Evaluation of the effects of tyrosine kinase inhibitors on the metabolism of human tumor cells using an NMR-based biochemical profiling strategy [Elektronische Ressource] / vorgelegt von Jelena Miljuš

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• Publié par: universitat_bremen
• Publié le: 01 janvier 2006
• Nombre de lectures: 19
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

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Evaluation of the Effects of Tyrosine Kinase
Inhibitors on the Metabolism of Human Tumor
Cells using an NMR-based Biochemical
Profiling Strategy




DISSERTATION
Zur Erlangung des Grades eines
Doktors der Naturwissenschaften
- Dr. rer. nat. -


Dem Fachbereich Biologie/Chemie der
Universität Bremen
vorgelegt von

Jelena Miljuš
Universität Bremen und University of Colorado Health Sciences Center,
Denver
April, 2006























ZA MOJU VOLJENU MAMU





DANKSAGUNG

Mein besonderer Dank gilt meinem Doktorvater Prof. Dr. Dieter Leibfritz und Frau
Dr. Natalie Serkova (UCHSC, Denver, USA).
Dank Prof. Leibfritz’s Betreuung und hilfreichen Diskussionen war es möglich diese
Arbeit anzufertigen. Vielen Dank für Ihr Vertrauen und Ermunterungen.
Bei Frau Dr. Serkova bedanke ich mich für die Themenstellung und freundliche
Betreuung dieser Arbeit. Ich möchte mich weiterhin für die guten Arbeitsbedingungen
und die finazielle Unterstützung bedanken.

Herrn Prof. Dr. Uwe Christians möchte ich für die Bereitschaft zur Begutachtung
meiner Arbei danken. Seine Diskussionsbereitschaft und Ratschläge haben mir oft weiter
geholfen.

Bei Herrn Johannes Stelten und Dr. Wieland Willker bedanke ich mich für deren
Hilfe bei Lösungen NMR relevanter Probleme. Ausserdem danke ich allen weiteren
Mitgliedern der Arbeitsgruppe von Prof. Dr. Leibfritz für die gute Zusammenarbeit.

I would like to thank Prof. Dr. Thomas Henthorn (chair) and all my other colleagues
from the Department of Anesthesiology (Clinical Research and Development) at the
UCHSC. My special thanks go to Dr. Saskia Trump, Jaimi Brown, Jamie Bendrick-
Peart, Dr. Yan-Ling Zhang and Jessica Collins for their help and support, and their
friendship.

Danke Jost ♥


MRS Magnetic Resonance ABBREVATIONS
Spectroscopy
MS Mass Spectrometry
Ab Antibody MUFA Monounsaturated Fatty
ADP Adenosine Diphosphate Acids
myo-Ins myo-Inositol
Ala Alanine n.s. not significant
Asp Aspartate +NAD Nicotineamide Adenine
ATP Adenosine Triphosphate Dinucleotide (oxd)
BSA Bovine Serum Albumin NADHmide
Cho Choline-containing Dinucleotide (red)
compounds NMR Nuclear Magnetic
Cho(l) Cholesterol Resonance
CML Chronic Myeloid Leukemia NSCLC Non-Small Cell Lung
Cr Creatine Cancer
COX-2 Cyclooxygenase-2 NTP Nucleotide Triphosphate
DAG Diacylglycerol PBS Phosphate Buffered Saline
EDTA Ethylendiaminetetraacetate PC Phosphocholine / Pyruvate
EGF(R) Epidermal Growth Factor Carboxylase
(Receptor) PCA Perchloric Acid
FA Fatty Acids PCr Phosphocreatine
FACS Flow Cytometry PDH Pyruvate Dehydrogenase
FBS Fetal Bovine Serum PE Phosphoethanolamine
FT Fourier Transformation P-gp P-glycoprotein
5-FU 5-Fluorouracil Ph Philadelphia Chromosome
Glc Glucose P Inorganic Phosphate i
Glu Glutamate PDE Phosphodiester
Gln Glutamine PME Phosphomonoester
GLT Glutathione ppm parts per million
GSH Reduced Glutathione PtdCho Phosphatidylcholine
GSSG Oxidized PUFA Polyunsaturated Fatty Acids
Gly Glycine ROS Reactive Oxygen Species
GPC Glycerophosphocholine RT-PCR Real-time Polymerase
GPE Glyospho- Chain Reaction
ethanolamine SD Standard Deviation
HPLC High Performance Liquid TAG Triacylglycerol
Chromatography TCA Tricarboxylic acid
HRP Horseradish Peroxidase TMSP Trimethylsilylpropionic-
HSQC Heteronuclear Single 2,2,3,3-d -acid 4
Quantum Correlation TK Tyrosine Kinase
Ile Isoleucine TKI Tyrosine Kinase Inhibitor
Lac Lactate Tyr Tyrosine
LDH Dehydrogenase VEGF(R) Vascular Endothelial
MDR Multi-drug Resistance Growth Factor
MRM Multiple Reaction
Monitoring

