Sample & Assay Technologies
Oxidative Stress and ROS Signaling
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Topics will be covered
� Overview
� Oxidants & antioxidant in biological systems
� Antioxidant defense mechanism
� Detection of oxidative stress
� Research Solutions @ QIAGEN
� Gene expression
� Epigenetics
� Functional studies
� Application Examples
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Oxidative stress and ROS
� Oxidative stress : imbalance between reactive oxygen species (ROS) and antioxidants.
� Under normal conditions, cells are able to balance the production of oxidants and antioxidants. Oxidative stress occurs when cells are subjected to excess levels of ROS or as a result of antioxidant depletion.
� ROS: chemically reactive molecules containing oxygen, highly reactive
� Under normal conditions, ROS are natural byproducts. It also can be generated by exogenous sources.
� Major sources of ROS: mitochondria, peroxisome, plasma membrane/cytoplasm.
Role of reactive oxygen species in cell signaling pathways, Biochemical and Biomedical Aspects of Oxidative Modification 2001
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The Biological Importance of ROS
Physiological positive effects of ROS
� Kill microorganism
� Second messengers
� Cellular differentiation & proliferation
� Regulate signal transduction & transcription� Cytokines and growth factor receptors (EGFR,
PDGFR, VEGFR, receptors of TNFα, IFNγ and IL-1β)
� Non-receptor tyrosine kinases (JAK, Src, MAPK and PI3K)
� Protein tyrosine phosphatases
� Serine/Threonine kinases (Akt, PKC and MAPK)
� Nuclear transcription factors (NFκ B, AP-1, NFAT, HIF-1 and Nrf2)
Oxidative Stress and Oxidative Damage in Carcinogenesis, Toxicologic Pathology, 38: 96-109, 2010
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Antioxidant Defense Mechanism
� Inhibit excess ROS formation, capture radicals, maintain redox homeostasis
and correct mechanism of destroyed biomolecules
� Mediated through Antioxidant Responsive Element (ARE) sequence
� Controlled by nuclear transcription factors Nrf2, AP-1, NFκB, p53, NFAT
ROS Transcription factors
Transcription
ARE AREAntioxidant response elements (AREs)
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Nrf2-mediated oxidative stress response
Nrf2: Nuclear factor-erythroid 2-related
factor is a basic leucine zipper (bZIP)
transcription factor that binds to ARE to
activate transcription.
Keap1: Kelch-like ECH-associated
protein 1. Keap1 interacts with Nrf2 in a
redox-sensitive manner.
Inactive Nrf2 is retained in the
cytoplasm by association with Keap1.
Upon exposure to oxidative stress, Nrf2
is phosphorylated in response to the
PKC, PI3K, and MAPK pathways. After
phosphorylation, Nrf2 is translocated to
the nucleus, binds AREs, and activates
detoxifying enzymes and antioxidant
enzyme.
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Antioxidants – maintain oxidative balance
� Non-enzymatic antioxidants
� Alpha tocopherol (vitamin E)
� Beta carotene
� Ascorbic acid (vitamin C)
� Antioxidant enzymes
� Superoxide dismutase (SOD) O2−● + O2
−● + 2H+ � O2 + H2O2
� Catalase (CAT) 2 H2O2 � 2 H2O + O2
� Glutathione peroxidase (GPx) 2 G-SH + R-O-OH � G-S–S-G + R-OH + H2O
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Antioxidant Action of Vitamin-C
Vitamin-C is a potent antioxidant and a regulator of redox-signal transduction
Under stress conditions Vitamin-C acts as a primary antioxidant in plasma and cells
Vitamin-C quenches ROS and serves as a cofactor of enzymes involved in the synthesis of collagen, neurotransmitters and Carnitine
https://www.qiagen.com/geneglobe/pathwayview.aspx?pathwayID=34&
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Oxidative damage of ROS
� Aggregation and fragmentation
� Enzyme inhibition
Proteins� DNA strand breaks� Mutations leading
to cancer
DNA� Lipid peroxidation� Damage to
membranes and lipoproteins
Membrane
ROS
Oxidative stress, Disease and aging
Review article: “Molecular targets of oxidative stre ss” Biochem. J. (2011) 434, 201
What is the cellular targets of ROS that account fo r the toxicity ?
