Transcription Factor NF Research Articles

Lymphocytes’ ‘last stand’ on the nuclear matrix after whole body exposure of rats to low-let ionizing radiation

We examined the functions of the rat lymphocyte nuclear matrix after a single exposure to total body irradiation with doses ranging from sublethal to lethal. Irradiation induced systemic oxidative stress, detected as increased activities of serum SOD and catalase, lymphocyte DNA damage, detected by the Comet assay, and apoptosis. After irradiation with lower doses, the recruitment of DNA repair centers on the matrix was observed by Western analysis as increased levels of matrix-associated PARP-1, p53 and PCNA. Augmented partitioning of the pro-survival transcription factor NF-?B on the matrix was also detected after irradiation. Exposure to a lethal dose caused breakdown of the matrix, observed as lamin B cleavage, and of the matrix-associated DNA repair centers, detected as caspase-mediated PARP-1 proteolysis and loss of protein associations with the matrix. These findings suggest that the nuclear matrix establishes functional 2 interactions in a defensive mechanism, integrated in a decision-making process that resolves cell fate.

P89 - Assessment of an in vitro model of lung epithelial cell stress responses

The ability of cells to adapt and survive environmental and physiological stress relies on activation of cellular stress responses. These include anti-oxidant, inflammatory and apoptotic responses. These cellular responses are mediated by cell signalling pathways, including those controlled by the transcription factors Nrf2 (antioxidant response) and NF-?ß (inflammatory response). As part of a suite of in vitro models for the comparison of different nicotine delivery products, an in vitro lung cell stress response model has been developed. Human bronchial epithelial cells were exposed to positive controls or cigarette smoke aqueous extracts (CSEaq) from 3R4F and 1R5F reference cigarettes for 4hours. Cellular responses were then measured as oxidative, pro-inflammatory, apoptotic and necrotic endpoints. Cellular oxidative stress was characterised by measurement of the intracellular glutathione ratio, intracellular ROS production and activation of the Nrf2-controlled Anti-oxidative Response Elements (ARE). The inflammatory response of the cells was determined through quantification of secreted cytokines, IL-1a, IL-6 and IL-8. Apoptotic and necrotic responses were characterised by measurements of Caspase 3/7 activity and live-cell protease activity. Stably transfected luciferase reporter cell lines were utilised to quantify the transcriptional control of anti-oxidant and inflammatory pathways. All cell stress response endpoints were activated by exposure to positive controls or CSEaq. The observed concentration-dependent lowering of the glutathione ratio and increase in intracellular ROS generation corresponded with an increase in Nrf2 transcriptional activation of the ARE. It has been demonstrated that this model was able to distinguish between CSEaq from the two different reference cigarettes. We propose that this model may be suitably sensitive for biological comparisons of cigarettes against different nicotine delivery products.

