Sensitive Transcription Factor Research Articles

O‐GlcNAc Plays a Role in Nrf2 Regulation in the Myocardium

The redox sensitive transcription factor NF‐E2–related factor 2 (Nrf2) protects against oxidative stress and its activity is aberrant in type 2 diabetes.. Acute increases in the protein posttranslational attachment of O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) to serine/threonine residues have been shown to mitigate oxidative stress responses in the heart. This suggests that O‐GlcNAc may play a role in Nrf2 signaling; however, the role of O‐GlcNAc modification on Nrf2 remains to be elucidated. We examined Nrf2‐mediated genes in a tamoxifen‐inducible cardiomyocyte‐specific OGT knockdown (OGTKD) mouse. We also augmented O‐GlcNAc in cardiomyocytes (H9C2 cell line) and examined Nrf2 protein levels and mRNA of Nrf2‐mediated genes. Immunoprecipitation demonstrated that Nrf2 is O‐GlcNAc modified in the heart. Our in vivo model showed that Nrf2 protein was increased (p<0.05) while mRNA of the Nrf2 gene‐product catalase was reduced in the OGTKD mouse(p<0.05). Conversely, our in vitro experiments demonstrated that increasing protein O‐GlcNAcylation in H9C2 cells increased transcription of the Nrf2‐mediate genes catalase, glutamate‐cysteine ligase (GCLM), and heme oxygenase 1 (HMOX1) (p<.05). These data provide evidence for O‐GlcNAc as novel post‐translational modification of Nrf2 and suggest that O‐GlcNAcylation plays a role in regulating Nrf2 signaling.

Expression of xCT and activity of system xc− are regulated by NRF2 in human breast cancer cells in response to oxidative stress

Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate by activating key transcription factors. One such master regulator, the redox sensitive transcription factor NF E2 Related Factor 2 (NRF2), controls the expression of cellular defense genes including those encoding intracellular redox-balancing proteins involved in glutathione (GSH) synthesis. Under basal conditions, Kelch-like ECH-associated protein 1 (KEAP1) targets NRF2 for ubiquitination. In response to oxidative stress, NRF2 dissociates from KEAP1, entering the nucleus and binding to the antioxidant response element (ARE) in the promoter of its target genes. Elevated reactive oxygen species (ROS) production may deplete GSH levels within cancer cells. System xc−, an antiporter that exports glutamate while importing cystine to be converted into cysteine for GSH synthesis, is upregulated in cancer cells in response to oxidative stress. Here, we provided evidence that the expression of xCT, the light chain subunit of system xc−, is regulated by NRF2 in representative human breast cancer cells. Hydrogen peroxide (H2O2) treatment increased nuclear translocation of NRF2, also increasing levels of xCT mRNA and protein and extracellular glutamate release. Overexpression of NRF2 up-regulated the activity of the xCT promoter, which contains a proximal ARE. In contrast, overexpression of KEAP1 repressed promoter activity and decreased xCT protein levels, while siRNA knockdown of KEAP1 up-regulated xCT protein levels and transporter activity. These results demonstrate the importance of the KEAP1/NRF2 pathway in balancing oxidative stress in breast cancer cells through system xc−. We have previously shown that xCT is upregulated in various cancer cell lines under oxidative stress. In the current investigation, we focused on MCF-7 cells as a model for mechanistic studies.

Dimethyl fumarate attenuates cerebral edema formation by protecting the blood–brain barrier integrity