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Disorders associated with oxidative stress
Diabetes
COPD
Neuro-degeneration
Asthma
Cardio-Vasculardisease
Dermatitis
Infertility
Athero-sclerosis
Cancer
OxidativeStress
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Overview of oxidative stress & ROS
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Crosstalk between oxidative Stress, mitochondria & autophagy
� In respond to cellular stress, necrosis,
apoptosis and autophagy are
activated.
� Oxidative stress is tightly linked to
mitochondrial dysfunction, as
mitochondria are both generators of
and targets of ROS
� Mitochondrial turnover is dependent
on autophagy
� Dysregulated redox signaling or
mitochondrial dysfunction can
influence autophagic activities
Review article: “Autophagy, mitochondria and oxidative stress: cross-talk and redox signaling” Biochem. J. 2012, 441, 523
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Detection of intracellular oxidative stress
� Direct approach� Detection of ROS / free radicals
� Colorimetric (ROS-dependent reactions), fluorometric (ROS-sensitive probes)
� Challenging: short-lived, highly reactive molecules
� Indirect approach� Antioxidant enzyme activity
� Detect oxidative stress damage products
� Measure cellular antioxidant capacity
� More simple: more stable modifications of macromolecules
� Wide range of techniques
Untreated Cells
Treated Cells
The cell-permeant dye H2-DCF-DA is non-fluorescent in a reduced state and exhibits fluorescence upon oxidation by ROS.
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Detection of oxidative damage to macromolecules
� Lipid markers of oxidative stress� 4-Hydroxynonenal (HNE) is a by-product of lipid peroxidation, widely used as a
marker of chronic oxidative stress
� Protein markers of oxidative stress� Protein carbonylation – formation of carbonyl derivatives of Pro, Lys, Arg and Thr
residues – is the most common product of protein oxidation (ELISA assay).
� Advanced oxidation protein products (AOPP) are created when proteins react with chlorinated oxidants.
� DNA markers of oxidative stress� 8-hydroxydeoxyguanosine (8-OHdG)
� Antioxidant assays� Catalase activity, SOD activity
� Total antioxidant capacity
� GSH levels
Avery SV. Molecular targets of oxidative stress. Biochem J. 2011 Mar 1;434(2):201-10.
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Oxidative stress studies: experiment design
Gene expression
Functional studies
Epigenetics
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Oxidative stress applications: Questions to be addressed
1. How is cell fate determined under oxidative
stress?
2. How to measure the degree of antioxidant
response?
3. The mechanism of Nrf2-dependent
antioxidant response in cancer?
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Application 1: gene expression in oxidative stress
.In response to cellular oxidative stress, pathways including necrosis, apoptosis, and autophagy will be activated.
.How is cell fate determined under oxidative stress?
FASEB J. 2011 October; 25(10): 3366FASEB J. 2011 October; 25(10): 3366FASEB J. 2011 October; 25(10): 3366
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Experiment design: oxidative stress and cell death Real-time RT-PCR (RT2 PCR) profile antioxidation autophagy and antiapoptosis focused genes
Treat with glucocorticoid
Isolate total RNA
Reverse transcription
(RT)
Data Analysis
cDNA synthesis
qPCR
Swiss-Webster mice MLO-Y4 cells
May 2, 9, 16, 23, Thursdays, qPCR Technology webina r serieshttps://www2.gotomeeting.com/register/716790538
Application 1: gene expression in oxidative stress
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Pathway-focused RT2 Profiler PCR Arrays
Apoptosis RT 2 Profiler PCR Array: 84 genes involved in programmed
cell death
Include: genes involved in induction of apoptosis, anti-apoptosis, regulation of apoptosis,
DEATH domain proteins, caspases and regulators
http://sabiosciences.com/rt_pcr_product/HTML/PAHS-012Z.html
Autophagy RT 2 Profiler PCR Array: 84 genes involved in autophagy
Include: autophagy machinery components, regulation of autophagy
http://sabiosciences.com/rt_pcr_product/HTML/PAHS-084Z.html
Oxidative stress RT 2 Profiler PCR Array: 84 genes involved in oxidative
stress
http://sabiosciences.com/rt_pcr_product/HTML/PAHS-087Z.html
Application 1: gene expression in oxidative stress
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Oxidative Stress RT2 Profiler PCR Array
- human, mouse, rat, and more
Antioxidants:Glutathione Peroxidases (GPx): GPX1, GPX2, GPX3, GPX4, GPX5, GPX6, GPX7, GSTP1, GSTZ1.Peroxiredoxins (TPx): PRDX1, PRDX2, PRDX3, PRDX4, PRDX5, PRDX6 (AOP2).Other Peroxidases: CAT, CYBB, CYGB, DUOX1, DUOX2, EPX, LPO, MGST3, MPO, PTGS1, PTGS2 (COX2), PXDN, TPO, TTN.Other Antioxidants: ALB, APOE, GSR, MT3, SELS, SOD1, SOD3, SRXN1, TXNRD1, TXNRD2.
