Transcription Factor NF-kappa Research Articles

Metabolite profiling of anticancer compounds in Saussure lappa based on UPLC-QToFMS/MS

Background: Saussure lappa is a plant of the Asteraceae family that has been known as a medicinal plant to treat cancer. However, there is still a lack of data about the metabolite profiling of this extract and target activity in the cancer signaling pathway. Objective: This study aimed to identify metabolites and evaluate the anticancer activity of Saussure lappa active ingredients. Method: The root sample was extracted using Ultrasound-Assisted Extraction (UAE) with 96% ethanol solvent. Evaluation of the anticancer activity of the compounds was carried out using the PASS SERVER tools. Result: About 17 metabolites have been characterised. Interestingly, Saussureamine B, C, D, costunolide, and dehydrocostus lactone showed highly antineoplastic potential properties with several possible activities such as apoptosis agonists, MMP9 expression inhibitors, and Transcription factor NF-kappa B inhibitors. Conclusion: The root extract of Saussure lappa is highly recommended to be developed as an anticancer drug candidate.

Koena, a Novel Compound That Dampens Inflammation in the Skin

Inflammatory skin diseases are characterized by the activation of the innate and adaptive immune system via the production of pro-inflammatory cytokines. The main proinflammatory cytokines responsible for this development include TNF-alpha, Interleukin 1, 6 and 17, also the transcription factor NF-kappa B. Small molecule anti-inflammatory compounds such as those detailed in the paper, show much promise in dealing with these skin disorders. Four nutraceutical molecules that are well characterized by their capacity to interact with many of the pro-inflammatory cytokines, importantly IL-17, are found in an Australian product, Koena. Koena has been shown to not only decrease these cytokines but also help increase the production of Interleukin 10 and important anti-inflammatory cytokines. This paper reports clinical benefits of Koena in a variety of circumstances; eczema and psoriasis; insect bites and sting; solar damaged skin and keratosis. An analysis of patient/purchaser reviews via the website Koena.com.au to ascertain what condition they purchased the product for, if any, and the satisfaction rating after use. More than 35% of reviewers reported purchasing Koena to help with inflamed skin. Those reporting being satisfied with the result was 95% for this use. This paper reports on the benefit of small molecule anti-inflammatories in modulating and not blocking an immune response. It also illustrates the benefits of multitherapy when dealing with complex inflammatory responses as opposed to monotherapy.

An interview with Nobel Laureate David Baltimore, PhD.

In an online interview, Nobel Laureate David Baltimore, Ph.D., reflected on his contributions to biomedical science that have had a major influence on the fields of molecular biology, virology, cancer, and immunology. Dr. Baltimore is President Emeritus and Distinguished Professor of Biology at the California Institute of Technology. Among other notable works, he discovered the critical nuclear transcription factor NF Kappa B and the Rag1 and Rag2 proteins that rearrange adaptive immune cell receptors.
 His career path, he says, evolved naturally, as math and science came easily to him. As a high school student, he participated in a summer program at the Jackson Lab in Bar Harbor, Maine, where he says he came away feeling that experimental biology was exciting and rewarding.
 “That's where I discovered that the frontiers of science, were not so distant; that I could actually make a discovery that nobody else in the world knew about,” he says. 
 And that he did. Independently, he and Howard Temin discovered the viral enzyme reverse transcriptase revising the canon of cellular information transfer. They published back-to-back papers in Nature demonstrating that this enzyme in virus particles could transcribe RNA to DNA. Both received a Nobel Prize for this work.
 In reflecting on his early experience evaluating how to work with recombinant DNA and how we should scientifically and safely approach gain of function research, he says, “We have to be very honest with ourselves about what might hold danger, and we have to control our instinct … to do anything we can to generate progress and understanding of life. …At the same time, we don’t want to hold back progress, and so there is a balancing.”
 Dr. Baltimore also discussed his optimism about vectored immunoprophylaxis as a strategy for prevention of HIV and his doubt that scalable strategies will be able to cure HIV. He also reflected on his philosophy for the training of young scientists and the successful training program that he developed at the Whitehead Institute.

