NRF2-mediated Oxidative Stress Response Research Articles

1 PATHWAYS AND CELLULAR FUNCTIONS INFLUENCED BY INSULIN TREATMENT DURING OOCYTE MATURATION – A TRANSCRIPTOME STUDY OF IN VITRO-PRODUCED BOVINE DAY 8 BLASTOCYSTS

Insulin as a key metabolic hormone has crucial functions in metabolic regulation in all mammals. Deviation of its physiological concentration occurs in metabolic disorders as obesity and diabetes in humans or negative energy balance and overfeeding in the cow. As these metabolic disorders are strongly correlated with reproductive disturbances, we investigated the effect of insulin during oocyte maturation on gene expression of bovine Day 8 blastocysts (BC8) by transcriptome analysis. Abattoir-derived oocytes (n = 882) were divided into 3 groups and in vitro matured for 22 h by adding insulin (H: High 10 µg mL–1; L: Low 0.1 µg mL–1 and Z: Zero, control). This was followed by standard in vitro production (IVP) and evaluation of developmental rates up to blastocyst stage. BC8 (n = 120) were pooled in groups of 10 and total RNA was extracted by parallel gDNA and total RNA-extraction (AllPrepDNA/RNA micro kit, cat no. 80284, Qiagen®, Valencia, CA, USA) for analyses of the transcriptome. All samples (4 biological replicates/group) resulted in RIN-values >7.5. RNA amplification, cDNA synthesis, purification, and labelling were performed and 825 ng of Cy3- and Cy5-labelled linearly amplified aRNA was hybridized on the Agilent-manufactured EmbryoGENE-slides in a 2-colour dye swap design. An empirical Bayes moderated t-test was applied to search for the differentially expressed transcripts (DET) between control and insulin-treated groups, using the ‘limma’ package in R (www.r-project.org). The DET were defined as having a 1.5-fold change difference between treatment and control and P < 0.05. Pathways and molecular functions influenced by insulin treatment were analysed by using Ingenuity Pathway Analysis (IPA; Ingenuity® Systems, www.ingenuity.com). As a global pattern, insulin treatment induced an up-regulation of genes. In total, 202 DET in the H and 142 DET in the L group were found where 104 DET were common in both insulin groups. Fifteen selected candidate genes chosen for qPCR validation and 12 (80%) showed similar expression patterns as the microarray data. DET relevant for following cellular functions were found in H: Cell Cycle, Cellular Compromise, Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry respective L: Cell Morphology, Cellular Growth and Proliferation, Cell Cycle, Carbohydrate Metabolism and Cellular Assembly and Organization. The top canonical pathways influenced were Epithelial Adherens Junction Signalling and Remodelling, Germ Cell Sertoli Cell Junction Signalling and NRF2-mediated Oxidative Stress Response. Correlatively, blastocyst rates on Day 8 were significantly lower in H and L v. Z (P < 0.05). The transcriptome data could explain the mechanisms behind the impaired development, as genes involved in cellular growth and energy metabolism in Day 8 blastocysts were affected. The fact that transcripts related to NRF2-mediated oxidative stress response and lipid metabolism are up-regulated suggests that insulin induces dysregulation of cellular functions and energy metabolism leading to impaired embryo developmental potential.Funded by FORMAS.

Proteomic analysis of the effects of aged garlic extract and its FruArg component on lipopolysaccharide-induced neuroinflammatory response in microglial cells.

Aged garlic extract (AGE) is widely used as a dietary supplement, and is claimed to promote human health through anti-oxidant/anti-inflammatory activities with hypolipidemic, antiplatelet and neuroprotective effects. Prior studies of AGE have mainly focused on its organosulfur compounds, with little attention paid to its carbohydrate derivatives, such as N-α-(1-deoxy-D-fructos-1-yl)-L-arginine (FruArg). The goal of this study is to investigate actions of AGE and FruArg on antioxidative and neuroinflammatory responses in lipopolysaccharide (LPS)-activated murine BV-2 microglial cells using a proteomic approach. Our data show that both AGE and FruArg can significantly inhibit LPS-induced nitric oxide (NO) production in BV-2 cells. Quantitative proteomic analysis by combining two dimensional differential in-gel electrophoresis (2D-DIGE) with mass spectrometry revealed that expressions of 26 proteins were significantly altered upon LPS exposure, while levels of 20 and 21 proteins exhibited significant changes in response to AGE and FruArg treatments, respectively, in LPS-stimulated BV-2 cells. Notably, approximate 78% of the proteins responding to AGE and FruArg treatments are in common, suggesting that FruArg is a major active component of AGE. MULTICOM-PDCN and Ingenuity Pathway Analyses indicate that the proteins differentially affected by treatment with AGE and FruArg are involved in inflammatory responses and the Nrf2-mediated oxidative stress response. Collectively, these results suggest that AGE and FruArg attenuate neuroinflammatory responses and promote resilience in LPS-activated BV-2 cells by suppressing NO production and by regulating expression of multiple protein targets associated with oxidative stress.

