View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by OTHES 1 DIPLOMARBEIT Titel der Diplomarbeit Influence of Indirubin-3´-monoxime on Redox Systems in PDGF-activated Vascular Smooth Muscle Cells Verfasser Alexander Heinrich angestrebter akademischer Grad Magister der Pharmazie (Mag. Pharm.) Wien, 2010 Studienkennzahl lt. A 449 Studienblatt: Studienrichtung lt. Pharmazie Studienblatt: Betreuerin / Betreuer: Univ. Prof. Dr. Verena Dirsch 2 3 A INTRODUCTION ................................................................................................................................. 5 1 INDIRUBIN ........................................................................................................................................... 5 1.1 Chemical properties, occurrence and history ........................................................................... 5 1.2 Medicinal use ............................................................................................................................ 6 1.3 Derivatives ................................................................................................................................ 7 1.4 Reported bioactivities of Indirubins ........................................................................................... 8 2 ATHEROSCLEROSIS ........................................................................................................................... 11 2.1 General overview .................................................................................................................... 11 2.2 Physiology ............................................................................................................................... 11 2.3 Pathological events that lead to atherosclerosis .................................................................... 13 2.4 Therapeutic options ................................................................................................................ 15 2.5 Restenosis .............................................................................................................................. 15 3 PDGF .............................................................................................................................................. 17 3.1 Structure and binding properties ............................................................................................. 17 3.2 Structure of the PDGF receptor (PDGFR) and ligand-induced autophosphorylation ............. 18 3.3 Signal transduction ................................................................................................................. 19 3.4 Important Intracellular Pathways, activated by PDGF ............................................................ 19 3.5 Distribution .............................................................................................................................. 25 4 ROS ................................................................................................................................................ 26 4.1 Oxidative Stress and Antioxidants .......................................................................................... 26 4.2 ROS Species .......................................................................................................................... 26 4.3 Genesis of ROS ...................................................................................................................... 27 4.4 Impact of ROS levels on cellular processes ........................................................................... 29 4.5 Regulation of PDGF-R signalling by ROS and phosphatases ............................................... 30 5 SPECIFIC BACKGROUND AND AIM OF THIS WORK ................................................................................. 32 B MATERIALS AND METHODS .......................................................................................................... 33 1 MATERIALS ................................................................................................................................... 33 1.1 Cell culture media and reagents ............................................................................................. 33 1.2 Growth factors and cytokines ................................................................................................. 33 1.3 Indirubin-3´monoxime ............................................................................................................. 33 1.4 Antibodies ............................................................................................................................... 34 1.5 Solutions ................................................................................................................................. 35 1.6 Other Equipment ..................................................................................................................... 38 2 METHODS ......................................................................................................................................... 39 2.1 Cell Culture ............................................................................................................................. 39 2.2 Preparation of cell lysates for Western Blotting ...................................................................... 41 2.3 Western blotting ...................................................................................................................... 43 2.4 Flow Cytometry ....................................................................................................................... 44 4 2.5 Statistics .................................................................................................................................. 46 C RESULTS .......................................................................................................................................... 47 1 IMPACT OF INDIRUBIN 3´- MONOXIME ON THE PHOSPHORYLATION OF THE KINASES ERK1,2 AND AKT ... 48 1.1 Stimulation with PDGF ............................................................................................................ 48 1.2 Treatment with I3MO .............................................................................................................. 48 2 INFLUENCE OF INDIRUBIN ON PDGF RECEPTOR PHOSPHORYLATION ................................................... 51 2.1 PDGF-induced autophosphorylation....................................................................................... 51 2.2 Treatment with I3MO .............................................................................................................. 51 3 INFLUENCE OF INDIRUBIN 3´-MONOXIME ON ROS MEDIATED SIGNALLING ............................................. 53 3.1 Impact of Indirubin 3´-monoxime on the kinases ERK1,2 compared to the Antioxidant NAC 54 3.2 Impact of Indirubin 3´-monoxime on AKT Kinase compared to the Antioxidant NAC ............ 56 3.3 Impact of Indirubin 3´-monoxime on STAT3 Kinase compared to the Antioxidant NAC ........ 58 3.4 Impact of Indirubin 3´-monoxime on PDGF receptor autophosphorylation compared to the Antioxidant NAC ............................................................................................................................ 60 3.5 Effect of Indirubin 3´-monoxime on the level of Reactive oxygen Species............................. 62 D DISCUSSION ..................................................................................................................................... 65 1 INFLUENCE OF I3MO ON PHOSPHORYLATION OF KINASES AKT AND ERK1,2 ....................................... 66 2 INFLUENCE OF I3MO ON PHOSPHORYLATION OF STAT3 TRANSCRIPTION FACTOR ................................ 66 3 INFLUENCE OF I3MO ON PHOSPHORYLATION OF THE PDGF RECEPTOR .............................................. 66 4 INFLUENCE OF INDIRUBIN 3´-MONOXIME ON ROS MEDIATED SIGNALLING ............................................. 67 5 INFLUENCE OF I3MO ON ROS PRODUCTION ...................................................................................... 68 6 INFLUENCE OF I3MO ON ROS PRODUCTION IN THE PRESENCE OF NAC .............................................. 68 7 CONCLUDING REMARKS AND OUTLOOK ............................................................................................... 69 E REFERENCES ................................................................................................................................... 70 APPENDIX ............................................................................................................................................ 80 APPENDIX ............................................................................................................................................ 80 A ABBREVIATIONS ............................................................................................................................... 81 B SOURCES OF GRAPHICS ................................................................................................................... 84 C CURRICULUM VITAE ......................................................................................................................... 85 D ACKNOWLEDGMENTS........................................................................................................................ 87 5 A Introduction 1 Indirubin 1.1 Chemical properties, occurrence and history Indirubin