Abstract 506: H2O2 upregulates xCT in human breast cancer cells via NRF-2

Abstract Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate, making the transcription factors involved in antioxidant defense regulation targets of interest. The transcription factor NF E2 Related Factor 2 (NRF-2) regulates cellular defense genes including those encoding intracellular redox-balancing proteins such as enzymes involved in glutathione metabolism. Glutathione in particular is an important intracellular antioxidant molecule. NRF-2 binds to the Antioxidant Response Element (ARE) in the promoter of its target genes. Under basal conditions, KEAP1 acts as an inhibitor that targets NRF-2 for ubiquitination. During oxidative stress, NRF-2 dissociates from KEAP1 and enters the nucleus to bind to the ARE sequence. It is hypothesized that the elevated Reactive Oxygen Species may be depleting the glutathione levels within the cancer cell. System xc- is a cysteine/glutamate antiporter that exports glutamate while importing cysteine to synthesize glutathione. In response to oxidative stress the cells increase system xc- activity in order to provide cysteine for glutathione synthesis. There is evidence that expression of xCT the specific subunit of System X is regulated by NRF-2. However this has not yet been demonstrated in human cancer cells, which is the focus of this project. Basal expression of NRF-2, Keap1 and xCT was characterized in two breast cancer cell lines (MDA MB 231 and MCF-7) compared to normal human breast epithelial 184B5 cells. Compared to 184B5 cells, Keap-1, NRF-2 and xCT were upregulated in MDA MB 231 cells, but down regulated in MCF-7 cells. MCF-7 cells were treated with H202 resulting in NRF-2 protein accumulation in the nucleus and decreased levels in the cytosol. Under the same treatment conditions, Keap-1 protein decreased in the nucleus and increased in the cytosol. With H202 treatment, xCT mRNA levels increased in MCF-7 cells. We have cloned approximately 2.6 kilobase (-2329 to +278 bp) of the human xCT promoter region from genomic DNA isolated from MDA MB 231 breast cancer cells. We found that the xCT promoter was responsive to treatment with H2O2, which increased transcriptional activity. This is consistent with results described for the mouse promoter, which is known to contain an antioxidant response element that can be bound by NRF-2 during oxidative stress. Co transfection of MDA MB 231 with the xCT promoter and an NRF-2 expression vector also increased promoter activity. This data suggests that under oxidative stress, NRF-2 is localized to the nucleus and transcriptionally upregulates xCT. This is the first study in which the regulation of xCT has been linked to oxidative stress via NRF-2 in human breast cancer cells. This work was supported by the Canadian Institutes for Health Research. Citation Format: Eric Habib, Katja Linher-Melville, Gurmit Singh. H2O2 upregulates xCT in human breast cancer cells via NRF-2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 506. doi:10.1158/1538-7445.AM2014-506

9: RAGE is a cell surface nucleic acid sensor

RAGE is a multi-ligand cell surface receptor of the Immunoglobulin super-family. It is composed of three extracellular immunoglobulin-like domains (V, C1 and C2), a single trans-membrane domain and a short cytoplasmic domain. RAGE is predominately expressed in the lung under normal conditions. However, during inflammation, RAGE expression increases dramatically in tissues like the vascular system, CNS and some hematopoietic cells. RAGE ligands are structurally heterogeneous and bind to RAGE through sequence nonspecific electrostatic interactions. They include advanced glycation end products, the DNA binding protein HMGB1, several proteins of the S100 family and certain types of β -sheet fibrils such as β -amyloid. Hence, RAGE is involved in many chronic inflammatory diseases such as diabetes, Alzheimer’s disease, asthma and systemic lupus erythematosus. Using EMSA and AlphaScreen, we showed that DNA and RNA bind to RAGE in a sequence nonspecific manner through electrostatic interaction of their phosphate backbone with the V and C1 domains. Human embryonic kidney (HEK) cells expressing RAGE bound more fluorescently labelled RNA and DNA than non-expressing cells as shown by microscopy and flow cytometry. In order to investigate the effect of RAGE expression on nucleic acid (NA) sensing by endosomal TLRs, we used HEK cells expressing TLR7, TLR8, TLR13 (ssRNA) or TLR9 (DNA) together with RAGE and found that RAGE increased NA induced activation of the transcription factor NF κ B. Finally, in a mouse model of acute lung inflammation, DNA inhalation induced a strong inflammatory response in WT mice, while RAGE knockout mice displayed dramatically reduced inflammation. We show that RAGE binds NAs at the cell surface, increases their uptake and thereby amplifies the activation of endosomal TLRs. This work helps to further elucidate the function of RAGE in the development and perpetuation of chronic inflammation and presents RAGE as a promising target in the development of anti-inflammatory treatments.