Impact of exposure to low concentrations of nitric oxide on protein profile in murine and human pancreatic islet cells

Homeostatic levels of nitric oxide (NO) protect efficiently against apoptotic death in both human and rodent pancreatic β cells, but the protein profile of this action remains to be determined. We have applied a 2 dimensional LC-MS-MALDI-TOF/TOF-based analysis to study the impact of protective NO in rat insulin-producing RINm5F cell line and in mouse and human pancreatic islets (HPI) exposed to serum deprivation condition. 24 proteins in RINm5F and 22 in HPI were identified to undergo changes in at least one experimental condition. These include stress response mitochondrial proteins (UQCRC2, VDAC1, ATP5C1, ATP5A1) in RINm5F cells and stress response endoplasmic reticulum proteins (HSPA5, PDIA6, VCP, GANAB) in HPI. In addition, metabolic and structural proteins, oxidoreductases and chaperones related with protein metabolism are also regulated by NO treatment. Network analysis of differentially expressed proteins shows their interaction in glucocorticoid receptor and NRF2-mediated oxidative stress response pathways and eNOS signaling. The results indicate that exposure to exogenous NO counteracts the impact of serum deprivation on pancreatic β cell proteome. Species differences in the proteins involved are apparent.

Abstract 3382: Nifurtimox enhances the therapeutic efficacy of radiation by inducing reactive oxygen species and related pathways: Pre-clinical study in medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children, accounting for about 20% of pediatric CNS tumors. Multimodality treatments including surgery, radiotherapy (XRT) and chemotherapy have greatly improved disease outcome but survivors often suffer from deleterious effects of aggressive treatment with XRT. The aim of this study is to use novel strategies to enhance the response of XRT in a pre-clinical model for MB. XRT is known to cause DNA damage and cell death by inducing reactive oxygen species (ROS). Induction of ROS causes perturbation in antioxidant defense system thereby causing cell damage and death. Therefore, induction of ROS may serve as a promising strategy in cancer therapy. Nifurtimox (Nfx), a nitrofuran compound used to treat the parasitic infection Chagas’ disease, is known to induce ROS in pre-clinical models for neuroblastoma (NB) and MB. It is currently in clinical trials for treating NB and MB in children. In this study, we investigated the effects of Nfx in augmenting the efficacy of XRT in MB cell lines. DAOY and D283 cells were treated with varying dose of Nfx (5-70 µg/ml) or XRT (2-10 Gy) and cell viability was assessed using CellTiter-Glo for up to 5 days. The effect of combination therapy on cell viability, apoptosis, ROS levels and catalase activity was evaluated using optimized dose of Nfx and XRT. Results revealed that combination treatment significantly inhibited MB cell growth when compared to the effect of either Nfx or XRT alone. This was accompanied by the activation of apoptotic cell death as determined by increased annexin V staining and caspase 3/7 activity. An increase in ROS was also observed following combination treatment with Nfx and XRT which correlated with cell growth inhibition. Interestingly, catalase activity was unaltered following the treatment with single or double agents suggesting a contribution of impaired antioxidant system in causing MB cell growth inhibition. To further delineate the underlying mechanisms and to identify other candidates modulated by this combination therapy, we undertook molecular profiling approach using Affimetrix gene expression arrays (Human Gene 2.0 ST Array). We examined differentially expressed genes and affiliated pathways, and biological processes underlying tumor cell responses to Nfx and XRT. Our results identified genes that are statistically enriched for biological functions related to tumorigenesis, including cancer cell death and survival, cell cycle, and other functions relating to cancer disease. Several transcription factors and their downstream targets were also found to be enriched including the NRF2-mediated oxidative stress response pathway that responds to various stimuli including ROS. In conclusion, this pre-clinical study demonstrates that Nfx has the potential to enhance the therapeutic efficacy of radiation in MB cell lines. Citation Format: Don Eslin, Umesh T. Sankpal, Chris M. Lee, Giselle Saulnier Sholler, Robert M. Sutphin, Paul Bowman, Jeffrey C. Murray, Riyaz M. Basha. Nifurtimox enhances the therapeutic efficacy of radiation by inducing reactive oxygen species and related pathways: Pre-clinical study in medulloblastoma. [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 3382. doi:10.1158/1538-7445.AM2014-3382

MDA-7/IL-24 inhibits Nrf2-mediated antioxidant response through activation of p38 pathway and inhibition of ERK pathway involved in cancer cell apoptosis.

