A STUDY TO INVESTIGATE THE INVOLVEMENT OF NADPH OXIDASE (NOX5) IN RESVERATROL (RSV) - INDUCED REACTIVE OXYGEN SPECIES (ROS) PRODUCTION IN U937 CELLS         BHUVANESHWARI D/O SHUNMUGANATHAN [BSc (Hons), NTU] A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PHYSIOLOGY, YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS Firstly I would like to express my gratitude to Dr Andrea Lisa Holme for her unwavering guidance and support throughout the course of my Masters project as my supervisor and her help with assays involving the LSC I would also like to thank Professor Shazib Pervaiz for his invaluable advice and input in this project as my co-supervisor My heartfelt gratitude also goes out to Dr Alan Premkumar and his students, Ms Hay Hui Sin, Ms Chen Luxi, Ms Loo Ser Yue and Ms Angele Koh They have always provided with unconditional moral support during the ups and downs of my graduate studies The time spent together will always be cherished I would also like to express my sincere appreciation to all members of the ROS ,apoptosis and cancer biology lab for accommodating me as a ‘surrogate/adpoted’ member of their lab In this ½ years I have not only enhanced my knowledge on scientific research but have also built friendships through my interaction with the people from this lab My sincere appreciation also goes out to the admin staff from the department of Physiology and also Cancer science institute (CSI), NUS for the use of their premises during the last half year Finally, but most importantly I would like to thank my family for being a strong pillar of support and above all God for guiding and bringing me through this phase of my life successfully TABLE OF CONTENTS ABSTRACT i LIST OF TABLES ii LIST OF FIGURES .iii ABBREVIATIONS USED vi INTRODUCTION 1.1: (a) Reactive oxygen species (ROS) (b) ROS as signaling molecules .3 (i) Cell signaling fate resulting from the alteration of cellular redox status (ii) The antioxidant enzyme systems as a means of maintaining homeostatic cellular redox balance 1.2: NADPH Oxidases (NOX) (i) Overview (ii) Sub-cellular localization of NOX enzymes .10 (iii) Physiological/ pathological functions of NOX enzymes generated ROS 13 1.3: NADPH Oxidase (NOX5) 19 1.4: Resveratrol (RSV) (i) Chemical structure and biological properties 24 (ii) Modulation of cellular function 25 (iii) Modulation of cellular redox status 26 (iv) Autophagy and metabolism (v) RSV and Reactive oxygen species (ROS)/NADPH Oxidase (NOX) 1.5: Aim of study 29 1.6: Mode of action of the classes of compounds used in the study…………………………29 MATERIALS AND METHODS 2.1: Materials/Reagents 30 2.2: Methods/Protocols/Plan of investigation 31 RESULTS 3.1: Resveratrol exerts dose-dependent cellular physiological effects in U937 39 a) MTT cell viability assay 40 b) Cell cycle analysis 42 3.2: Establishing working doses for treatment of U937 with ROS-producing compounds, ROS scavengers and NOX inhibitor via Cell viability MTT assay 44 3.3: Resveratrol increases NOX5 expression level in U937 50 3.4: Resveratrol produces ROS in U937 55 3.5: Scavenging of ROS produced by RSV abrogates the increase in NOX5 expression 57 ROS regulation of NOX5 expression 3.6: (i) The treatment of U937 with DDC shows a dose-dependent increase in NOX5 expression level 63 (ii) The treatment of U937 with hydrogen peroxide shows a dose-dependent increase in NOX5 expression level 65 (iii) a) The treatment of U937 with nitric oxide producing compounds such as SIN-1 chloride and DETA-NO not significantly alter NOX5 expression levels 66 b) NO producing compounds cause phosphorylation of c-Raf (positive control) .67 3.7: Resveratrol treatment results in the phosphorylation of CREB at early time points .68 3.8: Cyclosporin A inhibits CREB phosphorylation and abrogates the increase in NOX5 expression level following Resveratrol treatment 70 DISCUSSION/CONCLUSIONS/ FUTURE WORK 4.1: Overview on study 74 4.2: Resveratrol exerts several physiological effects in dose-dependent manner 75 4.3: Reseveratrol increases NOX5 expression level in U93 75 4.4: i) Resveratrol produces ROS 77 ii) The scavenging of ROS abrogates the increase in NOX5 expression level 77 4.5: i) ROS producing compounds such as DDC and Hydrogen Peroxide (H2O2) cause an increase in NOX5 expression 77 ii) ROS scavenging abrogates the increase in NOX5 expression 78 4.6: i) RSV treatment results in the phosphorylation of CREB 78 ii) The inhibition of CREB phosphorylation via pre-treatment