비만세포에서 연교(連翹) 추출물의 Th2 사이토카인 발현 및 신호전달 기전 억제 효과

Objectives Forsythiae Fructus treatment has been used for inflammatory and allergic diseases in Korean Medicine. Nevertheless, the mechanism of action and the cellular targets are not understood well. The pathogenesis of allergic diseases are associated with Th2 cytokines such as IL-13, MIP-<TEX>$1{\alpha}$</TEX>, IL-13, IL-5, GM-CSF, IL-4, TNF-<TEX>${\alpha}$</TEX> and IL-6, which are secreted by the mast cells. This study was conducted to investigate the effects of Forsythiae Fructus extracts (FF) on Th2 cytokines expression and signal transduction in MC/9 mast cells. Methods In the study, MC/9 mast cells were stimulated with DNP-IgE for 24 hours and then treated separately with CsA <TEX>$10{\mu}g/m{\ell}$</TEX> and varying doses of FF for one hour. MC/9 mast cells stimulated with DNP-IgE was the control group, a treatment with CsA was the positive control group and a treatment with varying doses FF was the experimental groups. The mRNA levels of IL-13, IL-5, GM-CSF, IL-4, TNF-<TEX>${\alpha}$</TEX>, IL-6 were analyzed with Real-time PCR. The levels of IL-13, MIP-<TEX>$1{\alpha}$</TEX> were measured using enzyme-linked immunosorbent assays(ELISA). NFAT, AP-1 and NF-<TEX>${\kappa}B$</TEX> p65 were examined by Western blot analysis. Results 1. FF were observed to suppress the mRNA expression of IL-13, IL-5, GM-CSF, IL-4, TNF-<TEX>${\alpha}$</TEX>, IL-6 in comparison to DNP-IgE control group. 2. FF also has inhibited the IL-13, MIP-<TEX>$1{\alpha}$</TEX> production significantly in comparison to DNP-IgE control group. 3. Western blot analysis of transduction factors involving Th2 cytokines expression has revealed a prominent decrease of the mast cell specific transduction factors including NFAT-1, NFAT-2, c-Jun, and NF-<TEX>${\kappa}B$</TEX> p65 but c-Fos. Conclusions In conclusion, the anti-allergenic activities of FF may be strongly related to the regulation of transcription factors NFAT-1, NFAT-2, c-Jun, and NF-<TEX>${\kappa}B$</TEX> p65 causing inhibition of Th2 cytokines in mast cells.

Analysis of JC virus DNA replication using a quantitative and high-throughput assay

Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication.

Differential Role of ERK and p38 on NF- κ B Activation in Helicobacter pylori-Infected Gastric Epithelial Cells.

Gastric cancer, as well as inflammation, caused by Helicobacter pylori, activates the production of chemokines by activation of redox-sensitive transcription factor NF-κB in gastric epithelial cells. Mitogen-activated protein kinases including extracellular signal-regulated kinase (ERK) and p38 kinase (p38) are activated by Helicobacter pylori, which may regulate NF-κB activation in the infected cells. However the mechanisms how ERK and p38 induce NF-κB activation have not been investigated. Present study aims to investigate the role of ERK and p38 on the activation of NF-κB in Helicobacter pylori-infected AGS cells. Western blot analysis was performed for determining the levels of IκB, p105, p50 and p65 in gastric epithelial cells infected with Helicobacter pylori and treated with ERK inhibitor U0126 and p38 inhibitor SB203580. Helicobacter pylori induced the degradation of IκBα and upregulation of p105, p50 and p65 in the infected cells. U0126 inhibited the degradation of IκBα while SB203580 suppressed expression of p105, p50 and p65 in Helicobacter pylori-infected cells. ERK and p38 differentially activate NF-κB; ERK induces degradation of IκBα while p38 upregulates the expression of p50 and p65, subunits of NF-κB in Helicobacter pylori-infected gastric epithelial AGS cells.

Aromadendrin Inhibits Lipopolysaccharide-Induced Nuclear Translocation of NF-κB and Phosphorylation of JNK in RAW 264.7 Macrophage Cells