Brain edema is a hallmark of various neuropathologies, but the underlying mechanisms are poorly understood. We aim to characterize how tissue hypoxia, together with oxidative stress and inflammation, leads to capillary dysfunction and breakdown of the blood–brain barrier (BBB). In a mouse stroke model we show that systemic treatment with dimethyl fumarate (DMF), an antioxidant drug clinically used for psoriasis and multiple sclerosis, significantly prevented edema formation in vivo. Indeed, DMF stabilized the BBB by preventing disruption of interendothelial tight junctions and gap formation, and decreased matrix metalloproteinase activity in brain tissue. In vitro, DMF directly sustained endothelial tight junctions, inhibited inflammatory cytokine expression, and attenuated leukocyte transmigration. We also demonstrate that these effects are mediated via activation of the redox sensitive transcription factor NF-E2 related factor 2 (Nrf2). DMF activated the Nrf2 pathway as shown by up-regulation of several Nrf2 target genes in the brain in vivo, as well as in cerebral endothelial cells and astrocytes in vitro, where DMF also increased protein abundance of nuclear Nrf2. Finally, Nrf2 knockdown in endothelial cells aggravated subcellular delocalization of tight junction proteins during ischemic conditions, and attenuated the protective effect exerted by DMF. Overall, our data suggest that DMF protects from cerebral edema formation during ischemic stroke by targeting interendothelial junctions in an Nrf2-dependent manner, and provide the basis for a completely new approach to treat brain edema.

Ca2+-dependent regulations and signaling in skeletal muscle: from electro-mechanical coupling to adaptation.

Calcium (Ca2+) plays a pivotal role in almost all cellular processes and ensures the functionality of an organism. In skeletal muscle fibers, Ca2+ is critically involved in the innervation of skeletal muscle fibers that results in the exertion of an action potential along the muscle fiber membrane, the prerequisite for skeletal muscle contraction. Furthermore and among others, Ca2+ regulates also intracellular processes, such as myosin-actin cross bridging, protein synthesis, protein degradation and fiber type shifting by the control of Ca2+-sensitive proteases and transcription factors, as well as mitochondrial adaptations, plasticity and respiration. These data highlight the overwhelming significance of Ca2+ ions for the integrity of skeletal muscle tissue. In this review, we address the major functions of Ca2+ ions in adult muscle but also highlight recent findings of critical Ca2+-dependent mechanisms essential for skeletal muscle-regulation and maintenance.

The disturbance of oxidant-antioxidant balance in rat colonic mucosa after antibiotic therapy

Antibiotic treatment increases susceptibility to development of inflammatory bowel diseases (IBD) both in children and adults. Oxidative stress plays a prominent role in IBD pathogenesis. The aim of present study was to test an interrelationship between the morphological changes in rat colonic mucosa, the levels of antioxidant enzymes and redox sensitive transcription factors Egr-1 and Sp-1 after treatment with cephalosporin antibiotic ceftriaxone (Cf) with broad spectrum of action. Study was performed on male Wistar rats (180–230 g). Cf (50 mg/kg, i.m.) were injected daily for 5 days. The colonic levels of catalase and superoxide dismutase activity were measured by the colorimetric assays and zymography; levels of Egr-1 and Sp-1 – by Western-blot analysis; histological chan­ges – by morphometric analysis. Body weight and diarrhea were recorded. Systemic administration of the ceftriaxone induced morphological and functional changes in rat colonic mucosa associated with initial stages of the acute inflammation. These changes were accompanied by a decrease of activity of superoxide dismutase and catalase. Levels of redox-sensitive transcription factors Egr-1 and Sp1 were significantly increased, which shows a disturbance of homeostasis of the intestinal barrier. Keywords: ceftriaxone, colon, antioxidants, transcription factors.