Genes Involved in ROS Metabolism:Superoxide Dismutases (SOD): SOD1, SOD2, SOD3.Other Genes Involved in Superoxide Metabolism: ALOX12, CCS, DUOX1, DUOX2, GTF2I, MT3, NCF1, NCF2, NOS2 (iNOS), NOX4, NOX5, PREX1, UCP2.Other Genes Involved in ROS Metabolism: AOX1, BNIP3, EPHX2, MPV17, SFTPD.Oxidative Stress Responsive Genes: APOE, ATOX1, CAT, CCL5 (RANTES), CYGB, DHCR24, DUOX1, DUOX2, DUSP1 (PTPN16), EPX, FOXM1, FTH1, GCLC, GCLM, GPX1, GPX2, GPX3, GPX4, GPX5, GPX6, GPX7, GSR, GSS, HMOX1, HSPA1A, KRT1, LPO, MBL2, MPO, MSRA, NQO1, NUDT1, OXR1, OXSR1, PDLIM1, PNKP, PRDX2, PRDX5, PRDX6 (AOP2), PRNP, RNF7, SCARA3, SELS, SEPP1, SIRT2, SOD1, SOD2, SQSTM1, SRXN1, STK25, TPO, TTN, TXN, TXNRD1, TXNRD2.
Oxygen Transporters: CYGB, MB.HKG: B2M, HPRT, RPL13A, GAPDH, HGDC
Application 1: gene expression in oxidative stress
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Results: Excess GC (glucocorticoid) decreased the expression of oxidative stress responsive genes
Application 1: gene expression in oxidative stress
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Results:• Excess GC increased osteocyte autophagy in vivo • Activation of the antioxidant genes positively correlated with activation of the autophagic
genes• No significant correlation between the activation of antioxidant and antiapoptosis genes
Application 1: oxidative stress & autophagy
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New ! Cell Death PathwayFinder PCR Array
Apoptosis:
Pro-Apoptotic: ABL1, APAF1, ATP6V1G2, BAX, BCL2L11, BIRC2 (c-IAP2), CASP1 (ICE), CASP3, CASP6, CASP7,
CASP9, CD40 (TNFRSF5), CD40LG (TNFSF5), CFLAR (CASPER), CYLD, DFFA, FAS (TNFRSF6), FASLG (TNFSF6),
GADD45A, NOL3, SPATA2, SYCP2, TNF, TNFRSF1A, TNFRSF10A (TRAIL-R), TP53.
Anti-Apoptotic: AKT1, BCL2, BCL2A1 (Bfl-1/A1), BCL2L1 (BCL-X), BIRC3 (c-IAP1), CASP2, IGF1R, MCL1, TNFRSF11B,
TRAF2, XIAP.
Autophagy: AKT1, APP, ATG12, ATG16L1, ATG3, ATG5, ATG7, BAX, BCL2, BCL2L1 (BCL-X), BECN1, CASP3, CTSB,
CTSS, ESR1 (ERa), FAS (TNFRSF6), GAA, HTT, IFNG, IGF1, INS, IRGM, MAP1LC3A, MAPK8 (JNK1), NFKB1, PIK3C3
(VPS34), RPS6KB1, SNCA, SQSTM1, TNF, TP53, ULK1.