Aromadendrin, a flavonol, has been reported to possess a variety of pharmacological activities such as anti-inflammatory, antioxidant, and anti-diabetic properties. However, the underlying mechanism by which aromadendrin exerts its biological activity has not been extensively demonstrated. The objective of this study is to elucidate the anti-inflammatory mechanism of aromadedrin in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Aromadendrin significantly suppressed LPS-induced excessive production of pro-inflammatory mediators such as nitric oxide (NO) and PGE2. In accordance, aromadendrin attenuated LPSinduced overexpression iNOS and COX-2. In addition, aromadendrin significantly suppressed LPS-induced degradation of IκB, which sequesters NF-κB in cytoplasm, consequently inhibiting the nuclear translocation of pro-inflammatory transcription factor NF- κB. To elucidate the underlying signaling mechanism of anti-inflammatory activity of aromadendrin, MAPK signaling pathway was examined. Aromadendrin significantly attenuated LPS-induced activation of JNK, but not ERK and p38, in a concentration-dependent manner. Taken together, the present study clearly demonstrates that aromadendrin exhibits anti-inflammatory activity through the suppression of nuclear translocation of NF-κB and phosphorylation of JNK in LPS-stimulated RAW 264.7 macrophage cells.

Abstract 2106: Gamma-tocotrienol upregulates the ceramide transporter, Arv-1, in pancreatic cancer cells.

Abstract Pancreatic cancer is the fourth leading cause of cancer-related death in the United States. Treatment options are limited and novel therapeutic agents that can inhibit signaling pathways implicated in the proliferation and survival of pancreatic cancer cells are of interest. Tocotrienols are members of the vitamin E family but, unlike tocopherols, possess an unsaturated isoprenoid side-chain that confers superior and mechanistically different anti-cancer properties on pancreatic cancer cells. The ability of tocotrienols to selectively inhibit the HMG CoA reductase pathway through post-translational degradation of HMG CoA reductase, inhibit the PI3/Akt, ERK pathways through downregulation of Her2/ErbB2 receptors as well as suppression of the activity of transcription factor NF kappa B, could be the basis for some of these properties. Recent studies have shown increase in cellular ceramide levels in cancer cells treated with tocopherols, leading to arrest in tumor proliferation and apoptosis. The actual mechanism of increase in ceramide levels is not clearly understood and is thought to be due to increased synthesis. We explored the hypothesis that the increase in ceramide on the cellular membrane could be due to increased transport from the ER to the membrane. The ARV1 (ACAT-related enzyme-2 required for viability) gene encodes an ER- localized protein involved in lipid homeostasis and ceramide transport in yeast and mammals. Deletion of the ARV-1 gene in yeast causes accumulation of ceramide in cytoplasm. Our previous work indicated that gamma-tocotrienol (GT3) has potent anti-proliferative activity in k-ras mutated pancreatic cancer cell (MIA-Paca-2) through the inhibitory effect on Akt, Ras/Raf/Erk and ErBb2 receptor as well as induction of apoptotic pathways through Fox-03 and GSK-3b. Our current data shows GT3 may decrease expression of caveolin, a cell survival marker, and cause up-regulation of ARV-1 that encodes a ceramide transport protein. We further show via HPLC (Babraham Bioscience Technologies Ltd, Cambridge, U.K) that total ceramide levels remain unchanged in the cell. There may be a change in ceramide (monoclonal anti-ceramide IgM, Sigma Chemicals) cellular distribution as indicated by immunofluorescence data. Upregulation of ARV-1 may be involved in increase in cellular ceramide transport or redistribution of ceramide to the cell membrane. Transport of ceramide to membrane lipid rafts or mitochondrial membrane channels may cause loss of survival signaling and induce apoptosis. Thus it is likely that GT3 apoptotic effect on k-ras mutated MIA-Paca2 cells is related not to changes in the total ceramide levels, but rather to its increased transport and redistribution. Further work is necessary in order to better understand the role of GT3 in ceramide redistribution in pancreatic cancer cells. Citation Format: Kanishka Chakraborty, Victoria P. Ramsauer, Janet W. Lightner, William L. Stone, Koyamangalath Krishnan. Gamma-tocotrienol upregulates the ceramide transporter, Arv-1, in pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2106. doi:10.1158/1538-7445.AM2013-2106

