Cancer therapy with metronomically scheduled cyclophosphamide [Elektronische Ressource] : experimental modalities within GDEPT and tumor escape mechanisms / Michael Günther

Michael Günther

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 28
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

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





Cancer Therapy with Metronomically Scheduled Cyclophosphamide:
Experimental Modalities within GDEPT and Tumor Escape Mechanisms


Michael Günther
aus
Buchloe

2006
Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung
vom 29. Januar 1998 von Dr. Manfred Ogris und Prof. Dr. Ernst Wagner betreut.






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




München, am 18.12.2006



__________________
(Michael Günther)


Dissertation eingereicht am 18.12.2006
1. Gutachter: Prof Dr. Ernst Wagner
2. Gutachter: Priv.-Doz. Dr. Carsten Culmsee

Mündliche Prüfung am 23.01.2007




















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(Heiner Neidig)
Table of contents 1
Table of contents
1 Introduction 6
1.1Novelapproaches for cancer treatment 6
1.1.1Antiangiogenic therapy 6
1.1.1.1Theangiogenic process and antiangiogenic approaches 7
1.1.1.2Metronomically scheduled chemotherapy 8
1.1.2Genetherapy 9
1.1.2.1Therapeutic concepts for cancer gene therapy 9
1.1.2.2CYP450/CPA combination10
1.1.2.3Targeting strategies within the GDEPT concept 12
1.1.2.4 PEI-polyplexes 13
1.2 Tumor environment 14
1.3 Resistance to chemotherapy 15
1.3.1.1 Multicellular tumor spheroids 16
1.3.1.2 NF-B 16
1.4 Objectives of this thesis 18
2 Materials and methods 19
2.1 Chemicals and reagents 19
2.1.1 Polyethylenimine (PEI) 19
2.1.2Plasmid DNA 19
2.1.3 Recombinant proteins 20
2.1.4Other reagents 20
2.1.5 Software 20
2.1.6Antibodies20
2.1.7 Measurement of protein concentration 21
2.2 Cell biological methods 21
2.2.1 Cell culture 21
2.2.2Agarose overlay technique 22
2.2.3 Multicellular spheroid culture (Agarose suspension culture) 22
2.2.4 Coculture in a transwell system 23
2.2.5 Storage of isolated and other cells 23
2.3 Molecular biological methods 23
2.3.1 Restriction digestion of plasmid DNA 23
2.3.2 Linearizing of plasmid DNA 23
2.3.3 Generation of stably transfected single cell clones 23
2.4 Polyplex formation and transfections 24
2.5 Gene expression assays 24
2.5.1 Luciferase reporter gene expression in vitro experiments 24
2.5.2 EGFP reporter gene expression 24
2.5.3CYP2B1 gene expression 25
2.5.3.1 CYP2B1 Transgene expression analysis after transient transfection 25
2.5.4 NF- B activity studies 25
2.6Assays for detection of CYP2B1 enzymatic activity 27
2.6.1 Resorufin assay 27
2.6.2Acroleinassa29
2.7 Hypoxia induced HRE-responsible gene expression 30
2.8 Angiogenesis assays 31
Table of contents 2
2.8.1 Migration assay wound healing (scratch wound assay) 31
2.8.2 Tube formation assay 31
2.8.3 Staining for integrin v3 32
2.8.4 Stainingfor F-actin32
2.8.5 ThrombospondinElisa33
2.9Proliferationandviability assays 33
2.9.1 Hoechst33258DNA contentassay 33
2.9.2 MTT assay 35
2.10Flowcytometric analysis andmicroscopy 36
2.10.1 Flow cytometric analysis ofEGF receptor
and CD71 on reisolated tumor cells 36
2.10.2 Flow cytometric analysis of integrin receptor
and aminopeptidase N on HUVEC cells 37
2.10.3 Flow cytometric analysis of integrin receptor status
on CT26 tumor cells 38
2.10.4 Transmission light and epifluorescence microscopy 38
2.11 In vivo/ex vivo experiments 39
2.11.1 Animals 39
2.11.2 Providing tumor cells for in vivo implantation 40
2.11.3 Tumor cell implantation and tumor models 40
2.11.4 Systemic application ofHoechst dye 33258 41
2.11.5 Intraperitoneal application of CPA 41
2.11.6 Isolation of tumors/organs for histology 41
2.11.7 Haematoxilin/Eosin stain 42
2.11.8 Vessel perfusion with fluorescent dye 42
2.11.9 Antibody stain 42
2.11.9.1 Staining for vascular markers 42
2.11.9.2 Staining for other epitopes 43
2.11.10 Reisolation of tumor cells 45
2.11.11 Isolation of PEC cells 46
2.11.12 Isolation of fibroblasts / NF- B Animals 46
3 Results 48
3.1Evaluation of tumor cells as producer cells inthestandard GDEPT concept 48
3.1.1Endogenous P450 activity of the used cancer cell lines 48
