Oxidative stress 2013


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Oxidative stress 2013

  1. 1. Oxidative Stress and ROS Signaling Any Questions ??? Ask now or contact Technical Support support@SABiosciences.com 1-888-503-3187 Webinar related questions: Qiawebinars@qiagen.com -1- Sample & Assay Technologies
  2. 2. 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 -2- Sample & Assay Technologies
  3. 3. 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 -3- Sample & Assay Technologies
  4. 4. 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 -4- Sample & Assay Technologies
  5. 5. 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 -5- Sample & Assay Technologies
  6. 6. 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. -6- Sample & Assay Technologies
  7. 7. 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 -7- O2 + H2O2 2 H2O + O2 G-S–S-G + R-OH + H2O Sample & Assay Technologies
  8. 8. 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& -8- Sample & Assay Technologies
  9. 9. 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 -9- Sample & Assay Technologies
  10. 10. Disorders associated with oxidative stress Diabetes Cancer Atherosclerosis COPD Oxidative Stress Neurodegeneration Asthma Infertility Dermatitis CardioVascular disease - 10 - Sample & Assay Technologies
  11. 11. Overview of oxidative stress & ROS - 11 - Sample & Assay Technologies
  12. 12. 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 - 12 - Sample & Assay Technologies
  13. 13. 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 - 13 - Sample & Assay Technologies
  14. 14. 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. - 14 - Sample & Assay Technologies
  15. 15. Oxidative stress studies: experiment design Gene expression Functional studies Epigenetics - 15 - Sample & Assay Technologies
  16. 16. 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? - 16 - Sample & Assay Technologies
  17. 17. 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 - 17 - Sample & Assay Technologies
  18. 18. Application 1: gene expression in oxidative stress 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 - 18 - Sample & Assay Technologies
  19. 19. Application 1: gene expression in oxidative stress 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 http://sabiosciences.com/rt_pcr_product/HTML/PAHS-084Z.html Oxidative stress RT2 Profiler PCR Array: 84 genes involved in oxidative stress http://sabiosciences.com/rt_pcr_product/HTML/PAHS-087Z.html - 19 - Sample & Assay Technologies
  20. 20. Application 1: gene expression in oxidative stress 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 - 20 - Sample & Assay Technologies
  21. 21. Application 1: gene expression in oxidative stress Results: Excess GC (glucocorticoid) decreased the expression of oxidative stress responsive genes - 21 - Sample & Assay Technologies
  22. 22. Application 1: oxidative stress & autophagy 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 - 22 - Sample & Assay Technologies
  23. 23. 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. http://sabiosciences.com/rt_pcr_product/HTML/PAHS-212Z.html - 23 - Sample & Assay Technologies
  24. 24. 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 - 24 - Sample & Assay Technologies
  25. 25. 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? - 25 - Sample & Assay Technologies
  26. 26. 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 - 26 - Sample & Assay Technologies
  27. 27. Application 2: reporter assays for oxidative stress 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. - 27 - Sample & Assay Technologies
  28. 28. Application 2: reporter assays for oxidative stress 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. - 28 - Sample & Assay Technologies
  29. 29. 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? - 29 - Sample & Assay Technologies
  30. 30. 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 - 30 - Sample & Assay Technologies
  31. 31. 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 - 31 - • • • • • • • • • • • miFinder Serum & Plasma Brain Cancer Breast Cancer Ovarian Cancer Prostate Cancer Cancer PathwayFinder Apoptosis New! Liver miFinder New! Cardiovascular Disease New! Sample & Assay Technologies
  32. 32. 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) - 32 - Sample & Assay Technologies
  33. 33. 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. - 33 - Sample & Assay Technologies
  34. 34. 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. - 34 - Sample & Assay Technologies
  35. 35. 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 - 35 - Sample & Assay Technologies
  36. 36. 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 - 36 - Sample & Assay Technologies
  37. 37. Pathway-focused solutions @ QIAGEN - Complete experimental systems: sample prep through data analysis - 37 - Sample & Assay Technologies
  38. 38. 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 - 38 - Sample & Assay Technologies