Reactive oxygen species (ROS) have a crucial role in melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24 (IL-24)-induced cancer cell apoptosis. However, cancer cell has a series of protective mechanisms to resist ROS damage. Nuclear factor erythroid 2-related factor 2 (Nrf2) activates antioxidant response element (ARE)-mediated gene expression involved in cellular protection against oxidative stress. As the Nrf2 repressor, Kelch-like ECH-associated protein-1 (Keap1) sequesters Nrf2 in cytoplasm to block Nrf2 nuclear translocation. In the present study, administration of MDA-7/IL-24 by means of tumor-selective replicating adenovirus (ZD55-IL-24) was used to investigate whether ZD55-IL-24 could attenuate Nrf2-mediated oxidative stress response in cancer cell. We found that ZD55-IL-24 effectively strengthened the association between Nrf2 and Keap1 to restrict Nrf2 nuclear translocation, thereby inhibiting ARE-dependent transcriptional response. To evaluate the detailed mechanism underlying the suppression of ZD55-IL-24 on Nrf2-mediated oxidative stress response, we further tested three different mitogen-activated protein kinase (MAPK) signaling pathways in A549 and HeLa cells transfected by ZD55-IL-24. Our data showed that ZD55-IL-24 inhibited extracellular signal-regulated kinase (ERK) signal pathway but activated p38 and c-Jun-NH2-kinase (JNK) signal pathways to exert the tumor-specific apoptosis. Moreover, ERK pathway inhibitor U0126 prevented Nrf2 phosphorylation at Ser40 to retard Nrf2 nuclear translocation, thus decreasing antioxidant gene transcription. In contrast, p38 pathway inhibitor SB203580 obviously promoted the dissociation of Nrf2 from Keap1 to promote antioxidant gene transcription. However, JNK pathway had no effect on Nrf2 subcellular localization or the association of Nrf2 with Keap1. Conclusively, our results indicate that ZD55-IL-24 inhibits Nrf2-mediated oxidative stress response not only by activating p38 signal pathway to potentiate the association of Nrf2 and Keap1 but also by suppressing ERK signal pathway to postpone Nrf2 nuclear translocation. Given the 'dark' side of Nrf2 on carcinoma cell survival and chemoresistance, our study provides a novel explanation about MDA-7/IL-24-induced cancer-specific apoptosis and therapeutic sensitization through suppression of the cytoprotective system.

Integrative biology identifies shared transcriptional networks in CKD.

A previous meta-analysis of genome-wide association data by the Cohorts for Heart and Aging Research in Genomic Epidemiology and CKDGen consortia identified 16 loci associated with eGFR. To define how each of these single-nucleotide polymorphisms (SNPs) could affect renal function, we integrated GFR-associated loci with regulatory pathways, producing a molecular map of CKD. In kidney biopsy specimens from 157 European subjects representing nine different CKDs, renal transcript levels for 18 genes in proximity to the SNPs significantly correlated with GFR. These 18 genes were mapped into their biologic context by testing coregulated transcripts for enriched pathways. A network of 97 pathways linked by shared genes was constructed and characterized. Of these pathways, 56 pathways were reported previously to be associated with CKD; 41 pathways without prior association with CKD were ranked on the basis of the number of candidate genes connected to the respective pathways. All pathways aggregated into a network of two main clusters comprising inflammation- and metabolism-related pathways, with the NRF2-mediated oxidative stress response pathway serving as the hub between the two clusters. In all, 78 pathways and 95% of the connections among those pathways were verified in an independent North American biopsy cohort. Disease-specific analyses showed that most pathways are shared between sets of three diseases, with closest interconnection between lupus nephritis, IgA nephritis, and diabetic nephropathy. Taken together, the network integrates candidate genes from genome-wide association studies into their functional context, revealing interactions and defining established and novel biologic mechanisms of renal impairment in renal diseases.

Cytoprotection of human endothelial cells against oxidative stress by 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im): Application of systems biology to understand the mechanism of action

Cellular damage from oxidative stress, in particular following ischemic injury, occurs during heart attack, stroke, or traumatic injury, and is potentially reducible with appropriate drug treatment. We previously reported that caffeic acid phenethyl ester (CAPE), a plant-derived polyphenolic compound, protected human umbilical vein endothelial cells (HUVEC) from menadione-induced oxidative stress and that this cytoprotective effect was correlated with the capacity to induce heme oxygenase-1 (HMOX1) and its protein product, a phase II cytoprotective enzyme. To further improve this cytoprotective effect, we studied a synthetic triterpenoid, 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), which is known as a potent phase II enzyme inducer with antitumor and anti-inflammatory activities, and compared it to CAPE. CDDO-Im at 200nM provided more protection to HUVEC against oxidative stress than 20μM CAPE. We explored the mechanism of CDDO-Im cytoprotection with gene expression profiling and pathway analysis and compared to that of CAPE. In addition to potent up-regulation of HMOX1, heat shock proteins (HSP) were also found to be highly induced by CDDO-Im in HUVEC. Pathway analysis results showed that transcription factor Nrf2-mediated oxidative stress response was among the top canonical pathways commonly activated by both CDDO-Im and CAPE. Compared to CAPE, CDDO-Im up-regulated more HSP and some of them to a much higher extent. In addition, CDDO-Im treatment affected Nrf2 pathway more significantly. These findings may provide an explanation why CDDO-Im is a more potent cytoprotectant than CAPE against oxidative stress in HUVEC.

Bovine embryo survival under oxidative-stress conditions is associated with activity of the NRF2-mediated oxidative-stress-response pathway.