3.1.1.1Acroleinassay 48
3.1.1.2Resorufin assay 49
3.1.1.3Comparison of the acrolein and the resorufin assay 50
3.1.2 Sensitivity of wt tumor cells against CPA treatment 51
3.1.2.1 Assays for determination of cell survival and proliferation 51
3.1.2.2 Survival and proliferation of CT26 and Neuro2A cells
after CPA or IFO treatment 51
3.1.3 Generation of CYP2B1 expressing tumor cells 52
3.1.3.1 Stable Transfection 52
3.1.3.1.1 Generation of the stable transfected X39 cell line 52
3.1.3.1.2 Pre-evaluation of CYP2B1 activity of the G418
resistant CT26 and Neuro2A clones 52
DETable of contents 3
3.1.3.1.3 Confirmation of continuity of CYP activity 52
3.1.3.1.4 Confirmation of CYP2B1 protein in X39 by antibody staining 53
3.1.3.1.5 Characterisation of the X39 clone for CYP2B enzymatic activity
by resorufin assay and CPA conversion 54
3.1.3.1.6 Suicidal effects of CYP2B1 expressing X39 and 9L-D2B1 tumor cells 55
3.1.3.1.7 Suicidal effects of CPA treatment in comparison to IFO treatment 57
3.1.3.1.8 Verification the presence of soluble cytotoxic metabolites
(Transwell system) 58
3.1.3.2 Transient transfection 59
3.1.3.2.1 Expression kinetics and expression levels after transient transfection 59
3.1.3.2.2 Evaluation of CYP2B1 dependent activation of CPA in vitro 60
3.1.3.2.3 Evaluation of sensitizing tumor cells against CPA
after transient transfection (conventional cell culture) 60
3.1.3.3 Bystander effect in a tumor environment mimicking
cell culture system: Agarose overlay technique 61
3.1.3.3.1 Diffusion in the agarose layer 61
3.1.3.3.2 Cell morphology under the agarose layer 62
3.1.3.3.3 Influence of the agarose layer on cell proliferation 63
3.1.3.3.4 Investigation of the agarose overlay technique in terms of
hypoxic stress 64
3.1.3.3.5 Dependency of the CYP2B1 on sufficient oxygen supply 65
3.1.3.3.6 CYP2B1 enzymatic activity under the agarose layer 65
3.1.3.3.7 Enhancement of bystander activity by the agarose
overlay technique in a coculture system 66
3.1.3.3.8 Kinetic of cell death induced by CPA treatment 68
3.1.3.3.9 Bystander effect in cocultures with a fixed ratio of CYP2B1
expressing cells 69
3.1.3.3.10 Enhancement of bystander activity by the agarose overlay
technique in transient transfection experiments 71
3.1.4 In vivo approach 71
3.1.4.1 Characterisation of tumor histology and tumor growth 72
3.1.4.1.1 Tumor histology 72
3.1.4.1.2 Tumor growth and body weight 72
3.1.4.2 Tumor response to metronomic scheduled CPA treatment 73
3.1.4.3 Metronomic scheduled CPA treatment combined with
precedent CYP2B1 gene transfer 76
3.2 Evaluation of tumor cells as producer cells
in the antiangiogenic GDEPT concept 77
3.2.1 Endogenous CYP activity in endothelial cells 77
3.2.2 Sensitivity of primary endothelial cells against CPA treatment 77
3.2.3 Coculture of endothelial cells with CYP2B1 expressing tumor cells 78
3.2.3.1 Evaluation of proliferation and survival 78
3.2.3.1.1 Proliferation of endothelial cells in a coculture 78
3.2.3.1.2 Proliferation and survival in a transwell system 80
3.2.3.2 Antimigrative effects in an endothelial cell – tumor cell
coculture system 81 Table of contents 4
3.2.3.3 Coculturing endothelial cells with X39 cells in the
presence of CPA disturbed the tube forming process 82
3.2.3.4 Coculturing of endothelial cells with CYP2B1 expressing tumor
cells in the presence of CPA results in modifications of F-actin 83
3.3 Role of acrolein in CPA therapy at metronomic schedule 85
3.3.1 Significant reduction in tumor blood flow and tumor growth
in CPA treated mice 85
3.3.2 Acrolein adducts in tumor tissue of treated mice 86
3.3.3 Antiangiogenic properties of acrolein in vitro 87
3.3.3.1Changes of cell morphology 87
3.3.3.2 Acrolein is antiproliferative on endothelial cells 88
3.3.3.3 Acrolein inhibits endothelial cell migration 89
3.3.3.4 Acrolein inhibits endothelial tube formation 90
3.3.3.5 Subacute cytotoxic acrolein concentrations disrupt
endothelial F-actin cytoskeleton 91
3.3.3.6 Acrolein inhibits integrin v 3 clustering on endothelium filopodiae 91
3.3.3.7 Acrolein modulates NF- B activity in cultured human endothelial cells 92
3.3.3.8 Acrolein modulates thrombospondin-1 levels
in primary endothelial cells (HUVECs) 93
3.3.4 Impact of a