“Kv1.3 inhibitors in the treatment of glioma and melanoma” Inaugural-Dissertation Zur Erlangung des Doktorgrades Dr. rer. Nat. der Fakultät für Biologie an der Universität Duisburg-Essen Vorgelegt von Elisa Venturini Aus Vicenza, Italy August 2015 Die der vorliegenden Arbeit zugrunde liegenden Experimente wurden am Institut für Molekularbiologie am Universitätsklinikum Essen durchgeführt. 1. Gutachter: Prof. Dr. Erich Gulbins 2. Gutachter: Prof. Dr. Shirley Knauer Vorsitzender des Prüfungsausschusses: Prof. Dr. Herbert de Groot Tag der mündlichen Prüfung: 25.11.2015 _______________________________ I E se avessi il dono della profezia e conoscessi tutti i misteri e tutta la scienza, e possedessi la pienezza della fede così da trasportare le montagne, ma non avessi l'amore, non sarei nulla. (Corinzi 13:2) II INDEX LIST OF FIGURES ABBREVIATIONS ................................................................................................................... I 1. INTRODUCTION .......................................................................................................... 1 1.1 Apoptosis ..................................................................................................................... 1 1.1.1 The extrinsic or ‘death receptor-mediated’ pathway ............................................ 1 1.1.2 The intrinsic or ‘mitochondrial’ pathway ............................................................. 2 1.2 Kv1.3 ............................................................................................................................ 5 1.2.1 Kv1.3 inhibitors .................................................................................................... 9 1.2.2 Kv1.3 ‘new inhibitors’: PAP-1 derivatives ........................................................ 10 1.2.3 Mitochondrial Kv1.3 and apoptosis .................................................................... 11 1.2.4 mtKv1.3 and cancer ............................................................................................ 14 1.3 Glioblastoma .............................................................................................................. 15 1.3.1 GBM and K+ channels ........................................................................................ 15 1.4 Aims of the study ....................................................................................................... 17 2 MATERIALS ................................................................................................................ 18 2.1 Chemicals ................................................................................................................... 18 2.2 Tissue culture ............................................................................................................. 21 2.3 Cell lines .................................................................................................................... 23 2.4 Equipment .................................................................................................................. 23 2.5 Buffers and solutions ................................................................................................. 25 2.6 Animals ...................................................................................................................... 27 3 METHODS .................................................................................................................... 28 3.1 Cell culture techniques ............................................................................................... 28 3.1.1 Culture and passage of established cell lines ...................................................... 28 3.1.2 Freezing and thawing of cells ............................................................................. 28 3.2 PAP-1 derivatives ...................................................................................................... 28 3.3 Cell viability assay ..................................................................................................... 28 3.3.1 Trypan blue ......................................................................................................... 28 3.3.2 MTT assay .......................................................................................................... 29 3.4 Biochemical techniques ............................................................................................. 29 3.4.1 Cell membrane fraction enrichment ................................................................... 29 3.4.2 Mitochondria isolation ........................................................................................ 29 III 3.4.3 Cytochrome c release assay ................................................................................ 30 3.4.4 Protein separation by SDS-PAGE ...................................................................... 30 3.4.5 High pressure liquid chromatography (HPLC) analysis ..................................... 32 3.5 Cytometry techniques................................................................................................. 34 3.5.1 Surface staining of cells ...................................................................................... 34 3.5.2 Detection of apoptosis by FITC-Annexin V staining ......................................... 34 3.5.3 Analysis of tumor immune cells ......................................................................... 35 3.6 Histology techniques .................................................................................................. 36 3.6.1 Paraffin embedding of organs ............................................................................. 36 3.6.2 Haematoxylin-eosin staining .............................................................................. 36 3.6.3 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining ................................................................................................................ 38 3.7 Mitochondrial membrane potential and reactive oxygen species (ROS) production measurements ........................................................................................................... 38 3.8 Transient transfection with siRNA ............................................................................ 39 3.9 Intracellular staining................................................................................................... 39 3.10 Immunogold electron microscopy.............................................................................. 39 3.11 In vivo techniques ....................................................................................................... 40 3.11.1 Mice .................................................................................................................... 40 3.11.2 Glioma injection ................................................................................................. 40 3.11.3 Melanoma flank injection ................................................................................... 40 3.11.4 Mice treatments .................................................................................................. 40 3.12 DNA techniques ......................................................................................................... 41 3.12.1 Mycoplasma PCR ............................................................................................... 41 3.12.2 Agarose gel electrophoresis ................................................................................ 41 3.13 Statistics ..................................................................................................................... 42 4 RESULTS ...................................................................................................................... 43 4.1 Kv1.3 is expressed in the plasma membrane of different glioma cell lines ............... 43 4.2 Kv1.3 is expressed in mitochondria of glioma cells .................................................. 45 4.3 Kv1.3 inhibitors PAP-1 and Psora-4 poorly induce cell death in glioma cells .......... 47 4.4 The ‘new’ Kv1.3 inhibitors reduce survival of different glioma cell lines ................ 49 4.5 The newly synthesized Kv1.3 inhibitors induce apoptosis and cytochrome c release of different glioma cell lines ..................................................................................... 52 4.6 PAP-1 derivatives induce apoptosis by specifically targeting Kv1.3 ........................ 56 4.7 Membrane permeant inhibitors cause ROS increase and mitochondrial depolarization in glioma cells ........................................................................................................... 59 IV 4.8 Clofazimine and PAP-1 derivatives do not reduce glioma growth in vivo ................ 62 4.9 In vivo accumulation of clofazimine and PAP-1 derivatives in different organs ...... 64 4.10 PAP-1 derivatives prevent melanoma growth in vivo ................................................ 65 4.11 Kv1.3 inhibitors synergize with chemotherapeutic treatment to reduce melanoma in vivo ....................................................................................................................... 70 4.12 Antioxidants prevent melanoma reduction induced by PAP-1 derivatives in vivo .... 71 4.13 PAP-1 derivatives do not change the composition of immune cell sub-population within the tumor .......................................................................................................