In present study, we sought to examine the ability of preimplantation bovine embryos to activate the NF-E2-related factor 2 (NRF2)-mediated oxidative-stress response under an oxidative stress environment. In vitro 2-, 4-, 8-, 16-cell-, and blastocyst-stage embryos were cultured under low (5%) or high (20%) oxygen levels. The expression of NRF2, KEAP1 (NRF2 inhibitor), antioxidants downstream of NRF2, and genes associated with embryo metabolism were analyzed between the embryo groups using real-time quantitative PCR. NRF2 and KEAP1 protein abundance, mitochondrial activity, and accumulation of reactive oxygen species (ROS) were also investigated in blastocysts of varying competence that were derived from high- or low-oxygen levels. The expression levels of NRF2 and its downstream antioxidant genes were higher in 8-cell, 16-cell, and blastocyst stages under high oxygen tension, whereas KEAP1 expression was down-regulated under the same conditions. Higher expression of NRF2 and lower ROS levels were detected in early (competent) blastocysts compared to their late (noncompetent) counterparts in both oxygen-tension groups. Similarly, higher levels of active nuclear NRF2 protein were detected in competent blastocysts compared to their noncompetent counterparts. Thus, the survival and developmental competence of embryos cultured under oxidative stress are associated with activity of the NRF2-mediated oxidative stress response pathway during bovine pre-implantation embryo development.

Physical activity and prostate gene expression in men with low-risk prostate cancer

Vigorous physical activity after diagnosis of localized prostate cancer may reduce the risk of disease progression and prostate cancer-specific mortality. The molecular mechanisms by which physical activity may exert protective effects in the prostate remain unknown. We examined the associations between self-reported physical activity and gene expression patterns in morphologically normal prostate tissue of 71 men with low-risk prostate cancer on active surveillance. Differential gene expression, gene set, and pathway analyses were conducted comparing dichotomous groups defined by type, intensity, and amount of physical activity reported. Cell cycling and DNA repair pathways were up-regulated in men who participated in ≥ 3 h/week vigorous activity compared with men who did not. In addition, canonical pathways involved in cell signaling and metabolism, the cellular effects of sildenafil (Viagra), and the Nrf2-mediated oxidative stress response were modulated in men who reported ≥ 3 h/week of vigorous activity. Differential expression analysis at the individual gene level revealed modest differences between men who performed vigorous activity for ≥ 3 h/week and those who did not. There were no differences in prostate gene expression in comparisons with exercise groupings that did not consider both duration and intensity of activity. Prostate gene expression and pathway analyses revealed sets of transcripts that may be modulated in normal prostate tissue by participating in ≥ 3 h/week of vigorous activity after diagnosis of low-risk prostate cancer. These findings suggest potential biological mechanisms by which vigorous activity may reduce risk of prostate cancer progression and warrant further study and validation.

N6-isopentenyladenosine and analogs activate the NRF2-mediated antioxidant response

N6-isopentenyladenosine (i6A), a naturally occurring modified nucleoside, inhibits the proliferation of human tumor cell lines in vitro, but its mechanism of action remains unclear. Treatment of MCF7 human breast adenocarcinoma cells with i6A or with three synthetic analogs (allyl6A, benzyl6A, and butyl6A) inhibited growth and altered gene expression. About 60% of the genes that were differentially expressed in response to i6A treatment were also modulated by the analogs, and pathway enrichment analysis identified the NRF2-mediated oxidative stress response as being significantly modulated by all four compounds. Luciferase reporter gene assays in transfected MCF7 cells confirmed that i6A activates the transcription factor NRF2. Assays for cellular production of reactive oxygen species indicated that i6A and analogs had antioxidant effects, reducing basal levels and inhibiting the H2O2- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced production in MCF7 or dHL-60 (HL-60 cells induced to differentiate along the neutrophilic lineage) cell lines, respectively. In vivo, topical application of i6A or benzyl6A to mouse ears prior to TPA stimulation lessened the inflammatory response and significantly reduced the number of infiltrating neutrophils. These results suggest that i6A and analogs trigger a cellular response against oxidative stress and open the possibility of i6A and benzyl6A being used as topical anti-inflammatory drugs.

Gene expressions changes in bronchial epithelial cells: Markers for respiratory sensitizers and exploration of the NRF2 pathway

For the classification of respiratory sensitizing chemicals, no validated in vivo nor in vitro tests are currently available. In this study, we evaluated whether respiratory sensitizers trigger specific signals in human bronchial epithelial (BEAS-2B) cells at the level of the transcriptome. The cells were exposed during 6, 10, and 24h to 4 respiratory sensitizers and 6 non-respiratory sensitizers (3 skin sensitizers and 3 respiratory irritants) at a concentration inducing 20% cell viability loss after 24h. Changes in gene expression were evaluated using Agilent Whole Human Genome 4×44K oligonucleotide arrays. A limited number of 11 transcripts could be identified as potential biomarkers to identify respiratory sensitizers. Three of these transcripts are associated to immune system processes (HSPA5, UPP1, and SEPRINE1). In addition, the transcriptome was screened for transcripts that are differentially expressed compared to vehicle control for each chemical. The results show that the NRF2-mediated oxidative stress response is activated in the cell line after stimulation with all of the chemicals that were selected in our study, and that – at the level of gene expression – this pathway shows no potential to discriminate between any of the three compound groups: respiratory sensitizers, skin sensitizers, or electrophilic respiratory irritants.

