF-α-activated NF-κB pathway, which was blocked by SO2. NF-κB inhibitors, PDTC or Bay 11-7082, abolished excessive p65 phosphorylation and adipocyte inflammation induced by AAT1 knockdown. This is the first report to suggest that endogenous SO2 is a novel adipocyte-derived inflammatory inhibitor.

Topics: 3T3-L1 Cells; Adipocytes; Adipose Tissue, Brown; Adipose Tissue, White; Animals; Anti-Inflammatory Agents; Aspartate Aminotransferases; Chemokine CCL2; Gene Expression Regulation; Interleukin-8; Isoenzymes; Male; Mice; NF-KappaB Inhibitor alpha; Nitriles; Phosphorylation; Proline; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Signal Transduction; Sulfones; Sulfur Dioxide; Thiocarbamates; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Nuclear factor-kappa B (NF-κB) signaling is implicated in both cancer development and inflammation processes. However, the roles and mechanisms of NF-κB signaling in the development of cancer-induced pain (CIP) remain unknown. This study was designed to investigate the roles of the p65 subunit of NF-κB in regulation of the purinergic receptor (P2X3R) plasticity in dorsal root ganglion (DRG) of CIP rats. We showed here that tumor cell injection produced mechanical and thermal hyperalgesia, and an enhanced body weight-bearing difference, which was correlated with an upregulation of p65 and P2X3R expression in lumber DRGs and a potentiation of ATP-evoked responses of tibia-innervating DRG neurons. Inhibition of NF-κB signaling using p65 inhibitor pyrrolidine dithiocarbamate, BAY-11-7082, or lentiviral-p65 short-hairpin RNA significantly attenuated CIP and reversed the activities of P2X3R. Interestingly, tumor cell injection led to a significant demethylation of CpG island in p2x3r gene promoter and enhanced ability of p65 to bind the promoter of p2x3r gene. Our findings suggest that upregulation of P2X3R expression was mediated by the enhanced binding capability of p65 with demethylated promoter of p2x3r gene, thus contributing to CIP. NF-κBp65 might be a potential target for treating CIP, a neuropathic pain generated by tumor cell-induced injury to nerves that innervate the skin.

Topics: Action Potentials; Animals; Antineoplastic Agents; Bone Neoplasms; Case-Control Studies; Disease Models, Animal; Enzyme Inhibitors; Female; Ganglia, Spinal; Hyperalgesia; Methylation; Neurons; NF-kappa B; Nitriles; Pain; Pain Management; Pain Threshold; Proline; Protein Binding; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2X3; Sulfones; Thiocarbamates

Tumor necrosis factor-α (TNF-α) is an established pro-atherosclerotic factor, but the mechanism is not completely understood. We explored whether TNF-α could promote atherosclerosis by increasing the transcytosis of lipoproteins (e.g., LDL) across endothelial cells and how NF-κB and PPAR-γ were involved in this process. TNF-α significantly increased the transcytosis of LDL across human umbilical vein endothelial cells (HUVECs) and stimulated an increase of subendothelial retention of LDL in vascular walls. These effects of TNF-α were substantially blocked not only by transcytosis inhibitors, but also by NF-κB inhibitors and PPAR-γ inhibitors. In ApoE(-/-) mice, both NF-κB and PPAR-γ inhibitors alleviated the early atherosclerotic changes promoted by TNF-α. NF-κB and PPAR-γ inhibitors down-regulated the transcriptional activities of NF-κB and PPAR-γ induced by TNF-α. Furthermore, cross-binding activity assay revealed that NF-κB and PPAR-γ could form an active transcription factor complex containing both the NF-κB P65 subunit and PPAR-γ. The increased expressions of LDL transcytosis-related proteins (LDL receptor and caveolin-1, -2) stimulated by TNF-α were also blocked by both NF-κB inhibitors and PPAR-γ inhibitors. TNF-α promotes atherosclerosis by increasing the LDL transcytosis across endothelial cells and thereby facilitating LDL retention in vascular walls. In this process, NF-κB and PPAR-γ are activated coordinately to up-regulate the expression of transcytosis-related proteins. These observations suggest that inhibitors of either NF-κB or PPAR-γ can be used to target atherosclerosis.

Topics: Anilides; Animals; Atherosclerosis; Benzamides; Caveolin 1; Caveolin 2; Cinchona Alkaloids; Filipin; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Lipoproteins, LDL; Mice; Mice, Knockout; NF-kappa B; Nitriles; PPAR gamma; Proline; Pyridines; Receptors, LDL; RNA, Small Interfering; Signal Transduction; Sulfones; Thiocarbamates; Transcytosis; Tumor Necrosis Factor-alpha