자초(紫草)가 HaCaT 세포의 사이토카인 유전자 발현에 미치는 영향

Objective : Propionibacterium acnes (P. acnes) is a major pathogenic bacteria for acne vulgaris. This study was performed to evaluate the effects of Lithospermum erythrorhizon extracts on the inflammatory cytokines gene expression by P. acnes in human keratinocytes, HaCaT cell line. Methods : Anti-bacterial activity and cytotoxicity of LE extracts was analyzed by agar plate culture and XTT assay. The cytokines gene expressions were assessed by real time RT-PCR for IL-8, MCP-1 and TNF-<TEX>${\alpha}$</TEX>. During the cell culture and treatments, amounts of secreted TNF-<TEX>${\alpha}$</TEX> were measured by ELISA. Translocation of transcription factor NF-<TEX>${\kappa}B$</TEX> from cytoplasm into nucleus was observed by immunocytochemistry and confocal microscopy. Results : There were no anti-bacterial effects and cytotoxicity as high as <TEX>$1,000{\mu}g/ml$</TEX> of LE extracts in XTT assay. Transcription levels of inflammatory cytokines, IL-8, MCP-1 and TNF-<TEX>${\alpha}$</TEX> were increased by P. acnes in HaCaT. LE extracts decreased the upregulated gene transcription levels. However, amounts of secreted TNF-<TEX>${\alpha}$</TEX> were similar in HaCaT cells with P. acnes and LE extracts. Translocation of NF-<TEX>${\kappa}B$</TEX> into nucleus by P. acnes was significantly inhibited by LE extracts. Conclusions : From the results of this study, LE extracts have anti-inflammatory effects on HaCaT cells by P. acnes that decreased the mRNA expressions of IL-8, MCP-1 and TNF-<TEX>${\alpha}$</TEX>. This anti-inflammatory effects of LE extracts could provide the potential of therapeutic substance for acne vulgaris.

Noisy transcription factor NF-κB oscillations stabilize and sensitize cytokine signaling in space

NF-κB is a major transcription factor mediating inflammatory response. In response to a pro-inflammatory stimulus, it exhibits a characteristic response-a pulse followed by noisy oscillations in concentrations of considerably smaller amplitude. NF-κB is an important mediator of cellular communication, as it is both activated by and upregulates production of cytokines, signals used by white blood cells to find the source of inflammation. While the oscillatory dynamics of NF-κB has been extensively investigated both experimentally and theoretically, the role of the noise and the lower secondary amplitude has not been addressed. We use a cellular automaton model to address these issues in the context of spatially distributed communicating cells. We find that noisy secondary oscillations stabilize concentric wave patterns, thus improving signal quality. Furthermore, both lower secondary amplitude as well as noise in the oscillation period might be working against chronic inflammation, the state of self-sustained and stimulus-independent excitations. Our findings suggest that the characteristic irregular secondary oscillations of lower amplitude are not accidental. On the contrary, they might have evolved to increase robustness of the inflammatory response and the system's ability to return to a pre-stimulated state.

The role of Qualea multiflora on Mammary Tumour Cells and Immune System

Article history: Received on: 21/08/2013 Accepted on: 14/09/2013 Available online: 30/09/2013 Species of the genus Qualea are used by the Brazilian public as a natural anti-inflammatory. Based on this evidence, we evaluated the effects of terpene fractions (F and TF) obtained from Qualea multiflora on nitric oxide production (Griess assay), cytokines (IL-1, IL-10, IL-12, and TNF-α) and the transcription factor NF-B by peritoneal macrophages. Since there is a relationship between inflammation and cancer, the cytotoxicity of F and TF against mammary tumoural cell lineage, and macrophages was evaluated. Inhibition levels close to 90% of the production of NO, IL-1, IL-12 and TNF-α; about 32% of NF-B; and a large stimulation of IL-10 production (close to the positive control) by peritoneal macrophages were observed in response to F and TF which are correlated with anti-inflammatory activity. Additionally, the samples showed exceptional cytotoxic activity against tumoural cells but not against macrophages. Since anti-inflammatory activity is important in tumour inhibition, further examination of potential anti-cancerous activity of Qualea multiflora is warranted.