Trauma‐associated human neutrophil alterations revealed by comparative proteomics profiling

Polymorphonuclear neutrophils (PMNs) play an important role in mediating the innate immune response after severe traumatic injury; however, the cellular proteome response to traumatic condition is still largely unknown. We applied 2D-LC-MS/MS-based shotgun proteomics to perform comparative proteome profiling of human PMNs from severe trauma patients and healthy controls. A total of 197 out of ~2500 proteins (being identified with at least two peptides) were observed with significant abundance changes following the injury. The proteomics data were further compared with transcriptomics data for the same genes obtained from an independent patient cohort. The comparison showed that the protein abundance changes for the majority of proteins were consistent with the mRNA abundance changes in terms of directions of changes. Moreover, increased protein secretion was suggested as one of the mechanisms contributing to the observed discrepancy between protein and mRNA abundance changes. Functional analyses of the altered proteins showed that many of these proteins were involved in immune response, protein biosynthesis, protein transport, NRF2-mediated oxidative stress response, the ubiquitin-proteasome system, and apoptosis pathways. Our data suggest increased neutrophil activation and inhibited neutrophil apoptosis in response to trauma. The study not only reveals an overall picture of functional neutrophil response to trauma at the proteome level, but also provides a rich proteomics data resource of trauma-associated changes in the neutrophil that will be valuable for further studies of the functions of individual proteins in PMNs.

Integrative analysis of proteomic and transcriptomic data for identification of pathways related to simvastatin‐induced hepatotoxicity

Hepatocytes are used widely as a cell model for investigation of xenobiotic metabolism and the toxic mechanism of drugs. Simvastatin is the first statin drug used extensively in clinical practice for control of elevated cholesterol or hypercholesterolemia. However, it has also been reported to cause adverse effects in liver due to cellular damage. In this study, for proteomic and transcriptomic analysis, rat primary hepatocytes were exposed to simvastatin at IC20 concentration for 24 h. Among a total of 607 differentially expressed proteins, 61 upregulated and 29 downregulated proteins have been identified in the simvastatin-treated group. At the mRNA level, results of transcriptomic analysis revealed 206 upregulated and 41 downregulated genes in the simvastatin-treated group. Based on results of transcriptomic and proteomic analysis, NRF2-mediated oxidative stress response, xenobiotics by metabolism of cytochrome P450, fatty acid metabolism, bile metabolism, and urea cycle and inflammation metabolism pathways were focused using IPA software. Genes (FASN, UGT2B, ALDH1A1, CYP1A2, GSTA2, HAP90, IL-6, IL-1, FABP4, and ABC11) and proteins (FASN, CYP2D1, UG2TB, ALDH1A1, GSTA2, HSP90, FABP4, and ABCB11) related to several important pathways were confirmed by real-time PCR andWestern blot analysis, respectively. This study will provide new insight into the potential toxic pathways induced by simvastatin.

Oral administration of pentachlorophenol induces interferon signaling mRNAs in C57BL/6 male mouse liver

Pentachlorophenol (PCP) was monitored for transcriptome responses in adult mouse liver at 2, 4, 8 and 24 hr after a single oral administration at four dose levels, 0, 10, 30 and 100 mg/kg. The expression data obtained using Affymetrix GeneChip MOE430 2.0 were absolutized by the Percellome method and expressed as three dimensional (3D) surface graphs with axes of time, dose and copy numbers of mRNA per cell. We developed the programs RSort, for comprehensive screening of the 3D surface data and PercellomeExploror for cross-referencing and confirmed the significant responses by visual inspection. In the first 8 hr, approximately 100 probe sets (PSs) related to PXR/SXR and Cyp2a4 and other metabolic enzymes were induced whereas Fos and JunB were suppressed. At 24 hr, about 1,200 PSs were strongly induced. We cross-referenced the Percellome database consisting of 111 chemicals on the liver transcriptome and found that about half of the PSs belonged to the metabolic pathways including Nrf2-mediated oxidative stress response networks shared with some of the 111 chemicals. The other half of the induced genes were interferon signaling network genes (ISG) and their induction was unique to PCP. Toll like receptors and other pattern recognition receptors, interferon regulatory factors and interferon alpha itself were included but inflammatory cytokines were not induced. In summary, these data indicated that functional symptoms of PCP treatment, such as hyperthermia and profuse sweating might be mediated by the ISG rather than the previously documented mitochondrial uncoupling mechanism. PCP might become a hint for developing low molecular weight orally available interferon mimetic drugs following imiquimod and RO4948191 as agonists of toll-like receptor and interferon receptor.