A Possible Role of Crustacean Cardioactive Peptide in Regulating Immune Response in Hepatopancreas of Mud Crab

Crustacean cardioactive peptide (CCAP), a cyclic amidated non-apeptide, is widely found in arthropods. The functions of CCAP have been revealed to include regulation of heart rate, intestinal peristalsis, molting, and osmotic pressure. However, to date, there has not been any report on the possible involvement of CCAP in immunoregulation in crustaceans. In this study, a CCAP precursor (designated as Sp-CCAP) was identified in the commercially important mud crab Scylla paramamosain, which could be processed into four CCAP-associated peptides and one mature peptide (PFCNAFTGC-NH2). Bioinformatics analysis indicated that Sp-CCAP was highly conserved in crustaceans. RT-PCR results revealed that Sp-CCAP was expressed in nerve tissues and gonads, whereas the Sp-CCAP receptor gene (Sp-CCAPR) was expressed in 12 tissues of S. paramamosain, including hepatopancreas. In situ hybridization further showed that an Sp-CCAPR-positive signal is mainly localized in the F-cells of hepatopancreas. Moreover, the mRNA expression level of Sp-CCAPR in the hepatopancreas was significantly up-regulated after lipopolysaccharide (LPS) or polyriboinosinic polyribocytidylic acid [Poly (I:C)] challenge. Meanwhile, the mRNA expression level of Sp-CCAPR, nuclear transcription factor NF-κB homologs (Sp-Dorsal and Sp-Relish), member of mitogen-activated protein kinase (MAPK) signaling pathway (Sp-P38), pro-inflammatory cytokines factor (Sp-TNFSF and Sp-IL16), and antimicrobial peptide (Sp-Lysozyme, Sp-ALF, Sp-ALF4, and Sp-ALF5) in the hepatopancreas were all up-regulated after the administration of synthetic Sp-CCAP mature peptide both in vivo and in vitro. The addition of synthetic Sp-CCAP mature peptide in vitro also led to an increase in nitric oxide (NO) concentration and an improved bacterial clearance ability in the hepatopancreas culture medium. The present study suggested that Sp-CCAP signaling system might be involved in the immune responses of S. paramamosain by activating immune molecules on the hepatopancreas. Collectively, our findings shed new light on neuroendocrine-immune regulatory system in arthropods and could potentially provide a new strategy for disease prevention and control for mud crab aquaculture.

INHIBITION OF TRANSCRIPTION FACTORS NF KAPPA B AND AP-1 LIMITS THE PROGRESSION OF OXIDATIVE-NITROSATIVE STRESS IN THE TISSUE OF CEREBRAL HEMISPHERES IN RATS AFTER MODELLED TRAUMATIC BRAIN INJURY

The study was aimed as investigating the effects of inhibitors of transcription factors NF kappa B and AP-1 activation on the development of oxidative-nitrosative stress in rat cerebral hemispheres following experimental traumatic brain injury (TBI). The study included 60 white Wistar male rats weighing 180-220 g, divided into 4 groups of 7 animals in each: the 1st group included pseudo-injured animals subjected to the same manipulations (ether anaesthesia, fixation) as the animals in the experimental groups, except for TBI modelling, the 2nd group included the animals exposed to modelled TBI, the 3rd and 4th groups involved the rats who received ammonium pyrolidine dithiocarbamate, the nuclear translocation inhibitor NF kappa B in a dose of 76 mg/kg and the inhibitor AP-1 SR 11302 in a dose of 1 mg/kg, respectively for 7 days following the TBI modelling. On the 7th day after the simulation of a moderate TBI in rat cerebral hemisphere tissue, the following signs of oxidative-nitrosative stress have been detected: increased production of superoxide radical anion by NADPH and NADH-dependent electron transport chains, increased activity of nitric oxide synthase (total and inducible), a decrease and impairment of the coupling of its constitutive isoform, growth in the concentration of peroxynitrite, the development of decompensated lipid peroxidation. The application of transcription factor inhibitors NF kappa B (PDTC) and AP-1 (SR 11302) significantly reduces the signs of oxidative-nitrosative stress in the tissue of rat cerebral hemispheres on the 7th day of the experiment, reduces the production of superoxide anion radical and the activity of nitric oxide synthase (total and inducible), improves the coupling of its constitutive isoform, limits the peroxynitrite concentration, and enhances the antioxidant potential.