with Cyclosporin abrogates the increase in NOX5 expression level 79 4.7: Conclusion 79 4.8: Future work .81 REFERENCES 83 SUPPLEMENTARY FIGURES .90 I ABSTRACT ROS are oxygen - derived small molecules The initial form of ROS is superoxide anion (O2.) which is produced by the gain of electrons Subsequent chemical reactions can lead to the formation of other forms of ROS species NOX enzymes are one of the major sources of ROS NOX5 belongs to the family of NOX enzymes NOX5 differs from the other members of the NOX family in aspects as such as its mode of activation and biophysical structure NOX5 comprises of membrane-bound subunits namely gp91 and p22 It does not require the recruitment of cytosolic subunits, unlike its counterparts The activity of NOX5 is regulated by elevations in cytosolic calcium levels and phosphorylation via kinases such as cAbl (Abelson murine leukemia viral oncogene) and PKC The N-terminus of NOX5 comprises EF-hands that serve as calcium binding domains It has been established so far that NOX5 is a growth signalling oxidase and its expression is regulated via growth regulatory agonists/triggers/signals NOX5 has been also shown to be expressed in a variety of tumour cell lines and is being explored as a cancer biomarker RSV a naturally occurring phenolic phytoalexin is currently being employed as a ‘parent’ compound in cancer drug development It has been well-studied that RSV is a ROS-modulating compound It can act as a pro/antioxidant depending on the concentration and cell line used Previous studies have shown that, RSV is able to alter the expression and activity of other NOX isoforms is a range of cell lines This study aimed to investigate the involvement of RSV-induced ROS production in regulating NOX5 expression in the U937 lymphoma cells The initial part of the study involved analyzing the expression of NOX5 following RSV treatment RSV was shown to increase NOX5 expression in U937 in a dose-dependent manner A time course was also performed and it showed that NOX5 expression starts to increase as early as 2hr ROS was being implicated as the possible factor involved in the regulation of NOX5 expression due to the nature of RSV The pre-treatment of U937 with ROS scavengers prior to RSV treatment abrogated the increase in NOX5 expression Further studies were performed and it showed ROS producing compounds such as DDC and H2O2 were able to increase NOX5 expression level However, NO-producing compounds did not significantly alter the NOX5 expression level CREB phosphorylation was observed post-RSV treatment and this was reversible via pre-incubation with Cyclosporin A CREB phosphorylation levels were also observed to precede the increase NOX5 expression level Thus CREB is being suggested as a possible transcription factor regulating NOX5 expression This study highlights the role of ROS producing compound RSV in regulating the expression level of NOX5 in U937 (442 words) i II LIST OF TABLES TABLE 1: A summary highlighting the cellular distribution, subcellular localization and the major physiological function of the NOX isoforms TABLE 2: Gene expression level of NOX 1-5, DUOX and DUOX2 relative to 18s rRNA expression (x10-8) in tumor cell lines ii III LIST OF FIGURES INTRODUCTION FIGURE 1: The main biochemical reactions involved in the formation of Reactive oxygen species (ROS) FIGURE 2: Diagram illustrating the importance of cellular homeostatic redox balance FIGURE 3: The intracellular sources of ROS The subcellular compartmentalization of ROS FIGURE 4: Pylogeny tree of the NOX family FIGURE 5: Schematic representation of NOX2 enzyme activation FIGURE 6: Schematic representation of the various mechanisms of activation The cofactors/subunits required for the activation are highlighted Diagrammatic representation of the transmembrane topology and domains of the NOX isoform FIGURE 7: Schematic representation of NADPH Oxidase (NOX5) FIGURE 8: A diagram representing the current research development on NOX5 FIGURE 9: Chemical structure of Resveratrol RESULTS FIGURE 1a: Cell viability assay of U937 in response to RSV treatment FIGURE 1b: FIGURE 1c: PI live cell cycle analysis assay of U937 cells following the indicated doses of RSV FIGURE 1d: Cellular morphology of cells for 24hrs with increasing doses of RSV and scanned at 0.25μM step size at 40x objective representative field images FIGURE 2a: Cell viability assay of U937 in response to tiron treatment FIGURE 2b: Cell