207 CIRCULATORY microRNA SIGNATURES IN FOLLICULAR FLUID IN RELATION TO THE GROWTH STATUS OF BOVINE OOCYTES

Cell-to-cell communication within the follicle involves many signalling molecules, and this process is believed to be mediated by secretion and uptake of exosomes that contained several bioactive molecules including circulatory microRNAs (miRNA). The present study was conducted to investigate the circulating miRNA expression pattern in exosome and nonexosomal portion of follicular fluid (FF) in follicles with fully grown or growing oocytes. For this, the FF and cumulus–oocyte complexes (COC) were retrieved from 5- to 8-mm individual follicles from ovaries obtained from local abattoir. Then, the oocytes were subjected to brilliant cresyl blue (BCB) staining and classified as BCB+ (fully grown oocytes) and BCB– (growing oocytes). Accordingly, the corresponding FF was classified as BCB+ and BCB– based on their oocyte source. Following this, the exosomes were trapped from each FF categories using ExoQuick™ (SBI System Bioscience). Thus, total RNA was isolated from exosomal and nonexosomal portion of the FF using miRNeasy mini kit (Qiagen) and subjected to miRNA expression studies. The human miRCURY LNA™ Universal RT miRNA PCR array system (Exiqon) was used for miRNA expression profiling. Data analysis was performed using a comparative threshold cycle (ΔCT) method. The results revealed that 26 miRNAs were found to be differentially expressed (fold change ≥2 and P < 0.05) between the exosomal portion of FF from fully grown and growing oocyte groups. Among these, 17 miRNA including miR-608, miR-654-5p, miR-640, miR-582-5p, miR-449b, miR-155 were upregulated and 9 miRNA including miR-373, miR-526b*, miR-33a*, miR-30b, miR-29a* were downregulated in exosomal portion of FF of growing oocyte groups. The ingenuity pathway analysis of genes predicted to be targeted by those miRNAs were found to be involved in WNT/β catenine, purine metabolism, protein ubiquitination, and cAMP-mediated signalling pathways. Similarly, 36 miRNA were differentially expressed between the non-exosomal portion of FF of fully grown and growing oocytes. From those, 27 miRNA including let-7i*, miR-328, miR-223, miR-19b-1*, miR-423-5p, miR-29c, miR-659 were upregulated, whereas the expression level of 9 miRNA including miR-381, miR-18a*, miR-30e*, miR-934, and miR-302c was downregulated in the nonexosomal portion of FF of growing oocyte groups. In addition, the ingenuity pathway analysis indicated that the genes predicted to be targeted by these miRNA were found to be involved in NRF2-mediated oxidative stress response, tight junction signalling, and protein ubiquitination pathways. In conclusion, this study detected the presence of exosome or non-exosome-mediated circulation of miRNA in the bovine follicular fluid and oocyte growth-dependent variation of circulatory miRNA in the follicular environment.

Transcriptome Fingerprint of Bovine 2-Cell Stage Blastomeres Is Directly Correlated with the Individual Developmental Competence of the Corresponding Sister Blastomere1

To date, gene expression profiles of bovine preimplantation embryos have only been indirectly related to developmental potential due to the invasive nature of such procedures. This study sought to find a direct correlation between transcriptome fingerprint of blastomeres of bovine 2-cell stage embryos with developmental competence of the corresponding sister blastomeres. Isolated blastomeres were classified according to the sister blastomere's development into three groups: two groups displayed developmental incompetency, including those blastomeres whose corresponding sister blastomeres either stopped cleaving after separation (2CB) or were blocked after two additional cleavages before embryonic genome activation (8CB). In the third group were competent blastomeres, which were defined as those whose sister blastomeres developed to the blastocyst stage (BL). As a result, developmental capacity of corresponding sister blastomeres was highly similar. Microarray analysis revealed 77 genes to be commonly differentially regulated among competent and incompetent blastomeres as well as blocked blastomeres. Clustering of differentially expressed genes according to molecular functions and pathways revealed antioxidant activity, NRF2-mediated oxidative stress response, and oxidative phosphorylation to be the main ontologies affected. Expression levels of selected candidate genes were further characterized in an independent model for developmental competence based on the time of first cleavage postfertilization. Moreover, overall results of this study were confirmed by higher developmental rates and more beneficial expression of CAT and PRDX1 when cultured in an antioxidative environment. These results will help us to understand molecular mechanisms defining developmental destination of individual bovine preimplantation embryos.

The Gold Compound Auranofin Induces Oxidative Stress and Apoptosis in Primary CLL Cells Independent of Classic Prognostic Markers and the Protective Effect of the Tissue Microenvironment