84-OR

Objectives Preeclampsia is a pregnancy-associated complex condition associated with inflammation, oxidative stress and angiogenic imbalance. Stress sensitive transcription factors nuclear factor-erythroid 2-like 1 (Nrf1) and nuclear factor-erythroid 2-like 2 (Nrf2) are involved in regulation of angiogenic, inflammatory and oxidative stress pathways. Recent evidence suggests a link between Nrf2 and angiogenic factor balance in preeclampsia though the degree to which maternal and placental DNA methylation contributes to disruption of Nrf pathways among women with preeclampsia is unknown. Our central hypothesis is that distinct patterns of DNA methylation in Nrf pathway and angiogenic genes contribute to altered angiogenic balance in preeclampsia. Methods Prospectively, we collected blood in each of three trimesters of pregnancy, maternal white blood cells in the first trimester of pregnancy and placental tissue at delivery from nulliparous women. We analyzed DNA methylation at individual CpG dinucleotides (Illumina Infinium) in Nrf and angiogenic (VEGFA, VEGFB, VEGC, FLT1, KDR, NRF1, PlGF) genes in maternal peripheral blood cells and placenta and circulating angiogenic factors (Millipore Bioplex) from women who developed preeclampsia and normotensive controls ( n = 6/group). Differences in average methylation beta scores (change in beta score >0.2 indicated significant gain of methylation; >−0.2 indicated significant loss of methylation) determined significant differences between groups. Results DNA methylation patterns in Nrf and angiogenic genes in maternal white blood cells and placenta were not significantly different in cases/controls. Among women with preeclampsia, PlGF was significantly lower across pregnancy and sFLT1 was significantly higher in the third trimester among women with preeclampsia. Conclusions DNA methylation in Nrf and angiogenic genes was not associated with altered angiogenic balance in women who developed late onset preeclampsia. Differences in DNA methylation may occur later in pregnancy when circulating factors differed between groups. Disclosures C.M. Anderson: None. J.L. Ralph: None. J.E. Ohm: None.

Nuclear Nox4 Role in Stemness Power of Human Amniotic Fluid Stem Cells.

Human amniotic fluid stem cells (AFSC) are an attractive source for cell therapy due to their multilineage differentiation potential and accessibility advantages. However the clinical application of human stem cells largely depends on their capacity to expand in vitro, since there is an extensive donor-to-donor heterogeneity. Reactive oxygen species (ROS) and cellular oxidative stress are involved in many physiological and pathophysiological processes of stem cells, including pluripotency, proliferation, differentiation, and stress resistance. The mode of action of ROS is also dependent on the localization of their target molecules. Thus, the modifications induced by ROS can be separated depending on the cellular compartments they affect. NAD(P)H oxidase family, particularly Nox4, has been known to produce ROS in the nucleus. In the present study we show that Nox4 nuclear expression (nNox4) depends on the donor and it correlates with the expression of transcription factors involved in stemness regulation, such as Oct4, SSEA-4, and Sox2. Moreover nNox4 is linked with the nuclear localization of redox sensitive transcription factors, as Nrf2 and NF-κB, and with the differentiation potential. Taken together, these results suggest that nNox4 regulation may have important effects in stem cell capability through modulation of transcription factors and DNA damage.

Ascorbic acid attenuates antineoplastic drug 5-fluorouracil induced gastrointestinal toxicity in rats by modulating the expression of inflammatory mediators

Damage to the mucous membrane is a serious issue associated with chemotherapy. Gastrointestinal (GI) toxicity is complex and multistep process and unregulated production of reactive oxygen species (ROS) and inflammatory mediators play vital role in the development of GI toxicity. In the present study we have investigated the attenuating potential of vitamin C (vit. C) on 5 fluorouracil (5-FU) induced GI toxicity by targeting oxidative stress and inflammatory markers in Sprague Dawley (SD) rats. Rats were gavaged with vit. C (500mg/kgb.wt.) or vehicle daily (day 1–10) and were given intraperitoneal injection of 5-FU (150mg/kgb.wt.) or saline (control) on day 8 to induce mucositis. We found that vit. C supplementation attenuated 5-FU induced lipid peroxidation, myeloperoxidase (MPO) activity, activation of NF-kB and expression of COX-2. Histological observations further supported the protective potential of vit. C against 5-FU induced intestinal anomalies such as neutrophil infiltration, loss of cellular integrity, villus and crypt deformities. Thus the biochemical, molecular and histological findings of the present study demonstrate that oxidative stress and inflammation play vital role in 5-FU induced GI toxicity and the inhibitory potential of vit. C is may be due to the modulation of oxidative stress, activation of redox sensitive transcription factor and also its downstream target molecules.