In vitro but not in vivo evidence indicates that blockade of NF-κB is effective in reducing inflammation and production of IL-8. We hypothesized that the failure of in vitro experiments to predict in vivo outcome was due to the use of short time periods of observation and the use of single cytokines to stimulate NF-κB.. HEK cells with a NF-κB reporter gene or CaCo-2 cells were stimulated with CM (IL-1-β; TNF-α, and IFN-γ) or individual cytokines in the presence and absence of NF-κB inhibitors, a STAT1 inhibitor, and/or a p38 MAPK inhibitor for periods up to 24 h. NF-κB activation, IL-8 production, and nitric oxide production were measured.. CM-induced IL-8 production in HEK cells was additive to synergistic. CM enhanced production of IL-8 at 24 h but not 4 h was independent of NF-κB. The p38 inhibitor SB203580 and the STAT1 inhibitor EGCG blocked CM-induced IL-8 production at both early and late time periods. The NF-κB inhibitors PDTC and BAY11-7082 were found to increase CM-stimulated IL-8 production in Caco-2 cells at 24 h.. Our data suggest an effective strategy to reduce IL-8 production is to block p38 or STAT1 rather than NF-κB.

Topics: Caco-2 Cells; Catechin; Cell Line; Cytokines; Genes, Reporter; Humans; Imidazoles; NF-kappa B; Nitric Oxide; Nitriles; p38 Mitogen-Activated Protein Kinases; Proline; Protein Kinase Inhibitors; Pyridines; STAT1 Transcription Factor; Sulfones; Thiocarbamates

Reactive oxygen species (ROS) are important signaling molecules in cells. Excessive ROS induce expression of inflammatory mediators, such as iNOS and COX2. Antioxidant enzymes, such as, heme oxygenase-1 (HO-1), tightly regulate ROS levels within cells. Here, we show that Bay 11-7082 (Bay) increased HO-1 mRNA and protein expression in human colon cancer HT29 cells. Bay induced translocation of NF-E2-related factor 2 (Nrf2) into nuclei and increased the binding activity of the antioxidant response element (ARE). In addition, PI3K/Akt inhibitor (LY294002) blocked Bay-induced HO-1 expression. Pretreatment with anti-oxidants (N-acetylcysteine (NAC) or glutathione) significantly reduced Bay-induced HO-1 mRNA/protein expression, nuclear translocation of Nrf2 and phosphorylation of Akt. However, PI3K/Akt signaling was independent of Bay-induced Nrf2 translocation and ARE binding activity. Furthermore, other NF-κB inhibitors, such as pyrrolidine dithiocarbamate (PDTC) and MG132, also increased HO-1 mRNA and protein expression. However, although overexpression of dominant negative inhibitory κB (IκB) reduced NF-κB-driven transcriptional activity, IκB overexpression did not increase HO-1 expression. Taken together, our results suggest that in human colon cancer HT29 cells, Bay induces HO-1 expression by increasing ROS production in an Nrf2-ARE and PI3K dependent manner, but Bay acts independently of NF-κB.

Topics: Analysis of Variance; Electrophoretic Mobility Shift Assay; Gene Expression Regulation; Glutathione; Heme Oxygenase-1; HT29 Cells; Humans; Immunohistochemistry; Leupeptins; NF-E2-Related Factor 2; NF-kappa B; Nitriles; Phosphatidylinositol 3-Kinases; Phosphorylation; Proline; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Response Elements; RNA, Messenger; Signal Transduction; Sulfones; Thiocarbamates; Transfection

The ability of human neutrophils to express a variety of genes encoding inflammatory mediators is well documented, and mounting evidence suggests that neutrophil-derived cytokines and chemokines contribute to the recruitment of discrete leukocyte populations at inflammatory sites. Despite this, our understanding of the signaling intermediates governing the generation of inflammatory cytokines by neutrophils remains fragmentary. Here, we report that inhibitors of the p38 MAPK and MEK pathways substantially diminish the release of (and in the case of p38 inhibitors, the gene expression of) several inflammatory cytokines in neutrophils stimulated with LPS or TNF. In addition, various NF-kappaB inhibitors were found to profoundly impede the inducible gene expression and release of inflammatory cytokines in these cells. The MAPK inhibitors did not affect NF-kappaB activation; instead, the transcriptional effects of the p38 MAPK inhibitor appear to involve transcriptional factor IID. Conversely, the NF-kappaB inhibitors failed to affect the activation of MAPKs. Finally, the MAPK inhibitors were found to prevent the activation a key component of the translational machinery, S6 ribosomal protein, in keeping with their post-transcriptional impact on cytokine generation. To our knowledge, this constitutes the first demonstration that in neutrophils, the inducible expression of proinflammatory cytokines by physiological stimuli largely reflects the ability of the latter to activate NF-kappaB and selected MAPK pathways. Our data also raise the possibility that NF-kappaB or MAPK inhibitors could be useful in the treatment of inflammatory disorders in which neutrophils predominate.

Topics: Boronic Acids; Cell Differentiation; Cytokines; Humans; Inflammation; Leupeptins; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Neutrophils; NF-kappa B; Nitriles; Proline; Protein Kinase Inhibitors; Structure-Activity Relationship; Sulfones; Thiocarbamates