iiCHAPTER 1 .........................................................................................................4
1. ZUSAMMENFASSUNG.......................................................................................................................... 4
1. SUMMARY............................................................................................................................................. 10
CHAPTER 2 .......................................................................................................15
2. INTRODUCTION .................................................................................................................................. 15
2.1. Blood cancer: an overview ............................................................................................................... 15
2.1.1. Chronic myeloid leukemia (CML): Background ...................................................................... 16
2.1.2. Regulation of normal tyrosine kinase activity........................................................................... 17
2.1.3. Mechanisms of TK dysregulation in cancer.............................................................................. 18
2.1.4. Strategies to target TKs in cancer therapy... 18
2.2. Imatinib Mesylate: The first successful small molecule TK inhibitor.............................................. 19
2.2.1. Imatinib: mechanism of action.................................................................................................. 19
2.2.2. Imatinib Resistance ................................................................................................................... 21
TM2.3. Gefitinib (Iressa ): Small molecule inhibitor of EGFR.................................................................. 25
2.3.1. Gefitinib: Mechanism of action................................................................................................. 25
2.3.2. Combination therapy: gefitinib with chemotherapeutics and COX-2 inhibitors....................... 27
2.4. Magnetic Resonance Spectroscopy (MRS) in biological systems.................................................... 28
2.5. Aims of the Study and Research Strategies ...................................................................................... 29
CHAPTER 3 .......................................................................................................31
3. RESULTS AND DISCUSSION............................................................................................................. 31
3.1. Imatinib resistance development: Long-term incubation of leukemia cells...................................... 31
3.1.1. Imatinib influence on cell proliferation and viability................................................................ 32
3.1.2. Glucose and energy metabolism in K562 leukemia cells / Changes after long-term imatinib
treatment ............................................................................................................................................. 34
3.1.3. Changes in lipids metabolism in K562 cells after long-term imatinib treatment ...................... 40
3.1.4. Changes in cell volume in K562 cells after long-term imatinib treatment................................ 44
3.1.5. Changes in amino acid concentrations rm iment............................... 45
3.1.6. Changes in Bcr-Abl protein expression and protein phosphorylation....................................... 45
3.1.7. Glucose uptake studies and Glut-1 transporter expression levels (total protein and m-RNA
expression) after long-term imatinib treatment................................................................................... 46
3.1.8. Changes in intracellular imatinib concentration and p-glycoprotein expression....................... 50
3.1.9. Discussion and Conclusions: Long-term imatinib treatment .................................................... 52
3.2. Imatinib effects: Short-term incubation of leukemia cells................................................................ 58
3.2.1. Imatinib influence on cell proliferation and viability 58
3.2.2. Glucose and energy metabolism in K562 leukemia cells / Changes after short-term imatinib
treatment ............................................................................................................................................. 59
3.2.3. Changes in lipids metabolism after short-term imatinib treatment ........................................... 61

3.2.4. Changes in amino acid concentrations after short-term imatinib treatment .............................. 63
3.2.5. Changes in Bcr-Abl protein expression and protein phosphorylation....................................... 63
3.2.6. Glucose uptake and Glut-1 transporter expression after short-term treatment.......................... 63
3.2.7. Changes in intracellular imatinib concentrations and p-glycoprotein expression ..................... 66
3.3. Imatinib-resistant cell lines: Metabolic profiling.............................................................................. 68
3.3.1. Imatinib resistant cell lines: proliferation and viability............................................................. 68
3.2.2. Glucose and energy metabolism in imatinib-resistant K562 and LAMA84 cells .....................