Characterization of new chemosensitizing agents [Elektronische Ressource] : spongistatin 1 and T8 / Lilianna Schyschka

Schyschka, Lilianna

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 26
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

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







Characterization of new chemosensitizing agents:
spongistatin 1 and T8




Lilianna Schyschka
aus Pyskowice


2009




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 19.01.2009

Lilianna Schyschka








Dissertation eingereicht am: 19.01.2009
1. Gutachter: Frau Prof. Dr. A. M. Vollmar
2. Gutachter: Herr Prof. Dr. F. Böckler
Mündliche Prüfung am: 13.02.2009


Contents

Table of contents
I Introduction ....................................................................................................... 1
1 Background ......................................................................................................... 1
2 Aim of the study .................................................................................................. 2
3 Spongistatin 1 ...................................................................................................... 3
4 Pharmacophore-based virtual screening for the discovery of target-specific
compounds .......................................................................................................... 5
5 Classification of cell death .................................................................................. 6
6 Apoptosis ............................................................................................................ 7
6.2 Caspases .............................................................................................................. 7
6.2.1 Caspase structure and classification .................................................................... 8
6.2.2 Caspase activation ............................................................................................... 9
6.2.3 Caspase substrates ............................................................................................. 10
7 Extrinsic apoptotic pathway .............................................................................. 10
8 Intrinsic apoptotic pathway ............................................................................... 12
9 Bcl-2 protein family .......................................................................................... 14
10 IAP family ......................................................................................................... 14
10.2 X-linked Inhibitor of Apoptosis Protein ........................................................... 16
10.2.1 Bir1 domain of XIAP ........................................................................................ 17
10.2.2 Bir2 and Bir3 domains of XIAP ....................................................................... 18
10.2.3 RING domain of XIAP ..................................................................................... 18
II Materials and Methods ................................................................................... 19
1 Materials ............................................................................................................ 19
1.1 Compounds ....................................................................................................... 19
1.2 Reagents ............................................................................................................ 19
1.3 Equipment ......................................................................................................... 20
2 Pharmacophore-based virtual screening for XIAP inhibitors ........................... 20
3 Cell culture ........................................................................................................ 21
3.2 Cell lines ........................................................................................................... 21
3.3 Primary cell isolation ........................................................................................ 22
3.4 Cell culture ........................................................................................................ 22
3.5 Splitting and seeding for experiments ............................................................... 23
3.6 Freezing and thawing ........................................................................................ 24
4 Cytotoxicity measurement (MTT assay) ........................................................... 25
5 Proliferation measurement ................................................................................ 25
6 Light and fluorescence microscopy .................................................................. 25
7 Flow cytometry ................................................................................................. 26
7.1 Nicoletti assay ................................................................................................... 27
8 Western blot ...................................................................................................... 28
I
Contents
8.2 Preparation of samples ...................................................................................... 29
8.2.1 Whole lysate preparation .................................................................................. 29
8.3 Preparation of cytosolic and mitochondrial fractions ....................................... 29
8.4 Protein quantification ........................................................................................ 30
8.5 SDS-PAGE ........................................................................................................ 31
8.6 Western blotting and detection ......................................................................... 32
8.7 Membrane stripping .......................................................................................... 34
8.8 Staining of gels and membranes ....................................................................... 34
9 Caspase activity assay ....................................................................................... 34
10 Clonogenic survival assay ................................................................................. 35
11 Reporter gene assay .......................................................................................... 35
12 TaqMan reverse-transcriptase-polymerase chain reaction ................................ 37
13 Transformation of bacteria and plasmid purification ........................................ 37
14 Fluorescence polarization assay ........................................................................ 38
15 Nuclear magnetic resonance analysis ................................................................ 39
16 Statistics ............................................................................................................ 39
III Results .............................................................................................................. 41
1 Spongistatin 1 .................................................................................................... 41
1.1 Spongistatin 1 induces cell death in Jurkat leukemia T cells ............................ 41
1.2 Spongistatin 1 induces cell death in primary leukemic cells ............................ 42
1.3 Spongistatin 1 inhibits clonogenic survival of Jurkat leukemic T cells ............ 44
1.4 Spongistatin 1 induce caspase-dependent apoptosis ......................................... 45
1.5 The extrinsic apoptotic pathway is not important for spongistatin 1 to induce
apoptosis ............................................................................................................ 47
1.6 Caspase-9 is essential for spongistatin 1 mediated apoptosis ........................... 48
1.7 Mitochondria play a pivotal role in spongistatin 1 induced cell death ............. 49
1.8 Bcl-2 and Bcl-xl overexpression saves Jurkat cells from spongistatin 1 induces
cell death ........................................................................................................... 50
1.9 Spongistatin 1 induces cell death in XIAP overexpressing Jurkat cells ........... 51
1.10 Spongistatin 1 reduces XIAP protein level ....................................................... 52
1.11 Spongistatin 1 sensitizes Jurkat cells for staurosporine treatment .................... 53
2 Search for XIAP inhibitors ............................................................................... 55
2.1 Virtual search for XIAP inhibitors – the pharmacophore model ...................... 55
2.2 Cellular models for the in vitro validation of virtual screening hits ................. 56
2.3 The synthetic compound T8 as XIAP inhibitor ................................................ 57
2.3.1 T8 sensitizes different Jurkat cell lines for etoposide treatment ....................... 59
2.3.2 T8 in combination with etoposide inhibits clonogenic growth of Jurkat cells . 59
2.3.3 T8 derivatives .......................................