Inhibitory Effects of Myrtucommuacetalone 1 (MCA-1) from Myrtus communis on Inflammatory Response in Mouse Macrophages.

(1) Introduction: Reactive oxygen species (ROS) and nitric oxide (NO) are key signaling molecules that play important roles in the progression of inflammatory disorders. The objective of this study was to explore the use of myrtucommuacetalone-1 (MCA-1), as a novel compound of natural origin and a potential anti-inflammatory agent. (2) Methodology: The anti-inflammatory potential of MCA-1, which was isolated from Myrthus communis Linn, was determined by assaying superoxide, hydrogen peroxide, and nitric oxide production in macrophages. Furthermore, the effects of the compound were analyzed via phosphorylation and translocation of the transcription factor NF kappa B, which is a key regulator of iNOS activation. The effect of MCA-1 on the inducible nitric oxide synthase (iNOS) enzyme was also examined using in silico docking studies. The anticancer potential for MCA-1 was evaluated with an MTT cytotoxic assay. (3) Results: In stimulated macrophages, MCA-1 inhibited superoxide production by 48%, hydrogen peroxide by 53%, and nitric oxide (NO) with an IC50 of <1 µg/mL. MCA-1 also showed a very strong binding pattern within the active site of the inducible nitric oxide synthase enzyme. Furthermore, 25 µg/mL of MCA-1 inhibited inducible nitric oxide synthase expression and abolished transcription factor (NFκB) phosphorylation and translocation to the nucleus. Cytotoxicity analyses of MCA-1 on 3T3 mouse fibroblasts, CC1 liver cell line, J774.2, macrophages and MDBK bovine kidney epithelial cell, yielded IC50 values of 6.53 ± 1.2, 4.6 ± 0.7, 5 ± 0.8, and 4.6 ± 0.7, µg/mL, respectively. (4) Conclusion: Our results suggest that MCA-1, a major phloroglucinol-type compound, shows strong anti-inflammatory activity and has a potential to be a leading therapeutic agent in the future.

Phytochemicals as potential IKK-β inhibitor for the treatment of cardiovascular diseases in plant preservation: terpenoids, alkaloids, and quinones.

Modulation of inhibitor kappa B kinase-beta (IKK-β) kinase activity could be useful for preventing inflammation that serves an efficient role in protection against cardiovascular diseases (CVDs). IKK-β induces inflammation by activating transcription factor NF-kappa B (NF-κB) through phosphorylation of IκB. Therefore, IKK-β is considered an interesting target for protecting and treating CVDs. The cardioprotective potential of terpenoids, alkaloids and quinines may be related to modulating inflammatory responses. In this study, the interactions between different classes of inhibitors and IKK-β were investigated, through the application of SystemsDock. Molecular docking results showed that Diosgenin and Ginsenoside Re were the top docking score compounds. Diosgenin and Ginsenoside Re are the most promising IKK-β inhibitors in terpenoids, alkaloids, and quinones. Diosgenin and Ginsenoside Re could be helpful to find the lead compounds on designing and developing novel cardioprotective agents.

Magnesium Chloride is an Effective Therapeutic Agent for Prostate Cancer

Background: Reduced levels of magnesium can cause several diseases and increase cancer risk. Motivated by magnesium chloride’s (MgCl2) non-toxicity, physiological importance, and beneficial clinical applications, we studied its action mechanism and possible mechanical, molecular, and physiological effects in prostate cancer with different metastatic potentials.Methods: We examined the effects of MgCl2, after 24 and 48 hours, on apoptosis, cell migration, expression of epithelial mesenchymal transition (EMT) markers, and V-H+-ATPase, myosin II (NMII) and the transcription factor NF Kappa B (NFkB) expressions.Results: MgCl2 induces apoptosis, and significantly decreases migration speed in cancer cells with different metastatic potentials. MgCl2 reduces the expression of V-H+-ATPase and myosin II that facilitates invasion and metastasis, suppresses the expression of vimentin and increases expression of E-cadherin, suggesting a role of MgCl2 in reversing the EMT. MgCl2 also significantly increases the chromatin condensation and decreases NFkB expression.Conclusions: These results suggest a promising preventive and therapeutic role of MgCl2 for prostate cancer. Further studies should explore extending MgCl2 therapy to in vivo studies and other cancer types.Keywords: Magnesium chloride, prostate cancer, migration speed, V-H+-ATPase, and EMT.