Recognition of lipid A variants by the TLR4-MD-2 receptor complex

Lipopolysaccharide (LPS) is a component of the outer membrane of almost all Gram-negative bacteria and consists of lipid A, core sugars, and O-antigen. LPS is recognized by Toll-like receptor 4 (TLR4) and MD-2 on host innate immune cells and can signal to activate the transcription factor NFκB, leading to the production of pro-inflammatory cytokines that initiate and shape the adaptive immune response. Most of what is known about how LPS is recognized by the TLR4-MD-2 receptor complex on animal cells has been studied using Escherichia coli lipid A, which is a strong agonist of TLR4 signaling. Recent work from several groups, including our own, has shown that several important pathogenic bacteria can modify their LPS or lipid A molecules in ways that significantly alter TLR4 signaling to NFκB. Thus, it has been hypothesized that expression of lipid A variants is one mechanism by which pathogens modulate or evade the host immune response. Additionally, several key differences in the amino acid sequences of human and mouse TLR4-MD-2 receptors have been shown to alter the ability to recognize these variations in lipid A, suggesting a host-specific effect on the immune response to these pathogens. In this review, we provide an overview of lipid A variants from several human pathogens, how the basic structure of lipid A is recognized by mouse and human TLR4-MD-2 receptor complexes, as well as how alteration of this pattern affects its recognition by TLR4 and impacts the downstream immune response.

Resveratrol inhibits Epstein Barr Virus lytic cycle in Burkitt’s lymphoma cells by affecting multiple molecular targets

Resveratrol (RV), a polyphenolic natural product present in many plants and fruits, exhibits anti-inflammatory, cardio-protective and anti-proliferative properties. Moreover, RV affects a wide variety of viruses including members of the Herpesviridae family, retroviruses, influenza A virus and polyomavirus by altering cellular pathways that affect viral replication itself. Epstein Barr Virus (EBV), the causative agent of infectious mononucleosis, is associated with different proliferative diseases in which it establishes a latent and/or a lytic infection. In this study, we examined the antiviral activity of RV against the EBV replicative cycle and investigated the molecular targets possibly involved. In a cellular context that allows in vitro EBV activation and lytic cycle progression through mechanisms closely resembling those that in vivo initiate and enable productive infection, we found that RV inhibited EBV lytic genes expression and the production of viral particles in a dose-dependent manner. We demonstrated that RV inhibited protein synthesis, decreased reactive oxygen species (ROS) levels, and suppressed the EBV-induced activation of the redox-sensitive transcription factors NF–kB and AP-1.Further insights into the signaling pathways and molecular targets modulated by RV may provide the basis for exploiting the antiviral activity of this natural product on EBV replication.

P147 The physiological role of PAI-2 in endotoxin-induced sepsis

Introduction Sepsis caused by uncontrolled inflammation is the leading cause of death in intensive care units today, but no effective therapeutic medication is available currently. During microbial infections, pathogen-associated molecular patterns (PAMPs) are recognized by the host innate immune system, which then mounts a defensive response. Myeloid cells play central roles in PAMP recognition and in the initiation and amplification of inflammatory cascades. Recent studies indicate that a novel system termed the inflammasome carries out processing of cytoplasmic stored pro-IL-1 β , and pro-IL-18. Both IL-1 β and IL-18 are highly potent proinflammatory cytokines. In addition, inflammasome activation has also been linked to an increase of host-cell death in certain cell types. Therefore the regulation of activation of the inflammasome is very important, but its mechanism is unclear. Plasminogen activator inhibitor 2 (PAI-2), a member of a large group of proteins that inhibit serine proteases, is regulated by IKK β -dependent activation of the transcription factor NF- κ B. Previous studies have shown that mice with Ikkβ deletion in myeloid cells are hypersusceptible to endotoxin (LPS)-induced septic shock, and increased LPS susceptibility is associated with elevated plasma IL-1 β . Reconstitution of Ikkβ -deficient macrophages with PAI-2 blocks apoptosis and IL-1 β release. Theseresults suggest that PAI-2 modulatesendotoxin-induced IL-1 β production and apoptosis in macrophages. Methods To determine the physiological role of PAI-2 in vivo , we generatedtransgenic mice that specifically express PAI-2 in macrophages and neutrophils, and crossed this transgenic mice with mutant mice with Ikkβ deletion in myeloid cells. Results We are currently examining if myeloid deletion Ikkb mice restored PAI-2 expression can resist LPS-induced septic shock. In addition, we are also investigating the underlying mechanism by which PAI-2 regulates LPS-induced IL-1 β production. Conclusion Our data would reveal the functional role of myeloid-specific expression of PAI-2 in LPS-induced septic shock in vivo .

