Oxidative stress 2013

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


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


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


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)


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.


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


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&


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 ?


Disorders associated with oxidative stress

Diabetes

COPD

Neuro-degeneration

Asthma

Cardio-Vasculardisease

Dermatitis

Infertility

Athero-sclerosis

Cancer

OxidativeStress


Overview of oxidative stress & ROS


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


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.


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.


Oxidative stress studies: experiment design

Gene expression

Functional studies

Epigenetics


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?


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


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



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


Oxidative stress RT 2 Profiler PCR Array: 84 genes involved in oxidative

stress




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



Results: Excess GC (glucocorticoid) decreased the expression of oxidative stress responsive genes



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


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.


Cell Death PathwayFinder PCR Array


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


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?


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


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.



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 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?


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


� 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!•


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)


� 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


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


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


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


Pathway-focused solutions @ QIAGEN- Complete experimental systems: sample prep through data analysis

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Oxidative stress 2013

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