Transcription factor NF-kappa B represses ANT1 transcription and leads to mitochondrial dysfunctions

Mitochondria are intracellular organelles involved in cell survival and death, and dysfunctions of mitochondria are related to neurodegenerative diseases. As the most abundant protein in the mitochondrial inner membrane, adenine nucleotide translocator 1 (ANT1) plays a critical role in mitochondrial function, including the exchange of adenosine triphosphate/adenosine diphosphate (ATP/ADP) in mitochondria, basal proton leak and mitochondrial permeability transition pore (mPTP). Here, we show that ANT1 transcription is regulated by transcription factor NF-kappa B (NF-κB). NF-κB is bound to two NF-κB responsive elements (NREs) located at +1 to +20 bp and +41 to +61 bp in the ANT1 promoter. An NF-κB signalling stimulator, tumour necrosis factor alpha (TNFα), suppresses ANT1 mRNA and protein expression. Activation of NF-κB by TNFα impairs ATP/ADP exchange and decreases ATP production in mitochondria. Activation of NF-κB by TNFα decreases calcium induced mPTP opening, elevates mitochondrial potential and increases reactive oxygen species (ROS) production in both T98G human glioblastoma cells and rat cortical neurons. These results demonstrate that NF-κB signalling may repress ANT1 gene transcription and impair mitochondrial functions.

The divergence between the virus and cellular oxidative stress as separate environmental agents that trigger autoimmunity originates from their different procedural mechanisms of activating the same molecular entity: the transcription factor NF-kappa B

To happen, autoimmunity in man requires triggering by environmental factors: the viruses and cellular stress, in genetically primed individuals. The viruses and stress are operatives in this scene as stimuli for the activation of the transcription factor (TF), NF-ΚB. NF-ΚB is unusually activated: the viral activation occurs via serine residues-phosphorylation by IKKβ and IKKe, while the activation by oxidative stress occurs via tyrosine phosphorylation of IΚBα. The phosphorylation of particular amino acid residues of a given protein molecule modulates that protein’s polymorphic conformations, appropriately. For a TF, a given conformation influences its choice of cognate DNA sequence recognition as well as its interactions with neighboring molecules. The TF NF-ΚB performs a battery of regulatory functions. Because it is variously phosphorylated as seen above this implies that NF-ΚB is capable of assuming a multitude of polymorphic conformations that we refer to as “ derivative isoforms ”. Thus the virus activation of NF-ΚB occurring by phosphorylation at serines S536 and S468 is observed to generate the isoforms with the potential to activate the transcription of viral latency genes, hereby installing a latent infection. But oxidative stress activation of NF-ΚB occurs via phosphorylation of Tyr42 of IΚBα and this yields isoforms that activate the transcription of replication and transcription activator (RTA), the master lytic switch, which thereby abrogates the latency. The steps involved are that the stress-activated NF-ΚB and the viral miRNAs conjoin in a regulatory circuitry identified as feedback and feed-forward network motifs that co-accomplish the switching on of RTA which in turn activates the transcription of the immediate early genes BZLF1 and BRLF1. These two latter genes together mobilize the expression of the set of lytic genes, resulting in a lytic cascade and consequentially set in trend the viral journey to the triggering of autoimmunity (Figure 1).

Expression of transcription factor NF-KAPPA B/P65 and cyclooxygenase-2 (COX-2) in testicular damage induced by Red Bull energy drink in rat

Expression of transcription factor NF-KAPPA B/P65 and cyclooxygenase-2 (COX-2) in testicular damage induced by Red Bull energy drink in rat

The Schizophrenia-Related Protein Dysbindin-1A Is Degraded and Facilitates NF-Kappa B Activity in the Nucleus.