Epigenetic regulation of inflammatory gene expression in macrophages by selenium

Acetylation of histone and non-histone proteins by histone acetyltransferases plays a pivotal role in the expression of proinflammatory genes. Given the importance of dietary selenium in mitigating inflammation, we hypothesized that selenium supplementation may regulate inflammatory gene expression at the epigenetic level. The effect of selenium towards histone acetylation was examined in both in vitro and in vivo models of inflammation by chromatin immunoprecipitation assays and immunoblotting. Our results indicated that selenium supplementation, as selenite, decreased acetylation of histone H4 at K12 and K16 in COX-2 and TNFα promoters, and of the p65 subunit of the redox sensitive transcription factor NFκB in primary and immortalized macrophages. On the other hand, selenomethionine had a much weaker effect. Selenite treatment of HIV-1-infected human monocytes also significantly decreased the acetylation of H4 at K12 and K16 on the HIV-1 promoter, supporting the down-regulation of proviral expression by selenium. A similar decrease in histone acetylation was also seen in the colonic extracts of mice treated with dextran sodium sulfate that correlated well with the levels of selenium in the diet. Bone-marrow-derived macrophages from Trspfl/flCreLysM mice that lack expression of selenoproteins in macrophages confirmed the important role of selenoproteins in the inhibition of histone H4 acetylation. Our studies suggest that the ability of selenoproteins to skew the metabolism of arachidonic acid contributes, in part, to their ability to inhibit histone acetylation. In summary, our studies suggest a new role for selenoproteins in the epigenetic modulation of proinflammatory genes.

A dumbell probe-mediated rolling circle amplification strategy for highly sensitive transcription factor detection

Highly sensitive detection of transcription factors (TF) is essential to proteome and genomics research as well as clinical diagnosis. We describe herein a novel fluorescent-amplified strategy for ultrasensitive, quantitative, and inexpensive detection of TF. The strategy consists of a hairpin DNA probe containing a TF binding sequence for target TF, a dumbbell-shaped probe, a primer DNA probe designed partly complementary to hairpin DNA probe, and a dumbbell probe. In the presence of target TF, the binding of the TF with hairpin DNA probe will prohibit the hybridization of the primer DNA probe with the “stem” and “loop” region of the hairpin DNA probe, then the unhybridized region of the primer DNA will hybridize with dumbbell probe, subsequently promote the ligation reaction and the rolling circle amplification (RCA), finally, the RCA products are quantified via the fluorescent intensity of SYBR Green I (SG). Using TATA-binding protein (TBP) as a model transcription factor, the proposed assay system can specifically detect TBP with a detection limit as low as 40.7fM, and with a linear range from 100fM to 1nM. Moreover, this assay related DNA probe does not involve any modification and the whole assay proceeds in one tube, which makes the assay simple and low cost. It is expected to become a powerful tool for bioanalysis and clinic diagnostic application.

Abstract 572: Transcriptional Regulation Of Renal Dopamine D1 Receptor Function During Oxidative Stress

There exists a strong link between Oxidative stress, renal dopaminergic system and hypertension. It is reported that reactive oxygen species attenuate renal proximal tubular dopamine receptor (D1R) function which disrupts sodium regulation and leads to hypertension. The mechanisms for renal D1R dysfunction, however are not clear. Here in, we investigated the role of redox sensitive transcription factors AP1 and SP3 in transcriptional suppression of D1R gene function and subsequent D1R signaling. Human kidney proximal tubular ( HK2 ) cell were treated with a pro-oxidant L-buthionine sulfoximine ( BSO , 50 mM for 72 hr) with and without a polyphenol resveratrol (250 μM). Treatment of HK2 cells with BSO caused oxidative stress (malondialdehyde [MDA], nmol/mg protein: control: 0.13 ± 0.01, BSO: 0.20 ± .02) and significantly reduced D1R mRNA and membrane receptor number (fmol/mg protein; control—140.1 ± 5.6, BSO—81.7 ± 4.4). Incubation of HK2 cells with SKF38393, a D1R agonist, caused a concentration dependent inhibition of Na/K-ATPase ( 86 Rb uptake, nmol/mg protein, control: basal—9.2 ± 0.2, SKF (1 μM)—6.3 ± 0.1). However, SKF38393 failed to inhibit Na/K-ATPase in BSO-treated cells (BSO: basal—9.5 ± 0.3, SKF—8.6 ± 0.2). Cells incubated with BSO exhibited increased AP1 (c-fos) and SP3 nuclear expression. Resveratrol per se had no effect on oxidative milieu (MDA: 0.11 ± 0.01) or D1R expression (143.3 ± 4.4), but it mitigated BSO-induced oxidative stress (MDA: 0.13 ± 0.02), AP1—SP3 up-regulation and D1R down-regulation (133.6 ± 6.2) and dysfunction (resveratrol + BSO [ 86 Rb uptake]: basal—9.7 ±0.3, SKF—6.7 ± 0.4). Incubation of cells with AP1 or SP3 specific siRNA reduced the expression of respective protein by ~85% and abolished BSO-induced D1R dysfunction. Down-regulation of SP3 had no effect on BSO-mediated AP1 activation, however, AP1 siRNA blocked BSO-dependent SP3 up-regulation. In cells transfected with Nrf2 siRNA, resveratrol failed to block BSO-induced AP1—SP3 activation and D1R dysfunction. These data shows that oxidative stress via AP1—SP3 activation suppresses D1R transcription which leads to D1R dysfunction. Resveratrol activates Nrf2, mitigates oxidative stress, blocks AP1—SP3 signaling and prevents D1R down-regulation and dysfunction.