viability assay of U937 in response to catalase treatment FIGURE 2c: Cell viability assay of U937 in response to NAC treatment FIGURE 2d: Cell viability assay of U937 in response to DPI treatment iii FIGURE 2e: Cell viability assay of U937 in response to DDC treatment FIGURE 2f: Cell viability assay of U937 in response to H2O2 treatment FIGURE 2g: Cell viability assay of U937 in response to SIN-1 Chloride treatment FIGURE 2h: Cell viability assay of U937 in response to DETA-NO treatment FIGURE 3: Analysis of NOX5 expression following 24hr RSV treatment FIGURE 4: Basal expression level of NOX5 in untreated U937 cells FIGURE 5: A time coure study of NOX5 expression in U937 following 25μM RSV treatment FIGURE 6: Analysis of NOX5 expression following 3hr RSV treatment FIGURE 7a: ROS production in U937 cells DCFDA assay was performed to measure the level of ROS produced following RSV treatment The percentage of cells in R2 region is a measure of fluorescence intensity FIGURE 7b: Pre-incubation of U937 with ROS scavengers and NOX inhibitor DPI abrogates the increase in NOX5 expression following 25μM RSV treatment FIGURE 8: RSV treatment does not significantly alter the antioxidant enzyme levels FIGURE 9: DDC treatment increaseS NOX5 expression in U937 cells FIGURE 10: Pre-incubation with Tiron abrogates the increase in NOX5 expression via DDC treatment FIGURE 11: H2O2 treatment increases NOX5 expression in U937 FIGURE 12: Pre-incubation with Catalase abrogates the increase in NOX5 expression via H2O2 treatment FIGURE 13: Nitric oxide producing compounds such as SIN-1 chloride and DETA-NO not significantly change the expression level of NOX5 FIGURE 14: RSV treatment results in the phosphorylation of CREB in a time-dependent manner FIGURE 15: Pre-treatment of Cyclosporin A inhibits the phosphorylation of CREB and abrogates the increase in NOX5 expression observed via RSV treatment iv DISCUSSION Figure 1: Diagram representing the summary of the study SUPPLEMENTARY FIGURES FIGURE 1a: PI cell cycle analysis of U937 cells following 12hr of RSV treatment FIGURE 1b: PI cell cycle analysis of U937 cells following 24hr of RSV treatment FIGURE 2a: DCFDA assay analyzing ROS production following 30 minutes of RSV treatment in U937 cells FIGURE 2b: DCFDA assay analyzing ROS production following 1hr of RSV treatment in U937 cells FIGURE 2c: DCFDA assay analyzing ROS production following 12hr of RSV treatment in U937 cells FIGURE 3a: DDC produces ROS FIGURE 3b: The ROS produced by DDC can be scavenged by tiron FIGURE 4a: H2O2 produces ROS FIGURE 4b: ROS produced by H2O2 can be scavenged by catalase v Discussion Besides Ca2+ flux, NOX5 is also regulated by phosphorylation at the serine residues Current studies have shown that PKC is involved in the phosphorylation of NOX5 (Serrander et al., 2007) The phosphorylation status of PKC and other kinases (Oliveira et al., 2003)such as p38, Erk, Jnk can be studied Thus other kinases that regulate NOX5 activity and expression can be identified The current data in this study shows that RSV treatment causes the phosphorylation of CREB and regulates NOX5 expression This can be further extended by using bioinformatics tools to study the promoter sequence of NOX5 Thus CRE element sequences can be identified to further strengthen the data that CREB is a possible transcription factor regulating NOX5 expression The factors that regulate CREB can also be identified to further support the evidence Previous investigations have highlighted that Protein kinase A (PKA) and members of the MAP Kinase family are able to activate CREB phosphorylation (Shaywitz and Greenberg, 1999) In addition other potential transcription factors can be identified via studying the promoter sequence Bringing forth the study in this direction would better project NOX5 as a cancer biomarker and pave for the way for therapeutic strategies in controling/preventing tumorigenesis 82 References REFERENCES Ahmad, K., Clement, 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Med 42, 1369-1380 89 Supplementary figures SUPPLEMENTARY FIGURES Figure 1a): PI cell cycle analysis of U937 cells following 12hr of RSV treatment PMA10ng/ml RSV 0.78125μM RSV 1.5625μM RSV 3.125μM RSV 6.25μM RSV 12.5μM RSV 25μM Figure 1a: Histograms and scatterplots representing the 12hr U937 cell cycle profile The U937 cells were treated with the indicated doses of RSV for 12hrs and the cell cycle was analyzed by PI assay using the LSC The