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English

Identification of novel mechanisms of action contributing to the biological activity of cytotoxic natural compounds [Elektronische Ressource] / Nancy López Antón

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Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Identification of novel mechanisms of action contributing to the biological activity of cytotoxic natural compounds Nancy López Antón aus Sabadell / Barcelona 2006 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 von Frau Prof. Dr. A. M. Vollmar betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet. München, am 02.03.2006 (Nancy López Antón) Dissertation eingereicht am: 02.03.2006 1. Gutachter: Frau Prof. Dr. A. M. Vollmar 2. Gutachter: Herr PD. Dr. C. Culmsee Mündliche Prüfung am: 03.04.2006 dedicated to my family “Life is pleasant. Death is peaceful. It’s the transition that’s troublesome” Isaac Asimov I. CONTENTS I. CONTENTS ................................................................................................. I II. ABBREVIATIONS ...................................................................................... 2 III. INTRODUCTION .....................

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Published 01 January 2006
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Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München





Identification of novel mechanisms of action
contributing to the biological activity of cytotoxic
natural compounds









Nancy López Antón
aus
Sabadell / Barcelona
2006













Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der
Promotionsordnung vom 29. Januar 1998 von Frau Prof. Dr. A. M. Vollmar
betreut.





Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.



München, am 02.03.2006




(Nancy López Antón)





Dissertation eingereicht am: 02.03.2006
1. Gutachter: Frau Prof. Dr. A. M. Vollmar
2. Gutachter: Herr PD. Dr. C. Culmsee
Mündliche Prüfung am: 03.04.2006

























































dedicated to my family











































“Life is pleasant. Death is peaceful.
It’s the transition that’s troublesome”
Isaac Asimov











I. CONTENTS
I. CONTENTS ................................................................................................. I
II. ABBREVIATIONS ...................................................................................... 2
III. INTRODUCTION ..................................................................................... 8
1 BACKGROUND – NATURAL PRODUCTS IN CANCER THERAPY .....................................8
2 CEPHALOSTATINS.....................................................................................................................9
3 SESQUITERPENE LACTONES ................................................................................................12
4 AIM OF THE WORK..................................................................................................................14
5 PROGRAMMED CELL DEATH, APOPTOSIS AND NECROSIS...........................................15
6 SIGNAL TRANSDUCTION IN APOPTOSIS............................................................................17
6.1 CASPASES ........................................................................................................................18
6.1.1 Classification and structure............................................................................................18
6.1.2 Substrate cleavage..........................................................................................................20
6.1.3 Mechanism of caspase activation and regulation...........................................................20
6.2 THE EXTRINSIC PATHWAY..........................................................................................22
6.3 INTRINSIC PATHWAYS .................................................................................................24
6.3.1 Mitochondria..................................................................................................................24
6.3.2 Endoplasmic reticulum ..................................................................................................27
6.3.3 Pathways originated in other organelles ........................................................................31
7 ELIMINATION OF APOPTOTIC CELLS .................................................................................32
7.1 PHAGOCYTOSIS..............................................................................................................32
7.1.1 Importance .....................................................................................................................32
7.1.2 “Eat-me” signals on the apoptotic cell...........................................................................33
7.1.3 Interaction phagocyte-target cell and phagocytosis .......................................................33
7.1.4 Inhibition of phagocytosis..............................................................................................36
7.2 CYTOKINE RESPONSE AND BIOLOGICAL CONSEQUENCES................................36
7.3 ROLES OF APOPTOTIC CELL CLEARANCE IN DISEASE ........................................39
IV. MATERIALS AND METHODS .............................................................. 42
1 MATERIALS...............................................................................................................................42
1.1 COMPOUNDS...................................................................................................................42
1.2 BIOCHEMICALS, INHIBITORS, DYES AND CELL CULTURE REAGENTS............42
1.3 TECHNICAL EQUIPMENT..............................................................................................43
2 CELL CULTURE.....44
2.1 CELL LINES......................................................................................................................44
2.2 CULTURE AND SPLITTING...........................................................................................44
2.3 SEEDING FOR EXPERIMENTS......................................................................................45

2.4 FREEZING AND THAWING ...........................................................................................45
3 FLOW CYTOMETRY.................................................................................................................46
3.1 INTRODUCTION..............................................................................................................46
3.2 DETERMINATION OF CELL VIABILITY (PI EXCLUSION ASSAY) ........................48
3.3 QUANTIFICATION OF DNA FRAGMENTATION BY PI STAINING (NICOLETTI
METHOD)........................................................................................................................................49
3.4 MEASUREMENT OF ROS GENERATION ....................................................................50
4 ANALYSIS OF OTHER CELL DEATH FEATURES ...............................................................51
5 MICROSCOPY............................................................................................................................52
5.1 LIGHT MICROSCOPY .....................................................................................................52
5.2 CONFOCAL LASER SCANNING MICROSCOPY.........................................................52
6 LUMINOMETRIC CASPASE-9 ACTIVITY ASSAY53
7 WESTERN BLOT .......................................................................................................................54
7.1 PREPARATION OF SAMPLES........................................................................................54
7.2 PROTEIN QUANTIFICATION56
7.3 SDS-PAGE.........................................................................................................................56
7.4 WESTERN BLOTTING AND DETECTION....................................................................58
7.5 MEMBRANE STRIPPING................................................................................................61
7.6 STAINING OF GELS AND MEMBRANES.....................................................................61
2+8 MEASUREMENT OF Ca .........................................................................................................62
9 CASPASE-4 siRNA.....................................................................................................................64
9.1 INSERT DESIGN AND CLONING INTO psiRNA-h7SKneo G1 VECTOR...................65
9.2 TRANSFORMATION OF LyoComp GT116....................................................................66
9.3 MINI PREPARATION OF PLASMIDS............................................................................66
9.4 SEQUENCING OF THE siRNA INSERT.........................................................................67
9.5 MAXI PREPARATION OF PLASMIDS ..........................................................................67
9.6 AMPLIFICATION OF psiRNA-h7SKneo AND psiRNA-h7SKnScr VECTORS.............67
9.7 TRANSFECTION AND SELECTION OF JURKAT CELLS...........................................68
9.8 EFFECT OF CASPASE-4 SILENCING IN CASPASE-9 ACTIVATION AND
APOPTOSIS .....................................................................................................................................68
10 PHAGOCYTOSIS ASSAY AND CYTOKINE RELEASE........................................................69
10.1 EVALUATION OF PHAGOCYTOSIS.............................................................................69
10.2 CYTOKINE QUANTIFICATION.....................................................................................70
10.3 ANALYSIS OF PHAGOCYTOSIS BY CONFOCAL MICROSCOPY ...........................71
11 STATISTICS ...............................................................................................................................72
V. RESULTS ................................................................................................. 74
A. CEPHALOSTATIN 1.....................................................................................................................74
1 IMPORTANCE OF CASPASE-9 IN CEPHALOSTATIN 1-INDUCED APOPTOSIS.............74
2 SIGNAL TRANSDUCTION IN CEPHALOSTATIN-INDUCED ENDOPLASMIC
RETICULUM (ER) STRESS AND APOPTOSIS ................................................................................75