All-optical regulation of gene expression in targeted cells.

Controllable gene expression is always a challenge and of great significance to biomedical research and clinical applications. Recently, various approaches based on extra-engineered light-sensitive proteins have been developed to provide optogenetic actuators for gene expression. Complicated biomedical techniques including exogenous genes engineering, transfection, and material delivery are needed. Here we present an all-optical method to regulate gene expression in targeted cells. Intrinsic or exogenous genes can be activated by a Ca2+-sensitive transcription factor nuclear factor of activated T cells (NFAT) driven by a short flash of femtosecond-laser irradiation. When applied to mesenchymal stem cells, expression of a differentiation regulator Osterix can be activated by this method to potentially induce differentiation of them. A laser-induced “Ca2+-comb” (LiCCo) by multi-time laser exposure is further developed to enhance gene expression efficiency. This noninvasive method hence provides an encouraging advance of gene expression regulation, with promising potential of applying in cell biology and stem-cell science.

Involvement of oxidative stress, Nuclear Factor kappa B and the Ubiquitin proteasomal pathway in dysferlinopathy

AimsDysferlinopathies are autosomal recessive neuromuscular disorders arising from mutations of the protein dysferlin that preferentially affect the limbs which waste and weaken. The pathomechanisms of the diseases are not known and effective treatment is not available. Although free radicals and upstream signaling by the redox sensitive transcription factor, NF-κB, in activation of the ubiquitin pathway are shown to occur in several muscle wasting disorders, their involvement in dysferlinopathy is not known. This study analyzed the role of oxidative stress, NF-κB and the ubiquitin pathway in dysferlinopathic muscle and in dysferlin knockdown human myoblasts and myotubes. Main methodsFourteen dysferlinopathic muscle biopsies and 8 healthy control muscle biopsies were analyzed for oxidative stress, NF-κB activation and protein ubiquitinylation and human primary myoblasts and myotubes knocked down for dysferlin were studied for their state of oxidative stress. Key findingsDysferlinopathic muscle biopsies showed NF-κB p65 signaling induced protein ubiquitinylation in response to oxidative stress. Dysferlin knock down primary muscle cell cultures confirmed that oxidative stress is induced in the absence of dysferlin in muscle. SignificanceAnti-oxidants that also inhibit nitrosative stress and NF-κB activation, might prove to be of therapeutic benefit in slowing the progression of muscle wasting that occurs with dysferlinopathy.

Extracellular redox state regulates catecholamine biosynthesis in PC12 cells with insulin resistance.