AbstractAbstract 865Chronic lymphocytic leukemia (CLL), remains incurable, except by allogeneic stem cell transplantation. While multiple chemotherapeutic options are available, relapse and eventually development of chemotherapy resistance are common. Drug resistance of CLL cells is mediated by genetic alterations including deletion of chromosomes 17p (p53 locus) and 11q (ATM locus), and by pro-survival signals emanating from the tissue microenvironment. Thus new therapies are needed that are active against tumor cells in protective tissue sites and those with adverse cytogenetic features. To identify possible new therapies for CLL, we conducted a high throughput screening assay of primary CLL cells exposed to 7 step-wise dilutions of each member of a 2,816 compound library (including FDA-approved drugs and known bio-actives from commercial suppliers). As a control for “unspecific” cytotoxicity, we used PBMCs from normal donors. Of 102 compounds efficacious against CLL cells of all 6 patients tested, 5 were not or minimally toxic in normal lymphocytes. One of these 5 compounds is Auranofin (AF), an FDA-approved oral disease-modifying anti-rheumatic drug.Next, we investigated the efficacy of AF against an expanded cohort of CLL samples (n=50) and investigated its mechanism of action. Samples were characterized for IGHV mutational status, ZAP70 expression, CD38 expression, and FISH cytogenetics. PBMCs were tested against serial dilutions of AF (final concentrations: 0.125–4μM) for 24 hours in flat bottom 96-well plates. MTS assay was performed to quantify cell viability. A dose-dependent response was observed in all samples. The inhibitory concentration at 50% (IC50) was calculated using GraphPad Prism Software. For a majority of samples, the IC50 was <1μM, a concentration easily achieved in vivo. AF cytotoxicity was independent of the IGHV mutational status, ZAP70 and CD38 expression, and cytogenetic subgroups (including 17p and 11q deletion). To assess whether AF is selectively cytotoxic for CLL cells, we determined drug-induced apoptosis of PBMCs of CLL patients for CLL cells (identified as CD19+/CD3-; >95% of those are CLL cells) and T-cells (CD19-/CD3+) separately using Annexin V staining. AF reduced viability of T-cells on average by 10% and of CLL cells by 50% (n=8; P<.05 for comparison between T and CLL cells). To determine the effect of the microenvironment on AF toxicity, we cultured primary CLL cells in the presence or absence of Nurse-Like Cells (NLC), a model of the protective effect of the microenvironment. NCLs enhance CLL cell viability and confer resistance to chemotherapeutic agents such as fludarabine. However, co-culture on NLCs did not protect CLL cells from the cytotoxic effect of AF. To investigate the effect of AF on tumor biology, total RNA (2.5 μg) from CLL PBMCs treated for 4 and 10 hours with AF in vitro was profiled on Human Genome U133 Plus 2.0 arrays (Affymetrix) and compared to untreated controls. There were 81 genes whose expression changed >2-fold at P<.01, most prominently several genes encoding heat-shock proteins and antioxidant enzymes such as heme oxygenase and thiroredoxin reductase. In Ingenuity Pathway analysis, the most significantly upregulated pathway in response to AF was the NRF2-mediated Oxidative Stress Response (P<.001). Combined, this suggests that AF induced oxidative stress resulting in the upregulation of a detoxifying response. To study the effect of AF on redox balance we used dihydroethidium (DHE) and concomitantly measured cell viability using 3,3′-dihexyloxacarbocyanine iodide (DiOC6) by flow cytometry. AF induced a time- and dose-dependent increase in ROS production in CLL cells that correlated with the onset of apoptosis. In conclusion, AF is selectively cytotoxic for CLL cells and its activity is independent of classic mechanisms of chemotherapy resistance. Based on these observations AF is now being studied in a clinical proof of concept phase IIa clinical trial for patients with relapsed/refractory CLL (NCT01419691). Disclosures:No relevant conflicts of interest to declare.

Gene expression profiling following NRF2 and KEAP1 siRNA knockdown in human lung fibroblasts identifies CCL11/Eotaxin-1 as a novel NRF2 regulated gene

BackgroundOxidative Stress contributes to the pathogenesis of many diseases. The NRF2/KEAP1 axis is a key transcriptional regulator of the anti-oxidant response in cells. Nrf2 knockout mice have implicated this pathway in regulating inflammatory airway diseases such as asthma and COPD. To better understand the role the NRF2 pathway has on respiratory disease we have taken a novel approach to define NRF2 dependent gene expression in a relevant lung system.MethodsNormal human lung fibroblasts were transfected with siRNA specific for NRF2 or KEAP1. Gene expression changes were measured at 30 and 48 hours using a custom Affymetrix Gene array. Changes in Eotaxin-1 gene expression and protein secretion were further measured under various inflammatory conditions with siRNAs and pharmacological tools.ResultsAn anti-correlated gene set (inversely regulated by NRF2 and KEAP1 RNAi) that reflects specific NRF2 regulated genes was identified. Gene annotations show that NRF2-mediated oxidative stress response is the most significantly regulated pathway, followed by heme metabolism, metabolism of xenobiotics by Cytochrome P450 and O-glycan biosynthesis. Unexpectedly the key eosinophil chemokine Eotaxin-1/CCL11 was found to be up-regulated when NRF2 was inhibited and down-regulated when KEAP1 was inhibited. This transcriptional regulation leads to modulation of Eotaxin-1 secretion from human lung fibroblasts under basal and inflammatory conditions, and is specific to Eotaxin-1 as NRF2 or KEAP1 knockdown had no effect on the secretion of a set of other chemokines and cytokines. Furthermore, the known NRF2 small molecule activators CDDO and Sulphoraphane can also dose dependently inhibit Eotaxin-1 release from human lung fibroblasts.ConclusionsThese data uncover a previously unknown role for NRF2 in regulating Eotaxin-1 expression and further the mechanistic understanding of this pathway in modulating inflammatory lung disease.

