Investigating the Role of Heme Oxygenase and Oxidative Stress in Oesophagogastric Cancer Oliver Hartwell Priest Department of Surgery and Cancer Imperial College London St Mary’s Campus London, UK A thesis submitted to Imperial College London for the degree of Doctorate of Philosophy Supervisors Professor George Hanna Department of Surgery and Cancer, Imperial College London St Mary’s Campus, London, UK Dr Nandor Marczin Department of Anaesthetics, Pain Medicine and Intensive Care Imperial College London, Chelsea and Westminster Campus, London, UK 2 ABSTRACT Background: Molecular mechanisms underlying gastric and oesophageal cancer include alterations in growth factors, cytokines and cell adhesion molecules. Heme oxygenase (HO) enzyme catalyses the degradation of heme and generates bilirubin and carbon monoxide that have antioxidant, anti-inflammatory and anti-apoptotic activities. HO enzyme is implicated in the biology of cancer by its effects on cell growth and resistance to apoptosis. The roles of HO-1 and HO-2 enzymes in cancer cell growth are poorly understood, with reports suggesting both anti-inflammatory, anti-tumour effects and tumour-protective mechanisms mediated by HO activity. The role of HO-2 in inflammation and cancer is largely unexplored. Further understanding the influence of the HO enzyme system may provide improved novel targets for oesophagogastric cancer therapy. Materials and Methods: The primary objective of the thesis was to characterise the role of the heme oxygenase pathway and modulation of HO activity in upper gastrointestinal cancer cell growth in vitro. Cell culture techniques included MTT growth assay, Western blotting protein analysis, pharmacological modulation of HO activity and targeted knockdown of HO mRNA. Apoptosis was studied with flow cytometry techniques. Enzymatic HO activity was measured by specific colorimetric assay. Results: Results showed that specific knockdown of HO-2 protein activity with induction of HO-1 enzyme caused a reduction in oesophageal and gastric cancer cell growth with cell cycle effects and an increase in apoptosis. Treatment with downstream products of HO activity carbon monoxide and bilirubin and the anti-inflammatory agent N-acetyl cysteine had similar effects and increased the response of cancer cells to chemotherapeutic agents. Conclusion: These studies further our understanding of the role of heme oxygenase in cancer, providing evidence that modification of HO activity in oesophagogastric cancer cells leads to reduced cell proliferation. The findings support future potential clinical applications of carbon monoxide therapy that require further experimental animal model and clinical studies in the context of oesophagogastric cancer. 3 Declaration of Originality: I hereby declare that I am the sole author of this thesis and that all work within it is my own. Any individuals who carried out work in collaboration with the author are appropriately credited and all reviewed literature appropriately referenced. Copyright Declaration: The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 4 Peer-Reviewed Publications and Presentations Published Abstracts: Priest OH, Bundy R, Marczin N, Hanna GB. Role of haem oxygenase in gastric cancer cell growth. Brit J Surg 2007; 94(S5): 55 Priest OH, Bundy R, Marczin N, Hanna GB. Role of Heme Oxygenase and Oxidative Stress in Gastric Cancer Cell Growth. Brit J Surg 2008; 95(S4): 31 – 33 Priest OH, Bundy RE, Kirolos A, Marczin NM, Hanna GB. N-acetyl cysteine inhibits human oesophageal adenocarcinoma cell growth and increases chemosensitivity via reduced HO activity and induction of apoptosis. Brit J Surg 2013; 100(S8): 58 – 62 Presentations: Priest OH, Agarwal R, Marczin NM, Hanna GB. Modification of heme oxygenase activity increases the response of gastric cancer cells to chemotherapy. 4th London Surgical Symposium, Imperial College London, September 2010 Priest OH, Motterlini R, Marczin NM, Hanna GB. Carbon monoxide releasing molecule-2 inhibits oesophageal adenocarcinoma cell proliferation by activating HO-1 signalling. 3rd London Surgical Symposium, Imperial College London, September 2009 Manuscripts in preparation: Priest OH, Bundy RE, Marczin NM, Hanna GB. Modification of heme oxygenase activity increases the response of gastric cancer cells to chemotherapy. Priest OH, Motterlini R, Marczin NM, Hanna GB. Carbon monoxide releasing molecule-2 inhibits oesophageal adenocarcinoma cell proliferation by activating HO-1 signalling. 5 Acknowledgements I am greatly indebted to my supervisors Professor George Hanna and Dr Nandor Marczin for their unfailing support, guidance and continued encouragement throughout the course of my study. I would also like to express my gratitude to the following people who have helped me during the course of my research: Dr Gretta Roberts – for guidance and training in cell culture methods Dr Ruth Bundy – for supervision and expertise in basic science experiments Dr Leanne Felkin – for real-time PCR protocols and supervision Dr Roberto Motterlini – for advice regarding the use of carbon monoxide-releasing molecules Dr Niga Nawroly – for training and support in flow cytometry techniques Mr Amir Kirolos – for assistance in cell culture experiments Cell culture experiments were supported by a grant from the European Union BAMOD Grant (framework 6), BAMOD, LSHC-CT-2005-019031. I would like to dedicate this work to my wife Lizzie whose grace, patience and equanimity made it all possible. 6 TABLE OF CONTENTS CHAPTER 1: INTRODUCTION .................................................................................................. 19 1.1 Oesophagogastric cancer – the clinical problem ............................................................ 19 1.1.1 Oesophageal carcinoma .................................................................................................. 19 1.1.2 Gastric carcinoma ............................................................................................................ 25 1.2 The Heme Oxygenase System ....................................................................................... 27 1.2.1 The physiological role of heme oxygenase ..................................................................... 27 1.2.2 Iron, Heme and Ferritin ................................................................................................... 28 1.2.3 Characterisation of the Heme Oxygenase Enzyme ......................................................... 35 1.2.4 Heme oxygenase isoenzymes ......................................................................................... 39 1.3 The consequences of heme breakdown......................................................................... 43 1.3.1 Heme oxygenase enzyme products ................................................................................ 43 1.3.2 Heme oxygenase and inflammation ............................................................................... 49 1.3.3 Heme oxygenase and cancer........................................................................................... 56 1.3.4 HO gene promoter polymorphism .................................................................................. 63 1.3.5 The role of the HO pathway in cancer cell biology ......................................................... 67 1.3.6 Heme oxygenase and anti-cancer therapy...................................................................... 73 1.3.7 Mechanisms of heme oxygenase activity in cancer ........................................................ 76 1.3.8 Inflammation and cancer ................................................................................................ 78 1.4 Study Aims and Objectives............................................................................................ 81 1.4.1 Hypotheses of study ........................................................................................................ 81 1.4.2 Aims of study ................................................................................................................... 81 CHAPTER 2: MATERIALS AND METHODS ................................................................................. 83 2.1 Cell Culture Experiments............................................................................................... 83 2.1.1 Cell Culture Techniques ................................................................................................... 83 2.1.2 Cell seeding and cell counting technique ........................................................................ 85 2.1.3 Measurement of cell growth: Cell counts and MTT assay technique ............................. 86 2.1.4 Obtaining growth curves for cell lines under different conditions ................................. 87 2.2. Investigating Protein Expression with Western Blotting ................................................ 87 2.2.1 Protein Assay ................................................................................................................... 87 2.2.2 Gel electrophoresis ........................................................................................................