Extracellular cysteine (Cys)/cystine (CySS) redox potential (Eh) plays a crucial role in maintaining redox homeostasis and an alteration of redox state occurs in various physiological conditions, including diabetes, cancer, and aging. This study was designed to determine whether a variation in extracellular redox state would alter the function of insulin-resistant PC12 cells. Various redox states were established by providing different extracellular Cys/CySS Eh to insulin-resistant PC12 cells. We intensively investigated the relationship between redox state and catecholamine biosynthesis in PC12 cells, and evaluated the changes in cellular reactive oxygen species (ROS), catecholamine (CA) synthesis, tyrosine hydroxylase (TH) expressions, and the activity of rate-limiting enzyme in CA synthesis by using DCF-fluorescence, HPLC, and the real-time PCR, respectively. We also determined the protein levels of NF-E2-related factor 2 (Nrf2), a redox sensitive transcription factor, using an ELISA assay. We found that the oxidized Cys/CySS Eh (0 mV) pretreatment decreased CA, TH, and Nrf2 levels, but induced ROS overproduction. Insulin induced a significant increase in CA synthesis and ROS production, blocked by more reducing redox conditions. The paradox of CA and TH alterations between insulin and 0 mV groups may be attributed to degree of redox imbalance as evidenced by different ROS levels in 2 groups, which is further confirmed by CA alterations in different concentrations of hydrogen peroxide. Additionally, dithiole-3-thione (D3T, an inducer of Nrf2) corrected 0 mV-induced TH inhibition. In conclusion, CA biosynthesis in insulin-resistant PC12 cells could be influenced by extracellular Cys/CySS redox effects on cellular redox sensitive transcription factors.

0020: Role of HIF-1 in the cardiac remodeling and inotropic effects of chronic intermittent hypoxia

Obstructive sleep apnea (OSA) is an important risk factor for cardiovascular morbidity and mortality. Chronic intermittent hypoxia (IH), a major component of OSA, has been shown to induce cardiovascular complications (hypertension, vascular remodelling, increased infarct size). We have shown that the hypoxia sensitive transcription factor HIF-1 is involved in the cardiovascular effects of IH. The α subunit of HIF-1 is degraded in normoxia (N) following prolyl hydroxylation by prolyl hydroxylase domain containing enzymes (PHD1, PHD2 and PHD3) while it is stabilized by PHD inactivation during hypoxia. The aim of the present study was to further explore the myocardial effects of IH and HIF-1 by assessing cardiac function and remodelling in mice models overexpressing or under expressing HIF-1α. For this, the response to IH was investigated in two strains of mice partially deficient for the genes encoding for PHD2 and HIF-1α, respectively. Mice were exposed in their cages, 8 h/day during 14 days, to 1-min cycles of IH (30s of 5% FiO2 followed by 30s of room air) or N (similar cycles of room air only). After exposure, the Fulton index (RV/LV+S) was evaluated in all experimental groups and cardiac contractility was assessed in heterozygous HIF-1α null mice and their respective controls. Exposure to IH induced right ventricular hypertrophy in control animals (Fulton index: 0.298 ± 0.010 vs 0.264 ± 0.036 in N mice). This effect of IH was enhanced by partial PHD-2 deletion (0.329 ± 0.014 vs 0.270 ± 0.014 in control mice) and abolished in heterozygous HIF-1α null mice (0.284 ± 0.008 vs 0.298 ± 0.012 in control mice). We also observed a significant increase in left ventricular developed pressure and maximal dP/dt after IH exposure in WT mice. This effect was absent in mice with partial HIF-1 deficiency.In conclusion, this study confirms the role of HIF-1 in the deleterious cardiovascular adaptation to IH leading to cardiac remodelling and increased contractility.

Regulation of iron acquisition and storage in yeast (360.1)

Iron is a required element for all eukaryotes, as the facile ability of iron to gain and loose electrons permits it to participate in a wide variety of oxidation‐reduction reactions. Iron can also donate electrons to oxygen or hydrogen peroxide resulting in the generation of toxic oxygen radicals such as, superoxide anion or hydroxyl radical. Organisms therefore tightly regulate cytosolic iron concentration through regulation of iron acquisition and storage. The budding yeast Saccharomyces cerevisiae has developed mechanisms to coordinate iron storage with iron acquisition. Under iron scarce conditions the transcriptional regulators Aft/2 induces the expression of a suite of genes that encode proteins involved in iron acquisition and utilization. Under iron sufficient conditions Aft1/2 regulated genes are no longer transcribed and a different transcription factor Yap5 induces a set of genes including CCC1, which encodes a vacuolar iron importer. Import of iron into the vacuole reduces cytosolic iron levels. Coordination of the products of these transcription factors is regulated by both transcriptional and post‐transcriptional mechanisms. Most notably, the high and low iron sensitive transcription factors both respond to the mitochondrial production of iron‐sulfur clusters. While there are differences in the molecules responsible for cytosolic iron regulation, coordination of iron acquisition and storage is a consistent finding in all species.