Gene expression analysis of toxicological pathways in TM3 leydig cell lines treated with Ethane dimethanesulfonate

Ethane dimethanesulfonate (EDS), a well-known alkylating agent, selectively destroys Leydig cells. To clarify the molecular pathways underlying EDS action on Leydig cells, we analyzed gene expression profiles of an EDS-treated TM3 Leydig cell line. In this study, we analyzed the representative canonical pathways and toxicity pathways/gene lists using the Ingenuity Pathways Analysis program. In TM3 cells, 677 and 6756 genes were identified as being up- or downregulated after 3 and 24 h EDS treatments, respectively, (>1.3-fold changes, p < 0.05). Toxicological pathway analysis revealed that expression of genes related to Nrf2-mediated oxidative stress response showed remarkable changes in early or later stage of EDS-treated TM3 cells. Several genes related to steroidogenesis and apoptosis were also differentially expressed at 24 h in EDS-treated TM3 cells. Overall, toxicological pathway analysis using gene expression profiling showed that oxidative stress might be an important factor in cell death in TM3 cells affected by EDS treatment.

Nrf2 Deficiency Leads to Altered Hematopoietic Stem Cell Function and Increased Sensitivity to Alkylating Agent Induced Myeloid Dysplasia,

Abstract 3828Therapy-related myelodysplasia or acute myeloid leukemia (t-MDS/AML) is a lethal complication of cancer treatment. In a previous study we conducted gene expression analysis comparing peripheral blood stem cell (PBSC) CD34+ cells from patients who subsequently developed t-MDS/AML after autologous hematopoietic cell transplantation (aHCT) for Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL) and matched controls that did not develop t-MDS/AML after aHCT. This study showed that the Nrf2-mediated oxidative stress response was one of the top ranked canonical pathways downregulated in t-MDS/AML cases compared to controls. Nrf2 is a basic region-leucine-zipper transcription factor that regulates expression of numerous detoxifying and antioxidant genes as well as ubiquitination and proteasomal degradation proteins. Since the Nrf2 pathway is a major cellular defense mechanism against oxidative and xenobiotic stresses, we hypothesized that downregulated Nrf2 response may cause increased toxicity after genotoxic therapeutic exposures and contribute to development of t-MDS/AML. To test this hypothesis, we studied Nrf2 knockout mice to determine the effects of Nrf2 deficiency on hematopoiesis under physiological conditions and after exposure to genotoxic stress. We did not observe significant differences in peripheral blood (PB) counts between Nrf2 knockout (KO) mice and wild-type (WT) mice. Analyses of hematopoietic cells from PB, bone marrow (BM) or spleen using multi-color flow cytometry demonstrated that the compositions of granulocyte, B cell and T cell lineages were similar between KO and WT mice. No differences in BM long-term hematopoietic stem cell (LT-HSC, Lin-c-Kit+Sca-1+ Flt3- CD48-CD150+), multi-potent progenitor (MPP, Lin-c-Kit+Sca-1+ Flt3- CD48+), common-myeloid progenitor (CMP, Lin-c-Kit+Sca-1-CD34+CD16/32-), granulocyte/monocyte progenitor (GMP, Lin-c-Kit+Sca-1-CD34+CD16/32+), or megakaryocyte/erythroid progenitor (MEP, Lin-c-Kit+Sca-1-CD34-CD16/32-) populations were seen between KO and WT mice. However, analysis of LT-HSC function using competitive repopulation assays demonstrated significantly reduced chimerism of donor derived cells in PB of recipient mice at 12 weeks and 16 weeks in KO group compared to WT control (42.49±3.09% vs. 54.75±2.13% at 12 weeks and 42.5±2.8% vs. 57.2±3.3% at 16 weeks for KO and WT respectively). Although we did not see significant differences in chimerism of donor-derived hematopoietic stem/progenitor cells in the BM of KO and WT groups at 16 weeks, we did observe significantly reduced contribution of donor-derived cells from the KO group compared to WT control in PB in secondary recipients as early as 8 weeks after secondary transplantation of BM cells (16.5±1.7% vs 61.9±1.5% for KO and WT respectively). These data indicate that Nrf2 deficiency causes cell-intrinsic functional defects in HSC self-renewal. In order to test if Nrf2 deficient hematopoietic cells are more sensitive to genotoxic stress compared to WT cells, we treated KO and WT mice with the alkylating agent ENU and monitored for hematological changes in PB. We did not observe significant differences in hematopoietic recovery after exposure to acute genotoxic stress between KO and WT mice. However, KO mice developed severe anemia at a significantly increased rate and with shorter latency compared to WT mice after ENU treatment (88% in KO vs. 28% in WT at 4 months, p=0.003). Anemic mice developed massive splenomegaly, and histological analysis showed loss of follicular structure and dramatic increase in erythropoiesis with areas of myeloid elements. Pathological examination of BM from anemic mice revealed normal cellularity but reduced erythropoiesis with expansion of myeloid cells. FACS analyses revealed expansion of myeloid (CD11b+, Gr-1+) and immature erythroid population (CD71+Ter119+/dim) in the spleen and BM, and increased CD11b+, Gr-1+ cells in PB. These findings are consistent with development of myeloid dysplasia in these mice. In conclusion, Nrf2 deficiency results in alterations in HSC self-renewal capacity under physiological condition, and leads to increased sensitivity to alkylator-induced myeloid dysplasia in mice. Disclosures:No relevant conflicts of interest to declare.