control (untreated) cells are represented in red as an overlay in the histograms 10ng/ml PMA was used as a positive control 90   Supplementary figures Figure 1b) PI cell cycle analysis of U937 cells following 24hr of RSV treatment PMA 10ng/ml RSV 6.25μM RSV 12.5μM RSV 25μM Control/Untreated   Figure 1b: Histograms representing the 24hr U937 cell cycle profile The U937 cells were treated with the indicated doses of RSV for 24hrs and the cell cycle was analyzed by PI assay using the LSC The control (untreated) cells are represented in red as an overlay in the histograms 10ng/ml PMA was used as a positive control 91   Supplementary figures Figure 2a) DCFDA assay analyzing ROS production following 30 minutes of RSV treatment in U937 cells 25μM RSV 12.5μM RSV   50μM RSV PMA 10ng/ml Figure 2a: Histograms representing the ROS production in U937 cells The U937 cells were treated with the indicated doses of RSV for 30 minutes and the ROS production was analyzed by the DCFDA assay using the LSC The control (untreated) cells are represented in red as an overlay in the histograms 10ng/ml PMA was used as a positive control 92   Supplementary figures Figure 2b) DCFDA assay analyzing ROS production following 1hr of RSV treatment in U937 cells 25μM RSV 12.5μM RSV    50μM RSV   25μM RSV      Figure 2b: Histograms representing the ROS production in U937 cells The U937 cells were treated with the indicated doses of RSV for 1hr and the ROS production was analyzed by the DCFDA assay using the LSC The control (untreated) cells are represented in red as an overlay in the histograms 10ng/ml PMA was used as a positive control 93   Supplementary figures Figure 2c) : DCFDA assay analyzing ROS production following 12hr of RSV treatment in U937 cells Untreated/control 3.125μM RSV 0.788125μM RSV 1.5625μM RSV 6.25μM RSV 12.5μM RSV 25μM RSV Figure 2c: Histograms representing the ROS production in U937 cells The U937 cells were treated with the indicated doses of RSV for 12hr and the ROS production was analyzed by the DCFDA assay using the LSC The control (untreated) cells are represented in green as an overlay in the histograms 10ng/ml PMA was used as a positive control 94   Supplementary figures Figure 3a: DDC produces ROS     17% 12% 27% Figure 3a: Histograms representing the ROS production in U937 cells The U937 cells were treated with the indicated doses of DDC and the ROS production was analyzed by the DCFDA assay using the LSC The control (untreated) cells are represented in blue as an overlay in the histograms 10ng/ml PMA was used as a positive control Figure 3b: The ROS produced by DDC can be scavenged by tiron 9% 8% 31% Figure 3b: Histograms representing the ROS production in U937 cells The U937 cells were pre-incubated with 3mM of tiron for an hour and treated with indicated doses of DDC for 1hour ROS production was analyzed by the DCFDA assay using the LSC The control (untreated) cells are represented in green as an overlay in the histograms 10ng/ml PMA was used as a positive control 95   Supplementary figures Figure 4a: H2O2 produces ROS    61%   75% 80% Figure 4a: Histograms representing the ROS production in U937 cells The U937 cells were treated with the indicated doses of H2O2 for 1hr and the ROS production was analyzed by the DCFDA assay using the LSC The control (untreated) cells are represented in red as an overlay in the histograms 10ng/ml PMA was used as a positive control Figure 4b: ROS produced by H2O2 can be scavenged by catalase   10% 27%   18% Figure 4b: Histograms representing the ROS production in U937 cells The U937 cells were pre-incubated with 1000U/ml of catalase for an hour and treated with indicated doses of DDC for 1hour ROS production was analyzed by the DCFDA assay using the LSC The control (untreated) cells are represented in blue as an overlay in the histograms 10ng/ml PMA was used as a positive control 96   ... esophageal cells resulting in esophageal adenocarcinoma (EA) Acid reflux damage is a major factor contributing to the transformation of EA NOX5-S was found to be the major NOX5 isoform present in. .. is able to activate/ modulate signal transduction pathways such as the MAP kinase signaling pathways, JAK/STAT signaling pathways, PI3K/Akt pathway and cAMP/cGMP signaling pathway (Holme and... death Platelet –activating factor (PAF), a pro-inflammatory, mediator was shown to regulate acid -induced NOX-5S expression (Si et al., 2008) NOX5-S increases upon PAF treatment and STAT5 was