Anticancer effects and antiangiogenic mechanisms of the marine compound spongistatin 1 [Elektronische Ressource] / Andrea Silvia Rothmeier

Andrea Silvia Rothmeier

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 32
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

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



Anticancer effects and antiangiogenic mechanisms
of the marine compound spongistatin 1




Andrea Silvia Rothmeier
aus München
2008



Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 der Promotionsordnung vom
29. Januar 1998 von Herrn PD Dr. Stefan Zahler am Lehrstuhl von Frau Prof. Dr. Angelika
M. Vollmar betreut.


Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig und ohne unerlaubte Hilfe erarbeitet.



München, den 09. Oktober 2008

______________________________ Andrea Rothmeier

Dissertation eingereicht am: 09. Oktober 2008
1. Gutachter: Herr PD Dr. Stefan Zahler
2. Gutachter: Herr Prof. Dr. Christian Wahl-Schott
Mündliche Prüfung am: 12. November 2008











Meinen Lieben

I. CONTENTI CONTENT
I. CONTENT........................................................................................I
II. ABBREVIATIONS...........................................................................1
III. INTRODUCTION.............................................................................4
1 BACKGROUND Marine Compounds ................................................................5
2 Spongistatin 1....................................................................................................6
3 Microtubule-Inhibiting Drugs in Cancer Therapy............................................7
3.1 Chemotherapy and the War on Cancer.................................................................... 7
3.2 Inhibition of Tumor Angiogenesis as an Anticancer Approach................................. 8
4 Microtubules ......................................................................................................9
4.1 The Structure of Microtubules ................................................................................ 10
4.1.1 The Tubulins ................................................................................................... 10
4.1.2 Protofilaments ................................................................................................. 10
4.2 Organization of Microtubules.................................................................................. 11
4.2.1 The MTOC 11
4.2.2 The Dynamics of Microtubules........................................................................ 12
4.3 Functions of Microtubules ...................................................................................... 13
4.3.1 The Mitotic Spindle.......................................................................................... 13
4.3.2 Cellular Transport............................................................................................ 14
5 Microtubule-Binding Compounds ..................................................................15
6 Tumor Angiogenesis .......................................................................................16
6.1 Initiation of Angiogenesis: Endothelial Proliferation ............................................... 17
6.1.1 Hypoxia-Induced Expression of VEGF............................................................ 17
6.1.2 The AKT and the ERK Pathways.................................................................... 18
6.1.3 The PKC pathway ........................................................................................... 20
6.2 Endothelial Migration.............................................................................................. 21
6.2.1 Matrix metalloproteinases ............................................................................... 21
6.2.2 Tip Cells and Stalk Cells ................................................................................. 21
6.2.3 Molecular Processes during Migration............................................................ 22
v I CONTENT
6.3 Vessel Formation ................................................................................................... 24
7 Aims of the Work .............................................................................................25
IV. MATERIALS AND METHODS......................................................26
1 Materials ...........................................................................................................27
1.1 Compounds............................................................................................................ 27
1.2 Biochemicals, Inhibitors, Dyes, and Cell Culture Reagents ................................... 27
1.3 Technical Equipment.............................................................................................. 28
2 Cell Culture.......................................................................................................29
2.1 HUVEC Isolation and Cultivation............................................................................ 29
2.2 L3.6pl Cultivation.................................................................................................... 29
3 Flow Cytometry................................................................................................30
3.1 Stimulation and Harvest ......................................................................................... 30
3.2 Cell Cycle Analysis................................................................................................. 30
4 Cell Viability Measurements ...........................................................................31
5 Microscopy.......................................................................................................31
5.1 Immunohistochemistry ........................................................................................... 31
5.2 Live-Cell Imaging.................................................................................................... 32
5.2.1 Mitochondria Staining...................................................................................... 32
5.2.2 Visualization of Membrane Traffic................................................................... 32
5.2.3 Expression of Recombinant Proteins.............................................................. 32
6 Protein Sample Preparations..........................................................................33
6.1 Total Cell Lysates................................................................................................... 33
6.2 Microtubule Fractionation....................................................................................... 34
6.3 Membrane Fractionation ........................................................................................ 35
6.4 Protein Isolation of Tissue Sections ....................................................................... 35
7 Western Blot Transfer .....................................................................................36
7.1 Protein Quantification............................................................................................. 36
7.2 SDS-PAGE............................................................................................................. 36
7.3 Tank-Blotting .......................................................................................................... 37
7.4 Detection ................................................................................................................ 38
vi I CONTENT
7.4.1 Enhanced Chemiluminescence....................................................................... 38
7.4.2 LI-COR............................................................................................................ 38
7.5 Staining Gels and Membranes............................................................................... 40
8 Kinome Array (PepChip) .................................................................................40
9 PKC In Vitro Assay ..........................................................................................41
10 Angiogenic In Vitro Assays ............................................................................41
10.1 Proliferation Assay ................................................................................................. 41
10.2 Migration Assay...................................................................................................... 41
10.3 Chemotaxis Assay.................................................................................................. 42
10.4 Tube Formation Assay ........................................................................................... 43
11 Angiogenic Ex/In Vivo Assays........................................................................43
11.1 Mice........................................................................................................................ 43
11.2 Mouse Aortic Ring Assay ....................................................................................... 44
11.3 Mouse Cornea Pocket Assay....................................................