Mitochondrial superoxide mediates TNF‐α‐induced myokine release from C2C12 myotubes (1153.4)

TNF‐α is a key inflammatory mediator and is proposed to induce transcriptional responses via the generation of Reactive Oxygen Species (ROS). The aim of this study was to determine the role of mitochondrial superoxide generation in TNF‐α ‐ induced generation of myokines by skeletal muscle. Significant elevation in the release of IL‐6 (control untreated: 20.1pg/ml ± 2.3; TNF‐α treated: 73.8pg/ml ± 9.3), CCL2/MCP‐1 (980pg/ml ± 74.8; 31194pg/ml ± 5409), CCL5/RANTES (22.4pg/ml ± 2.09; 604pg/ml ± 93.4) and CXCL1/KC (236pg/ml ± 32.6; 3477pg/ml ± 383) and in superoxide, localised to the mitochondria were seen in response to TNF‐α treatment of muscle cells and this was associated with activation of NFκB. The TNF‐α mediated changes in superoxide, activation of NFκB and increased release of myokines were attenuated following pre‐treatment with the mitochondria targeted antioxidant, SS‐31 peptide (IL‐6, 11.9pg/ml ± 1.83; MCP‐1, 11322pg/ml ± 1124; RANTES, 252pg/ml ± 51.4; KC, 1166pg/ml ± 204) indicating that the ability of TNF‐α to induce myokine release is mediated through mitochondrial superoxide, which is, at least in part, associated with activation of the redox sensitive transcription factor NFκB. These novel findings provide evidence that targeting mitochondrial ROS could provide a potential for therapy for inflammatory myopathies. The authors thank the Medical Research Council and National Institute on Aging (grant AG‐020591) for their generous financial support and Dr M.B Reid (University of Kentucky Center for Muscle Biology) for the kind gift of the SS‐31 peptide.

HIF-1α- and hypoxia-dependent immune responses in human CD4+CD25high T cells and T helper 17 cells

The central oxygen sensitive transcription factor HIF-1α has been implicated in the differentiation of n(T(reg)) and Th17 cells and to orchestrate metabolic changes of activated T cells. However, data on the functional relevance of HIF-1α and Hox, in general, for nT(reg)-suppressive activity and T cell function in primary human cells are still missing. Therefore, we analyzed the effect of Hox and HIF-1α on human T(res), n(Treg), and Th17 cells. Under Hox, nT(reg)-mediated suppression of T(res) proliferation, CD25 expression, and secretion of IFN-γ were significantly reduced, whereas expression levels of VEGF, TNF-α, and IL-10 were significantly increased. In contrast to observations in mice, Th17 lineage commitment, as determined by RORγt expression, was not affected by activation or inhibition of HIF-1α expression using DMOG or YC-1 treatment, respectively. Nevertheless, the secretion of IL-17A was increased by DMOG and reduced by YC-1 under Th17-skewing conditions in a dose- dependent manner. In conclusion, Hox and HIF-1α substantially influence human T cell-mediated immune responses by modulation of nT(reg)-suppressive function and IL-17A secretion by Th17 cells.

