CONTENTS

List of Figures

i-ii

Abbreviations

iii

Introduction

1-32

1.1. Arachidonic acid metabolism

1.2. Lipoxygenases
1.2.1. Occurrence
1.2.2. Classification and Nomenclature
1.2.3. Enzyme Structure
1.2.4. The mechanism of the lipoxygenase reaction
1.3. 5-Lipoxygenase
1.4. 8-Lipoxygenase
1.5. 12-Lipoxygenase
1.6. 15-Lipoxygenase
1.6.1. Enzymatic properties of 15-LOX
1.6.2. Regulation of cellular LOX activity
1.6.3. Biological role of 15-LOXs
1.7. LOX metabolism, polyunsaturated fatty acids and carcinogenesis
1.7.1. Procarcinogenic LOX metabolism of arachidonic acid
1.7.2. Anticarcinogenic LOX metabolism of arachidonic and
linoleic acids
1.8. Apoptosis
1.9. Oxidative stress & apoptosis
1.9.1. Sources of ROS/RNI production
1.9.2. Relevance of oxidative stress to apoptosis
1.9.3. Models for oxidative stress-induced apoptosis
1.9.4. Antioxidants and protection against apoptosis

Scope & Objectives

33-35

Materials & Methods

36-47

3.1 Reagents
3.2. Preparation and separation of 15-LOX metabolites - HPLC analysis
3.3. LC-MS analysis
3.4. Cell culture and treatment
3.5. Cell viability and cytotoxicity
3.6. Morphological differentiation and analysis of nuclear morphology
3.7. DNA fragmentation assay
3.8. Quantification of apoptosis by flow cytometry
3.9. Preparation of whole cell extracts and immunoblot analysis
3.10. Detection of cytochrome c release using Western blot analysis
3.11. Measurement of reactive oxygen species (ROS)
3.12. Estimation of cellular glutathiones (GSH & GSSG)
3.13. Caspase-3 activity assay
3.14. Antioxidant enzyme assays
3.14.1. Assay of superoxide dismutase (SOD)
3.14.2. Assay of catalase
3.14.3. Assay of glutathione peroxidase (GPx)
3.15. Electrophoretic mobility shift assay (EMSA)

Results

48-78

4.1. HPLC and LC-MS analysis

4.2. Effects of 15-LOX metabolites on the growth of K-562 cell line
4.3. Phase contrast microscopy
4.4. Fluorescence microscopic studies
4.5. Flow cytometric analysis of 15-LOX-2 metabolite-induced apoptosis
4.6. 15-(S)-HPETE and 15-(S)-HETE treatment evoke cytochrome c release
4.7. Caspase-3 activation in response to 15-(S)-HPETE and 15-(S)-HETE
treatment
4.8. Z-VAD-FMK, a broad spectrum caspase inhibitor, prevents 15-LOX-2
metabolite-induced apoptosis

4.9. PARP cleavage in response to 15-LOX-2 metabolite treatment

4.10. 15-LOX-2 metabolites induced DNA fragmentation in K-562 cells
4.11. Reactive oxygen species (ROS) mediate 15-LOX-2 metabolite - induced
cell death
4.12. 15-LOX-2 metabolites activate NADPH Oxidase to generate ROS and
subsequently induce apoptosis
4.13. Role of calcium channel blockers in 15-LOX-2 metabolite-induced apoptosis
4.14. Effect of 15-LOX-2 metabolites on antioxidant enzymes
4.15. Cellular glutathione peroxidase levels and induction of apoptosis by
15- LOX-2 metabolites
4.16. Effect of 15-LOX-2 metabolites on transcription factors AP-1 and NF-kB

Discussion

79-93

5.1. Differential effects of 15-LOX-1 and 15-LOX-2 metabolites on the

growth of chronic myeloid leukemia cell line - K562
5.2. 15-LOX-2 metabolites induce apoptosis in K-562 cells
5.3. 15-LOX-2 metabolites induce apoptosis through intrinsic death pathway
5.4. ROS mediates 15-LOX-2 metabolite-induced apoptosis in K-562 cells
5.5. NADPH Oxidase mediates ROS generation in K-562 cells exposed to
15-LOX-2 metabolites
5.6. Calcium plays a key role in 15-LOX-2 metabolite-induced apoptosis
5.7. Catalase and ERK signaling in 15-LOX-2 metabolite induced apoptosis in
K-562 cells
5.8. Cellular Peroxidase is critical in 15-LOX-2 metabolite-induced cell death
5.9. A hypothesis in making

Summary

94-99

References

100-123