Oxidative stress 2013

Oxidative Stress and ROS Signaling

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Sample & Assay Technologies

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 IL1β)
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

Antioxidant response elements (AREs)

ARE

ARE

Transcription
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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+

Catalase (CAT)

2 H2O2

Glutathione peroxidase (GPx)

2 G-SH + R-O-OH

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O2 + H2O2

2 H2O + O2
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/pathwa
yview.aspx?pathwayID=34&
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Oxidative damage of ROS
ROS

Membrane
Lipid peroxidation
Damage to
membranes and
lipoproteins

Proteins

DNA
DNA strand breaks
Mutations leading
to cancer

Aggregation and
fragmentation
Enzyme inhibition

Oxidative stress, Disease and aging
What is the cellular targets of ROS that account for the toxicity?
Review article: “Molecular targets of oxidative stress” Biochem. J. (2011) 434, 201
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Disorders associated with oxidative stress
Diabetes
Cancer

Atherosclerosis

COPD

Oxidative
Stress

Neurodegeneration

Asthma

Infertility

Dermatitis

CardioVascular
disease
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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
Untreated Cells

Direct approach
Detection of ROS / free radicals
Colorimetric (ROS-dependent reactions),
fluorometric (ROS-sensitive probes)
Challenging: short-lived, highly reactive
molecules

Treated Cells

Indirect approach
Antioxidant enzyme activity
Detect oxidative stress damage products
Measure cellular antioxidant capacity
More simple: more stable modifications of
macromolecules

The cell-permeant dye H2-DCF-DA is
non-fluorescent in a reduced state and
exhibits fluorescence upon oxidation by
ROS.

Wide range of techniques

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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): 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

cDNA synthesis

Swiss-Webster mice

MLO-Y4 cells

Isolate total RNA

Reverse
transcription
(RT)

Treat with glucocorticoid

Data Analysis

qPCR

May 2, 9, 16, 23, Thursdays, qPCR Technology webinar series
https://www2.gotomeeting.com/register/716790538
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Pathway-focused RT2 Profiler PCR Arrays
Apoptosis RT2 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 RT2 Profiler PCR Array: 84 genes involved in autophagy
Include: autophagy machinery components, regulation of autophagy

Oxidative stress RT2 Profiler PCR Array: 84 genes involved in oxidative
stress

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Antioxidants:

Oxidative Stress RT2 Profiler PCR Array

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.

- human, mouse, rat, and more

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

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

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Application 1: oxidative stress & autophagy
Results:
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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

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New ! Cell Death PathwayFinder PCR Array
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.

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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 JulAug;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 Biol
Res. 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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Application 2: reporter assays for oxidative stress
Cignal Reporter Assays & Arrays
■ Dual-luciferase format
■ GFP format
■ DNA and Lenti assay
■ 45 signaling pathways

http://sabiosciences.com/cellassay.php
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45-pathway array & 10-Pathway Arrays
Cignal Finder Stress & Toxicity 10-pathway reporter Kit

Single Pathway Assay
Antioxidant Response Reporter

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.

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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 endothelial protection in response
to diabetic condition both in vitro and in vivo.

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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
miRNeasy
Isolation

Target Identification

miRNA
Studies

Expression
miScript miRNA PCR Arrays

RT² Profiler™ microRNA
Targets PCR Arrays

Function
miRNA mimics & inhibitors
May 2013: 4 part miRNA focused webinar series, monthly
https://www2.gotomeeting.com/register/789549898
Online Webinar: http://sabiosciences.com/seminarlist.php
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miScript miRNA PCR Arrays
miFinder miRNA PCR Array
1 hour

• Profiles the expression of the 84
most abundantly expressed and
best characterized miRNAs in
miRBase (www.miRBase.org);

2 minutes

• Maximize the likelihood of
discovering miRNAs whose
expression patterns correlate with
the biological phenotypes;

2 hours

• Available in Human, Mouse, Rat,
Dog, Rhesus Macaque, more

miRNA PCR Array

15 minutes

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•
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•
•
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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, nontumorigenic MCF-10A cells
Screened the expressions of 84 miRNAs using Human miFinder Array (MIHS001Z)

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Application 3: miRNA expression in cancer research

Luciferase reporter assay to measure Nrf2 ARE promoter activity
Epigenetic therapy can restore miR-200a expression and reduce Keap1 level

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 Nrf2dependent antioxidant pathway in breast cancer.
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Oxidative stress and DNA methylation
EpiTect Methyl qPCR Array workflow
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.
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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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Thank You for Attending!

Are you ready to try?
Call 1-888-503-3187 for more information
Email: support@SABiosciences.com

Wei Cao
Wei.Cao@QIAGEN.COM
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Oxidative stress 2013

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