Phospho-aspirin (MDC-22) inhibits breast cancer in preclinical animal models: an effect mediated by EGFR inhibition, p53 acetylation and oxidative stress

BackgroundThe anticancer properties of aspirin are restricted by its gastrointestinal toxicity and its limited efficacy. Therefore, we synthesized phospho-aspirin (PA-2; MDC-22), a novel derivative of aspirin, and evaluated its chemotherapeutic and chemopreventive efficacy in preclinical models of triple negative breast cancer (TNBC).MethodsEfficacy of PA-2 was evaluated in human breast cancer cells in vitro, and in orthotopic and subcutaneous TNBC xenografts in nude mice. Mechanistic studies were also carried out to elucidate the mechanism of action of PA-2.ResultsPA-2 inhibited the growth of TNBC cells in vitro more potently than aspirin. Treatment of established subcutaneous TNBC xenografts (MDA-MB-231 and BT-20) with PA-2 induced a strong growth inhibitory effect, resulting in tumor stasis (79% and 90% inhibition, respectively). PA-2, but not aspirin, significantly prevented the development of orthotopic MDA-MB-231 xenografts (62% inhibition). Mechanistically, PA-2: 1) inhibited the activation of epidermal growth factor receptor (EGFR) and suppressed its downstream signaling cascades, including PI3K/AKT/mTOR and STAT3; 2) induced acetylation of p53 at multiple lysine residues and enhanced its DNA binding activity, leading to cell cycle arrest; and 3) induced oxidative stress by suppressing the thioredoxin system, consequently inhibiting the activation of the redox sensitive transcription factor NF-κB. These molecular alterations were observed in vitro and in vivo, demonstrating their relevance to the anticancer effect of PA-2.ConclusionsOur findings demonstrate that PA-2 possesses potent chemotherapeutic efficacy against TNBC, and is also effective in its chemoprevention, warranting further evaluation as an anticancer agent.

Tinospora cordifolia extract modulates COX-2, iNOS, ICAM-1, pro-inflammatory cytokines and redox status in murine model of asthma

Ethnopharmacological relevanceTinospora cordifolia (Willd.) Miers is an important constituent of several ayurvedic medicinal preparations. In Ayurveda it is mentioned as “rasayan” and traditionally used for the treatment of asthma, chronic cough besides other ailments. This study was carried out to study the mechanisms involved in protection accorded by extract of Tinospora cordifolia (Tc) stem to asthmatic mice by regulation of oxidative stress, pro-inflammatory mediator release and redox signaling involving NFκB. Materials and methodsBALB/c mice were sensitized with intraperitoneal (i.p.) Ovalbumin (Ova) on days 0 and 14, followed by intranasal Ovalbumin (Ova) challenge on days 24 and 27 to generate an in vivo asthma model. Tc extract (hydroalcoholic, 100mg/kg) and dexamethasone (1mg/kg) were given orally from day 15 to 23 to the Tc+Ova treated group and Dex+Ova treated group respectively. Oxidative stress parameters e.g. activity of superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx) and catalase, lipid peroxidation, GSH/GSSG ratio, protein carbonyl content, eosinophil peroxidase, myeloperoxidase activity, and NO release were measured in tissue, blood and bronchoalveolar lavage fluid (BALF). Estimation of cytokines was done in BALF. Western blot analysis was done for IκB α, iNOS, COX-2, iCAM-1 and pJNK MAPKs along with histopathology. ResultsTc extract treated mice showed decreased airway hyper-responsiveness, eosinophil count and IgE content in blood as compared to Ova treated asthmatic mice. Increase in activities of SOD, catalase, glutathione reductase, glutathione peroxidase as well as GSH/GSSG ratio was observed while a decrease in MDA formation, protein carbonyl content, eosinophil peroxidase, myeloperoxidase activity and NO release in BALF was seen in Tc treated mice. In BALF, levels of cytokines IL-4 and TNF-α were reduced and IFN-γ levels increased in extract treated mice. At the same time Tc treatment of Ova-challenged mice significantly increased the level of IκB α, cytosolic inhibitor of redox sensitive transcription factor NFκB. Immunoblot analysis revealed considerable decrease in the levels of COX-2, ICAM-1, iNOS, and pJNK. Histopathology and PAS staining also indicate a protective effect of Tc extract in inflammation and mucus hyper-secretion due to goblet cell hyperplasia. ConclusionThe results suggest a protective effect of Tc extract against oxidative stress, pro-inflammatory mediator release and redox signaling in the murine model of asthma. The Tc extract shows therapeutic potential for management of asthmatic inflammation and other lung inflammatory conditions.