Dystrobrevin-binding protein 1 (DTNBP1), a gene encoding dysbindin-1, has been identified as a susceptibility gene for schizophrenia. Functioning with partners in synapses or the cytoplasm, this gene regulates neurite outgrowth and neurotransmitter release. Loss of dysbindin-1 affects schizophrenia pathology. Dysbindin-1 is also found in the nucleus, however, the characteristics of dysbindin in the nucleus are not fully understood. Here, we found that dysbindin-1A is degraded in the nucleus via the ubiquitin-proteasome system and that amino acids 2-41 at the N-terminus are required for this process. By interacting with p65, dysbindin-1A promotes the transcriptional activity of NF-kappa B in the nucleus and positively regulates MMP-9 expression. Taken together, the data obtained in this study demonstrate that dysbindin-1A protein levels are highly regulated in the nucleus and that dysbindin-1A regulates transcription factor NF-kappa B activity to promote the expression of MMP-9 and TNF-α.

Olfactomedin-4 is a candidate biomarker of solid gastric, colorectal, pancreatic, head and neck, and prostate cancers.

Olfactomedin-4 (OLFM4, OLM4) is a 72 kDa secreted glycoprotein belonging to the olfactomedin family. The OLFM4 gene expression is regulated by the transcription factors NF-kappa B and AP-1, and the OLM4 functions are poorly understood. OLM4 has been described as being able to interact with cell surface proteins such as lectins and concanavalin-A suggesting that one function of OLM4 is to regulate cell adhesion and migration. OLM4 is a marker for intestinal stem cells and is expressed at the bottom of the intestinal crypts. Expression of OLM4 during tumor development showed that OLM4 expression is increased in the early stages of tumor initiation. As OLM4 is a secreted protein, it is a prime candidate for biomarker research for tumor detection or progression. Levels of circulating OLM4 were significantly higher in patients with gastric, colorectal, and pancreatic cancers than in healthy subjects.

Toll-like receptor 3 inhibits Newcastle disease virus replication through activation of pro-inflammatory cytokines and the type-1 interferon pathway.

Newcastle disease virus (NDV) is an avian paramyxovirus that can selectively replicate in and destroy human tumor cells. In this report, we demonstrate that NDV infection in HeLa cells leads to the activation of the pattern recognition Toll-like receptor 3 (TLR3). Overexpression of TLR3 enhanced the activity of the IFN-β promoter and the transcription factor NF-kappa B (NF-κB), thereby decreasing viral protein synthesis and the virus titer. In addition, the reduction of endogenous TLR3 by small interfering RNA (siRNA) increased NDV replication. Similar anti-NDV effects were observed in DF-1 chicken fibroblast cells with the exogenous expression of chicken TLR3 (cTLR3). Immunofluorescence staining of HeLa cells indicated that the dsRNA generated during NDV replication colocalized with TLR3 in punctate subcellular structures. Altogether, our results strongly suggest that TLR3 actively participates in the recognition of the innate pro-inflammatory response after NDV infection and leads to the consequent antiviral cytokine/interferon secretion.

Triptolide induces apoptosis through the SERCA 3 upregulation in PC12 cells

Diterpenoid triepoxide - Triptolide (TTL) - increased protein levels of the noradrenaline transporter in three pheochromocytoma cell lines. This transporter is involved in the apoptosis induction through the inhibition of a transcription factor NF-kappa B. Nevertheless, calcium release from the endoplasmic reticulum can also induce inner mitochondrial pathway of apoptosis in variety of cells. Therefore, the aim of this work was to evaluate an involvement of calcium and, more specifically, the intracellular calcium transport systems in the apoptosis induction in pheochrocytoma cell line PC12. We observed significantly increased amount of reticular calcium in TTL-treated cells compared to control, untreated cells. Surprisingly, gene expression of the IP3 receptors was not changed after the TTL treatment, but ryanodine receptor of the type 2 (RyR2) was downregulated and sarco/endoplasmic reticulum calcium ATPase type 3 (SERCA 3) was upregulated in TTL- treated cells, compared to untreated controls. SERCA 3 blocking with the specific blocker thapsigargin prevented increase in apoptosis observed by the TTL treatment. Decrease in the ATP production by a replacement of glucose in the cultivation medium for its nonutilizable analog 2-deoxyglucose also prevented induction of the apoptosis in TTL-treated PC12 cells. Thus, these results suggest that upregulation of the SERCA 3 is ultimately involved in the TTL-induced apoptosis in PC12 cells.