Abstract C39: Catalytic inhibition of human DNA topoisomerase II α by salicylate and related nonsteroidal anti-inflammatory drugs

Abstract Salicylate-based drugs, including aspirin (acetylsalicylic acid), have long been used in the treatment of mild to moderate cancer pain and have been shown in population-based studies to have cancer chemopreventive properties. Several mechanisms have been hypothesized to underlie these effects, including the inhibition of cyclooxygenases and the inhibition of the transcription factor NF-\#954;B. We have recently demonstrated, however, that salicylate, the primary metabolite of aspirin, is a novel catalytic inhibitor of human DNA topoisomerase IIα (topo II), a nuclear enzyme essential for cell proliferation and division and the target of several widely used anti-cancer chemotherapeutics. As a consequence of this inhibition, and independent of its capacity to inhibit cyclooxygenases and NF-\#954;B, we have demonstrated that a brief pretreatment of human breast cancer cells with salicylate attenuates the cytotoxicity of the topo II poisons, doxorubicin and etoposide. We have observed that salicylate prevents doxorubicin-induced DNA double-strand break generation, which is attributable to salicylate-mediated inhibition of doxorubicin-stabilized topo II-DNA cleavable complex formation in vivo. Together, these data suggest that co-administration of salicylates could negatively impact the efficacy of cancer treatment regimens incorporating topo II-targeting therapeutics. We have now extended our investigation of salicylate, using a biochemical approach to determine the mechanism whereby salicylate inhibits the catalytic activity of topo II. As the inhibition of topo II can occur at one of several stages in its catalytic cycle, we have undertaken multiple independent approaches, including an examination of DNA binding, DNA intercalation, measurement of ATPase activity and evaluation of salicylate's capacity to stabilize topo II in a closed clamp formation without causing DNA double-strand breaks. In addition to delineating the mechanism of salicylate-mediated inhibition of topo II, we have investigated whether common salicylate- and non-salicylate-based non-steroidal anti-inflammatory drugs (NSAIDs) possess similar topo II inhibitory properties, initially by examining the effects of short-term exposure on doxorubicin-induced DNA damage signaling followed by a direct examination of their effects on topo II catalytic activity. Our experiments demonstrate that inhibition of topo II is readily observed with multiple salicylate-based therapies at clinically achieved concentrations. These investigations identify a novel cellular target of salicylate and will inform future studies that may reveal evidence warranting the discouragement of NSAID co-administration in patients undergoing treatment for any of the broad-reaching malignancies using topo II poisons in their therapeutics regimen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C39.

Epithelial cells modulate genes associated with NF kappa B activation in co-cultured human macrophages

Macrophages located in airways and the alveolar space are continually exposed to different signals from the respiratory mucosa. In this respect, epithelial cells represent an important source of cytokines and mediators modulating the state of activation and/or differentiation of mononuclear phagocytes. Many of the proinflammatory genes induced in macrophages during immune and immunopathological reactions are regulated by transcription factor NF kappa B. The aim of our study was to characterize changes in the expression of genes associated with NF kappa B activation and signalling in THP-1 human macrophages co-cultured with A549 respiratory epithelial cells. At least 4-fold upregulation of mRNA level was found in 29 of 84 tested genes including genes for multiple cytokines and chemokines, membrane antigens and receptors, and molecules associated with NF kappa B signalling. The mRNA induction was confirmed at the level of protein expression by evaluating the release of IL-6 and IL-8 and by ICAM-1 expression. Blocking of one NFκB subunit by p65 siRNA inhibited the production of IL-6 in both cell types while IL-8 release from THP-1 cells did not seem to be affected. We conclude from our data that unstimulated respiratory epithelial cells regulate genes associated with NF kappa B dependent immune responses in human macrophages and that these interactions may play a key role in immediate responses in the respiratory mucosa.