Necrosis: ATP6V1G2, BMF, C1orf159, CCDC103, COMMD4, CYLD, DEFB1, DENND4A, DPYSL4, EIF5B, FOXI1,
GALNT5, GRB2, HSPBAP1, JPH3, KCNIP1, MAG, OR10J3, PARP1 (ADPRT1), PARP2, PVR, RAB25, S100A7A,
SPATA2, SYCP2, TMEM57, TNFRSF1A, TXNL4B.
http://sabiosciences.com/rt_pcr_product/HTML/PAHS-212Z.html
Cell Death PathwayFinder PCR Array
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Applications of oxidative stress PCR Arrays
Publications Citing the Oxidative Stress PCR Array
• Susceptibility to Inhaled Flame-Generated Ultrafine Soot in Neonatal and Adult Rat Lungs, TOXICOLOGICAL SCIENCES 124(2), 472–486 (2011), Jackie K. W. Chan,* Michelle V, etc.
• Adaptive induction of NF-E2-related factor-2-driven antioxidant genes in endothelial cells in response to hyperglycemia, Am J Physiol Heart Circ Physiol 300: H1133–H1140, 2011, Zoltan Ungvari,1 Lora Bailey-Downs, etc.
• Tryptophan from human milk induces oxidative stress and upregulates the Nrf-2-mediated stress response in human intestinal cell lines. J Nutr. 2011 Aug;141(8):1417-23. Elisia I, Tsopmo A, Friel JK, etc.
• Age-related gene response of human corneal endothelium to oxidative stress and DNA damage. Invest Ophthalmol Vis Sci. 2011 Mar 1;52(3):1641-9. Joyce NC, Harris DL, Zhu CC
• Gene expression analysis of oxidative stress and apoptosis in proton-irradiated rat retina. In Vivo. 2010 Jul-Aug;24(4):425-30. Mao XW, Green LM, Mekonnen T, Lindsey N, Gridley DS
• Increased ANG II sensitivity following recovery from acute kidney injury: role of oxidant stress in skeletal muscle resistance arteries. Am J Physiol Regul Integr Comp Physiol. 2010 Jun;298(6):R1682-91. Phillips SA, etc.
• Effects of spaceflight on innate immune function and antioxidant gene expression. J Appl Physiol. 2009 Jun;106(6):1935-42. Baqai FP, Gridley DS, Slater JM, Luo-Owen X, Stodieck LS, Ferguson V, etc.
• Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defenses. Braz J Med BiolRes. 2005 Jul;38(7):995-1014. Scandalios JG
• Oxidative stress and gene regulation. Free Radic Biol Med. 2000 Feb 1;28(3):463-99. Allen RG, Tresini M
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Application 2: gene functions in oxidative stress
Transcription factors Nrf2 and Nrf1 bind to the antioxidant response elements (ARE) and regulate genes involved in protecting cells from oxidative damages.
.How to measure the degree of
antioxidant response?
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Cignal Reporter Assays & Arrays
■ Dual-luciferase format
■ GFP format
■ DNA and Lenti assay
■ 45 signaling pathways
http://sabiosciences.com/cellassay.php
Application 2: reporter assays for oxidative stress
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45-pathway array & 10-Pathway Arrays Single Pathway Assay
Antioxidant Response ReporterCignal Finder Stress & Toxicity 10-pathway reporter Kit
Measure the transcriptional activity of the Nrf2 (NF-E2-related factor 2) and Nrf1 transcription factors. Nrf2 and Nrf1 bind to the antioxidant response elements (AREs) and regulate genes involved in protecting cells from oxidative damage.
Application 2: reporter assays for oxidative stress
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Goal: answer two questions: 1) whether hyperglycemia activates Nrf2 in endothelial cells; 2) whether induction of Nrf2-regulated ROS detoxification systems protect endothelial function in type 2 diabetes.
Experiment: treat wild type and Nrf2 knockout mouse with a high-fat diet (HFD) and standard diet; use reporter assay to validate Nrf2 activity in cultured endothelial cell model.
Conclusion: Hyperglycemia induces oxidative stress; adaptive activation of the Nrf2/ARE pathway has a critical role in endothe lial protection in response to diabetic condition both in vitro and in vivo .
Application 2: reporter assays for oxidative stress
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Application 3: antioxidant response mechanism
miRNAs play a regulatory role in Nrf2/Keap1 pathway in mammary epithelium
.How is Keap1 regulated?