Nuclear Factor κ-B Is Activated in the Pulmonary Vessels of Patients with End-Stage Idiopathic Pulmonary Arterial Hypertension

ObjectivesTo assess activation of the inflammatory transcription factor NF-kappa B (NF-κB) in human idiopathic pulmonary arterial hypertension (PAH).BackgroundIdiopathic PAH is a severe progressive disease characterized by pulmonary vascular remodeling and excessive proliferation of vascular cells. Increasing evidence indicates that inflammation is important in disease pathophysiology.MethodsNF-κB-p65 and CD68, CD20 and CD45 were measured by immunohistochemistry and confocal microscopy on lung specimens from patients with idiopathic PAH (n = 12) and controls undergoing lung surgery (n = 14). Clinical data were recorded for all patients including invasive pulmonary hemodynamics for the PAH patients. Immunohistochemical images were analyzed by blinded observers to include standard pulmonary vascular morphometry; absolute macrophage counts/mm2 and p65-positivity (p65+) using composite images and image-analysis software; and cytoplasmic:nuclear p65+ of individual pulmonary arterial endothelial and smooth muscle cells (PASMC) in 10–20 pulmonary arteries or arterioles per subject. The expression of ET-1 and CCL5 (RANTES) in whole lung was determined by RT-qPCR.ResultsMacrophage numbers were increased in idiopathic PAH versus controls (49.0±4.5 vs. 7.95±1.9 macrophages/100 mm2, p<0.0001): these macrophages demonstrated more nuclear p65+ than in macrophages from controls (16.9±2.49 vs. 3.5±1.25%, p<0.001). An increase in p65+ was also seen in perivascular lymphocytes in patients with PAH. Furthermore, NF-κB activation was increased in pulmonary arterial endothelial cells (62.3±2.9 vs. 14.4±3.8, p<0.0001) and PASMC (22.6±2.3 vs. 11.2±2.0, p<0.001) in patients with PAH versus controls, with similar findings in arterioles. Gene expression of both ET-1 mRNA ((0.213±0.069 vs. 1.06±0.23, p<0.01) and CCL5 (RANTES) (0.16±0.045 vs. 0.26±0.039, p<0.05) was increased in whole lung homogenates from patients with PAH.ConclusionsNF-κB is activated in pulmonary macrophages, lymphocytes, endothelial and PASMC in patients with end-stage idiopathic PAH. Future research should determine whether NF-κB activation is a driver or bystander of pulmonary vascular inflammation and if the former, its potential role as a therapeutic target.

Abstract A30: NF-kB activating peptides identified in genetic screen cooperate with Ras transformation by suppression of p53 linking inflammation to carcinogenesis

Abstract Chronic inflammatory conditions are frequently linked to increased risk of carcinogenesis. The transcription factor NF-kappa B acts as a central component of signaling pathways governing the inflammatory response. NF-kB is also constitutively activated in several tumors. While the tumor promoting inflammatory effects are largely attributed to activation of NFκB signaling in tumor milieu, it is unclear whether activation of inflammatory pathway alone can initiate tumorigenesis. The mechanism by which constitutive activation of NF-kB contributes to carcinogenesis remains elusive. We hypothesized that constitutive activation of NFkB by proinflammatory agents renders p53 incapable of opposing oncogene induced transformation. As pharmacological activation of NF-kB in normal cells is prone to transient effects due to feedback regulation, we performed an unbiased genetic screen for proinflammatory peptides in an NFκB-GFP reporter cell line using a lentiviral trimer peptide library spanning the coding regions of 800 extracellular ligands. We identified several pro-inflammatory peptides whose expression lead to robust activation of NFκB signaling. Suppression of p53 has been shown to be the only criteria to be met to override ras oncogene induced senescence in rodent fibroblasts, which prompted us to test the NFκB activating peptides in this model. Here, we show that indeed, these NF-kB activating peptides cooperate with oncogenic RasV12 to induce transformation in both in rat fibroblast cell line REF52 and primary mouse embryonic fibroblasts (MEFs) through r p53. Further, these Ras transformed fibroblast cells formed tumors in nude mice. Thus, pro-inflammatory genetic elements that activate NF-kappa B pathway can override Ras induced senescence to facilitate cellular transformation underscoring the importance of NFκB inhibitors in preventing initiation of tumerigenesis. Citation Format: Venkatesh Natarajan, Andrei P. Komarov, Alex Chenchik, Andrei V. Gudkov. NF-kB activating peptides identified in genetic screen cooperate with Ras transformation by suppression of p53 linking inflammation to carcinogenesis. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A30.