Abstract 2645: PEITC induces cell death in melanoma cells through redox mechanisms

Abstract Melanoma is one of the deadliest types of cancers accounting for about 80% of skin cancer deaths. Reactive oxygen species (ROS), produced by the mitochondrial respiratory chain and other biochemical mechanisms, play a critical role in melanoma progression through activation of transcription factors AP-1 and NF- κB and loss of E-cadherin. However, a further elevated level of ROS in cells, beyond a certain cellular threshold, may lead to mitochondrial damage, cytotoxicity and subsequent cell death. Hence, increasing ROS levels by either activating a ROS-generating enzyme or by depletion of antioxidants is a plausible strategy to induce cell killing in melanoma cells. The natural compound β-phenylethyl isothiocyanate (PEITC) found in cruciferous vegetables exhibits significant antitumor activity. Recent studies demonstrated that PEITC selectively induced cancer cell death by disabling the glutathione antioxidant system. Based on these observations, we hypothesize that PEITC may preferentially eliminate melanoma cells through ROS-mediated mechanism due to the increased basal ROS levels in melanoma cells and their high dependence on glutathione. To test this hypothesis, the cytotoxic effect of PEITC was tested in a panel of melanoma cell lines in comparison with normal melanocytes. Our results demonstrated that PEITC had time- and concentration-dependent antiproliferative effect in melanoma cells. The inhibitory effect is melanoma specific, since PEITC had minimum cytotoxic activity in normal melanocytes. Further analysis of ROS by FACS using the fluorescent dye DCFDA revealed that this cell death was primarily due to increased ROS accumulation triggered by PEITC-induced depletion of cellular glutathione. This aberrant increase in ROS caused mitochondrial membrane damage, as determined by biochemical analysis using Rhodamine 123, and subsequent cell death through apoptosis. Importantly, PEITC was very effective in melanoma cells, which exhibited resistance to standard anti-tumor drugs like temozolomide (TMZ) and carmustine (BCNU). Our study suggests that PEITC may be an effective anti-melanoma therapeutic agent that warrants further pre-clinical and clinical investigation. Our work is now directed towards identifying other ROS related cellular mechanisms that can be exploited to increase the therapeutic specificity and sensitivity of PEITC in melanoma cells. 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 2645. doi:10.1158/1538-7445.AM2011-2645

Thymocytes are activated by toluene inhalation through the transcription factors NF‐κB, STAT5 and NF‐AT

Toluene has been extensively examined for effects on the central nervous system. To investigate the influence of low-level inhalation of toluene on the naive immune cells, male C3H/HeN mice were exposed to filtered air (control) and 50 ppm of toluene for 3 weeks. Low-level exposure resulted in (1) increased proliferation of thymocytes, (2) IL-2 production induced in thymocytes and (3) activation of the transcription factors NF-κB, STAT5 and NF-AT in thymocytes. These results suggest that thymocytes are sensitive cells and T cell activators are candidates for biomarkers for low-level exposure to toluene on naive immune cells.

Targeting Inflammatory Pathways by Flavonoids for Prevention and Treatment of Cancer

Observational studies have suggested that lifestyle risk factors such as tobacco, alcohol, high-fat diet, radiation, and infections can cause cancer and that a diet consisting of fruits and vegetables can prevent cancer. Evidence from our laboratory and others suggests that agents either causing or preventing cancer are linked through the regulation of inflammatory pathways. Genes regulated by the transcription factor NF- kappaB have been shown to mediate inflammation, cellular transformation, tumor cell survival, proliferation, invasion, angiogenesis, and metastasis. Whereas various lifestyle risk factors have been found to activate NF- kappaB and NF- kappaB-regulated gene products, flavonoids derived from fruits and vegetables have been found to suppress this pathway. The present review describes various flavones, flavanones, flavonols, isoflavones, anthocyanins, and chalcones derived from fruits, vegetables, legumes, spices, and nuts that can suppress the proinflammatory cell signaling pathways and thus can prevent and even treat the cancer.