.What is the mechanism of Nrf2-dependent antioxidant
response in breast cancer?
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miRNA assays and reagents from QIAGEN
Target Identification Expression
Isolation
Function
miRNA mimics & inhibitors
miScript miRNA PCR Arrays
miRNeasy
miRNAStudies
May 2013: 4 part miRNA focused webinar series, monthlyhttps://www2.gotomeeting.com/register/789549898
Online Webinar: http://sabiosciences.com/seminarlis t.php
RT² Profiler™ microRNA Targets PCR Arrays
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� 1 hour
� 2 minutes
� 2 hours
� 15 minutes
miScript miRNA PCR Arrays
miFinder miRNA PCR Array
• Profiles the expression of the 84 most abundantly expressed and best characterized miRNAs in miRBase (www.miRBase.org);
• Maximize the likelihood of discovering miRNAs whose expression patterns correlate with the biological phenotypes;
• Available in Human, Mouse, Rat, Dog, Rhesus Macaque, more
miRNA PCR Array• miFinder• Serum & Plasma• Brain Cancer• Breast Cancer• Ovarian Cancer• Prostate Cancer • Cancer PathwayFinder• Apoptosis New!• Liver miFinder New!• Cardiovascular Disease New!•
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Application 3: miRNA expression in cancer research
Experiments: Human breast cancer cell lines MDA-MB-231 and Hs578T, non-tumorigenic MCF-10A cells
� Screened the expressions of 84 miRNAs using Human miFinder Array (MIHS-001Z)
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� Conclusion: miR-200a regulates the Keap1/Nrf2 pathway in mammary epithelium and epigenetic therapy can restore the miR-200a regulation of Keap1 expression, therefore to reactivate Nrf2-dependent antioxidant pathway in breast cancer.
Luciferase reporter assay to measure Nrf2 ARE promoter activityEpigenetic therapy can restore miR-200a expression and reduce Keap1 level
Application 3: miRNA expression in cancer research
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Oxidative stress and DNA methylation
Stress & Toxicity PathwayFinder DNA Methylation PCR Array
Oxidative Stress: BNIP3, CYP1A1, GPX3, PRDX2, PTGS2 (COX2), SCARA3.
DNA Damage: ATM, BRCA1, CCND1, GADD45A, GADD45G, MLH1, MSH2, TP53, XPC.
Unfolded Protein Response: INSIG1, UBE2G2.Hypoxia Signaling: CSTB, RARA.Heat Shock Protein: DNAJC15.Growth Arrest & Senescence: ATM, CDKN1A (p21CIP1/WAF1), GADD45A, GDF15 (PLAB), MSH2, TP53.Inflammation: PRDX2, PTGS2 (COX2).
Anti-Apoptosis: BNIP3, CDKN1A (p21CIP1/WAF1), PRDX2.Proliferation & Carcinogenesis: BRCA1, CCND1, CDKN1A (p21CIP1/WAF1), CYP1A1, PRDX2, PTGS2
(COX2), TP53.
EpiTect Methyl qPCR Array workflow
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We Provide Service – Send Samples to Us & Receive Results!
. Whole Genome� Illumina Gene Expression Profiling� Illumina Genotyping
. Pathway / Focused Panel� Mutation Profiling� Methylation� PCR Array� miRNA PCR Array
. Individual Gene / Locus� Mutation Detection� Methylation� qPCR
. Sample Preparation – DNA, RNA Extraction and Purification� Cells, Tissue or Biofluids� Fixed Tissue� Small Sample
. http://www.sabiosciences.com/servicecore.php
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Summary
.QIAGEN offers many products for oxidative stress studies
� Gene Expression� RT2 Profiler PCR Arrays & Assays� Custom PCR Arrays
� Epigenetics� miScript miRNA PCR Arrays & Assays� EpiTect Methyl qPCR Arrays & Assays
� Functional Studies� Cignal Reporter Assays� SureSilencing shRNA Plasmid� Flexitube siRNA� Flexiplate siRNA, AllStar Control
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Pathway-focused solutions @ QIAGEN- Complete experimental systems: sample prep through data analysis
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Are you ready to try?Call 1-888-503-3187 for more information
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