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

The Effect of Mtor-Inhibition on Inflammation in Kidney Ischemia-Reperfusion Injury in Mice

Introduction: Ischemia-reperfusion injury (IRI) plays an important role in acute renal transplant rejection and delayed allograft function and also long-term graft survival. Multiple stimuli initiating inflammation, including ROS and cytokines, participate in pathogenesis of IRI and are at some level controlled through activation of transcription factor NF-kappa B. We therefore determined the influence of mTOR-inhibition (Everolimus; eve) on inflammation and NF-kappa B activity after induction of IRI. Method: C57/BL6 mice were subjected to IRI by clamping both renal pedicles for 45 minutes. Application of eve started one day before IRI induction in a dose of 1.5 mg/kg b.w. subcutaneously daily. Both eve treated and non-treated mice were sacrificed 30 min, 1h, 2h, 6h, 12h, 1d, 2d, 3d and 7d after IRI. Kidneys were examined by morphology and standard molecular biology techniques including EMSA. Results: Eve treatment significantly increased at early (2d) (p< 0.05), however, significantly reduced kidney injury index at later (7d) time points (p< 0.004) compared with non-treated mice after IRI induction. Interstitial infiltration of F4/80-positive cells after IRI was significantly reduced at later time points in eve treated animals (p< 0.01), which is associated with a decreased expression in TGF-ß and iNOS. NF-kappa B activity was significantly increased in non-treated animals at early (6h) and also later (3d and 7d) time points, while eve treatment of mice significantly increased NF-kappa B binding activity at all time points, showing increased nuclear expression of phospho-p65 at all time points (p< 0.01) and Rel B at later time points (p< 0.01), when compared with non-treated animals. Biphasic increase in TNF-alpha and chemokine CCL2, CCL5 and CXCL10 expression, both in treated and non-treated animals, was observed. The second peak of TNF-alpha expression was significantly increased while CCL2 and CCL5 expression was significantly reduced in eve treated animals. The expression of the anti-inflammatory cytokine IL-10 was lower at later time points in eve treated mice. Conclusion: Expression profiles showed a biphasic expression of TNF-α and chemokines coinciding with the early inflammatory and late repair phase after IRI induction. The beneficial role of eve treatment in late repair phase could be partly reflected by decreased numbers of infiltrating macrophage. The pattern of NF-κB activation under m-TOR inhibition reflects activation via canonical and also non-canonical pathway in IRI which warrants further clarification.

Thioredoxin regulates NF-kappa B activity through cytokine-mediated denitrosylation of p65

Activation of the pro-inflammatory transcription factor NF-Kappa B (p50–p65) is inhibited via S-nitrosylation of the p65 subunit (SNO-p65). In the respiratory epithelium, cytokine stimulation induces rapid denitrosylation of p65 concomitant with NF-Kappa B activation and initiation of the pulmonary immune response. However, the mechanism for NF-Kappa B denitrosylation has remained unclear. We hypothesize that thioredoxin (Trx), an oxidoreductase with denitrosylating activity, mediates this response. We utilized cytokine-stimulated respiratory epithelial cells (A549) and C57BL6 mice exposed to aerosolized LPS (5 mg/m 3 ) to test this hypothesis. Trx reductase (TrxR) activity was inhibited in mice and in cells by pretreatment with the organic gold compounds auranofin and aurothioglucose, respectively. Trx protein expression, SNO-p65 formation and NF-Kappa B activity were quantified in cell lysates and in lung homogenates. Bronchoalveolar lavage fluid was analyzed for markers of airway inflammation (WBC count and cytokines). We found that TrxR inhibition prevented cytokine-mediated denitrosylation of p65, NF-Kappa B activation, and LPS-induced airway inflammation. Trx1 co-immunoprecipitated with p65 at baseline, but this interaction was not increased with cytokine stimulation nor did cytokines alter TrxR activity. Cytokine-induced and Trx-dependent denitrosylation of p65 was, however, dependent upon the ERK MAP kinase pathway, and the regulatory mechanisms for ERK-dependent denitrosylation will be discussed.