Investigation of helenalin-induced cell death in Bcl-2 overexpressing Jurkat cells [Elektronische Ressource] / Ruth Hoffmann

Ruth Hoffmann

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 55
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München



Investigation of helenalin-induced cell death in Bcl-2
overexpressing Jurkat cells






Ruth Hoffmann
(geb. Meßmer)
aus Spaichingen
2010 Erklärung
Diese Dissertation wurde im Sinne von §13 Abs. 3 bzw. 4 der Promotionsordnung vom 29.
Januar 1998 von Frau Prof. Dr. Angelika M. Vollmar am Lehrstuhl für Pharmazeutische
Biologie betreut.




Ehrenwörtliche Versicherung
Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.


München, am 20.09.2010




Ruth Hoffmann (geb.Meßmer)




Dissertation eingereicht am: 20.09.2010
1. Gutachter: Prof. Dr. Angelika M. Vollmar
2. Gutachter: PD Dr. Manfred Ogris
Mündliche Prüfung am: 29.10.2010 CONTENTS 5
CONTENTS 6 CONTENTS
1 CONTENTS
1 CONTENTS..............................................................................................................6
2 INTRODUCTION ....................................................................................................10
2.1 Background and aim of the study ...........................................................................10
2.2 Sesquiterpene lactones ..........................................................................................11
2.2.1 Helenalin.................................................................................................................11
2.3 Bcl-2 .......................................................................................................................13
2.3.1 The Bcl-2 family......................................................................................................13
2.3.2 Regulation of Bcl-2 by phosphorylation ..................................................................13
2.4 Cell death ...............................................................................................................14
2.5 Apoptosis................................................................................................................14
2.6 Necrosis..................................................................................................................16
2.7 Autophagy ..............................................................................................................18
2.7.1 Crosstalk between apoptosis and autophagy.........................................................19
2.8 ER stress and autophagy .......................................................................................19
2.8.1 ER stress19
2.8.2 ER stress - autophagy link......................................................................................20
2.9 Mechanism how Bcl-2 protects from cell death ......................................................21
2.9.1 Bcl-2-mediated regulation of mitochondrial membrane permeabilization...............21
2.9.2 n of calcium flux in the ER ...............................................22
2.10 Significance to overcome Bcl-2-mediated resistance.............................................24
2.10.1 Bcl-2 inhibitors in cancer therapy ...........................................................................24
2.10.2 Overcoming cell death resistance by Bcl-2 overexpression using helenalin ..........25
3 MATERIALS AND METHODS...............................................................................28
3.1 Materials .................................................................................................................28
3.1.1 Biochemicals, inhibitors, dyes, buffers and cell culture reagents ...........................28
3.1.2 Technical equipment ..............................................................................................30
3.2 Cell Culture.............................................................................................................30
3.2.1 Cell lines30
3.2.2 Cell culture..............................................................................................................31
3.2.3 Seeding for experiments.........................................................................................32
3.2.4 Freezing and thawing .............................................................................................32
3.3 Flow cytometry .......................................................................................................32
3.3.1 Quantification of Cell death ....................................................................................33
3.3.1.1 Nicoletti assay ........................................................................................................33
3.3.1.2 PI exclusion assay..................................................................................................33 CONTENTS 7
3.3.1.3 Annexin-V/PI double staining .................................................................................33
3.3.2 Measurement of ROS generation...........................................................................34
3.3.3 nt of mitochondrial potential dissipitation.............................................34
3.4 Clonogenic assay ...................................................................................................34
3.5 Western blot ...........................................................................................................35
3.5.1 Whole lysate preparation........................................................................................35
3.5.2 Preparation of cytosolic and mitochondrial fractions ..............................................36
3.5.3 Immunoprecipitation ...............................................................................................36
3.5.4 Protein quantification ..............................................................................................37
3.5.5 SDS-PAGE .............................................................................................................37
3.5.6 Tank electroblotting ................................................................................................38
3.5.7 Protein detection.....................................................................................................39
3.5.7.1 Enhanced chemiluminescence...............................................................................40
3.5.7.2 Infrared imaging......................................................................................................40
3.5.8 Staining of gels and membranes ............................................................................41
3.6 Transfection of cells41
3.6.1 with Apaf-1 and AIF siRNA ................................................................41
3.6.2 Transfection with plasmids .....................................................................................41
3.7 Reporter gene assay ..............................................................................................41
3.8 Electrophoretic mobility shift assay (EMSA)...........................................................42
3.8.1 Praparation of nuclear extracts...............................................................................42
3.8.2 Binding reaction and electrophoretic separation ....................................................43
3.9 Caspase activity assay ...........................................................................................44
3.10 Calcium measurement............................................................................................45
3.11 Transmission Electron Microscopy.........................................................................45
3.12 Statistical Analysis..................................................................................................45
4 RESULTS...............................................................................................................48
4.1 Helenalin overcomes Bcl-2-mediated resistance....................................................48
4.2 Helenalin does not abrogate mitochondrial function of Bcl-2 and acts
independently of the mitochondria and the apoptosome........................................51
4.2.1 Mitochondrial function.............................................................................................51
4.2.2 Caspase dependency54
4.3 Mechanisms of helenalin’s bypass of Bcl-2-mediated cytoprotection.....................58
4.3.1 ER stress, autophagy and necroptosis...................................................................58
4.3.2 Helenalin inhibits Bcl-2-induced NF- κB activity ......................................................63
4.3.3 duces cell death by induction of ROS..................................................66 8 CONTENTS
5 DISCUSSION .........................................................................................................70
5.1 Untypical signaling of helenalin-induced cell death ................................................70
5.1.1 Apoptosis................................................................................................................70
5.1.2 Autophagy .....................................