Employing Pancreatic Tumour Γ-Glutamyl Transferase for Therapeutic Delivery
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Employing pancreatic tumour γ-glutamyl transferase for therapeutic delivery Emma E Ramsay A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Prince of Wales Clinical School Lowy Cancer Research Centre Faculty of Medicine University of New South Wales March 2014 List of publications Ramsay, E. E., Hogg, P. J., & Dilda, P. J. (2011). Mitochondrial metabolism inhibitors for cancer therapy. Pharmaceutical Research, 28, 2731–2744. Ramsay, E.E., Decollogne, S., Joshi, S., Corti, A., Apte, M., Pompella, A., Hogg, P.J. & Dilda, P.J. (2014). Employing pancreatic tumour γ-glutamyltransferase for therapeutic delivery. Molecular Pharmaceutics, (in press). Ramsay, E.E., Hogg, P.J. & Dilda, P.J. Glutathione-S-conjugates as potential prodrugs to target γGT-expressing, drug-resistant tumours. Frontiers in Pharmacology, (in preparation). List of published abstracts Decollogne, S., Ramsay, E. E., Joshi, S., Corti, A., Pompella, A., Apte, M., Hogg, P. J., Dilda, P. J. (2012). Both pancreatic cancer and pancreatic stellate cells express high levels of gamma- glutamyl transferase that may be employed to deliver a metabolism inhibitor to the tumour mass. EACR 22nd Biennial Meeting, Barcelona, European Journal of Cancer, 48(s5), 251, abstract 1041. List of presentations Dilda, P. J., Decollogne, S., Ramsay, E. E., Park, D., & Hogg, P. J. (2010). A tumour marker for selection of patients that should better respond to treatment with the Australian anti-cancer drug, GSAO. In The Lowy Symposium. Sydney, Australia. Ramsay, E. E., Decollogne, S., Joshi, S., Hogg, P. J., & Dilda, P. J. (2011). Identification of a tumour enzyme responsible for the activation of the metabolism inhibitor, GSAO. In 23rd Lorne Cancer Conference. Lorne, Australia. Decollogne, S., Ramsay, E. E., Joshi, S., Hogg, P. J., & Dilda, P. J. (2011). Tumour cell expression of γ-glutamyl transpeptidase positively correlates with the anti-tumour efficacy of the metabolism inhibitor, GSAO. In 23rd Lorne Cancer Conference. Lorne, Australia. Ramsay, E. E., Decollogne, S., Joshi, S., Corti, A., Pompella, A., Apte, M., Hogg, P.J. & Dilda, P. J. (2012). Pancreatic tumor stromal cells express high levels of γ-glutamyl transferase that may iii be employed to deliver a metabolism inhibitor to the tumor mass. In 103rd Annual Meeting of the American Association for Cancer Research. Chicago, Illinois. Ramsay, E. E., Decollogne, S., Joshi, S., Corti, A., Pompella, A., Apte, M., Hogg, P.J. & Dilda, P. J. (2012). Tumour expression of γ-glutamyl transferase may be utilised to predict patient response to γ-glutamyl prodrugs. In ASMR 20th New South Wales Scientific Meeting. Sydney, Australia. Ramsay, E. E., Decollogne, S., Joshi, S., Corti, A., Pompella, A., Apte, M., Hogg, P.J. & Dilda, P. J. (2012). Pancreatic tumour gamma-glutamyl transferase expression to predict patient response to the metabolism inhibitor GSAO. In Sydney Cancer Conference. Sydney, Australia. (Oral presentation). List of scholarships and awards (2013) European Association for Cancer Research Travel Fellowship Award (2013) Australian Society for Medical Research Research Award (International) (2012-2013) Pfizer Oncology Research Unit Scholarship (2010-2014) NHMRC Biomedical Scholarship (2010-2012) The Cancer Institute of NSW Research Scholar Award (2012) Translational Cancer Research Network Top-Up Scholarship (2011-2012) The Commercialisation Training Scheme Scholarship (2011) Lorne Cancer Bursary iv Acknowledgments I would like to thank and acknowledge the contributions of Doctor Stephanie Decollogne from the Lowy Cancer Research Centre, Sydney, who performed the in vivo experiments described in Chapter 7 with my assistance. Thank you for your time and patience in showing me these skills. I would also like to thank Mrs Swapna Joshi for her assistance with the immunohistochemistry presented in Chapter 2 and 7. I would like to thank and acknowledge the work of the Pancreatic Research Group, University of New South Wales, in particular Professor Minoti Apte and Ms Eva Fiala-Beer for the primary human pancreatic stellate cells. I wish to acknowledge Mrs Rabeya Akter from the Mark Wainwright Analytical Centre at the University of New South Wales for elemental analysis. I would like to acknowledge the work of the Infection and Immunity Research Group of the University of New South Wales, in particular Professor Andrew Lloyd, Mrs Koko Bu and Doctor Hoai Nguyen for provision of the plasma samples used in Chapter 6. I would like to acknowledge Doctor Amber Johns of the New South Wales Pancreatic Cancer Network for providing human pancreatic sections in Chapter 2. I would like to acknowledge those who funded me through my PhD: the National Health and Medical Research Council, the Cancer Institute of New South Wales, and the Translational Cancer Research Network. I would like to thank my supervisors, Professor Philip Hogg and Doctor Pierre Dilda. Their support and encouragement made this experience much easier than I had ever imagined. They have generously allowed me to experience so much of research life; giving me opportunity for an internship, collaborations, conferences and courses, all of which have given me many insights into the facets of biomedical research. In particular, I’d like to thank them for the opportunity to travel to Sweden and the USA to experience research life in different cultures and contexts. I would like to thank the Australian Society for Medical Research and the European Association for Cancer Research for the opportunity to travel to Sweden for collaboration with Professor Matthias Löhr and Doctor Rainer Heuchel at the Karolinska Institutet. Thank you, Matthias and Rainer, for accepting me into your lab and giving me time and guidance throughout my stay. I would also like to thank Pfizer Inc. and the Cancer Institute of New South Wales for the opportunity to undertake a six month internship with Pfizer under the supervision of Doctor v Julie (Xie) Zhi. Both trips were enriching experiences, and I am very thankful to have had these opportunities. Thank you to everyone who contributed in their own way to this thesis: family, friends and colleagues. Your encouragement and support throughout have been a huge blessing. Finally, to my Dad, thank you for all your love and support and thank you for always believing in me, no one could ask for a better father. vi Abbreviations 5FU 5-fluorouracil ABBA L-2-amino-4-boronobutanoic acid ADEPT Antibody-directed enzyme prodrug therapy ADP Adenine dinuceleotide phosphate ALK Anaplastic lymphoma kinase ANT Adenine nucleotide translocase APC Adenomatous polyposis coli As Arsenic ATP Adenine trinucleotide phosphate BAE Bovine aortic endothelial bFGF Basic fibroblast growth factor BMI Body mass index BxPC-3/vector BxPC-3 transfected with empty vector BxPC-3/γGT BxPC-3 transfected with the human γGT gene C Control CAO 4-(N-(S-cysteinylacetyl)amino)phenylarsonous acid Cdkn2A Cyclin-dependent kinase inhibitor 2A DMEM Dulbecco’s modified eagle’s media DNA Deoxyribonucleic acid DOPA Dihydroxyphenylalanine DPC Deleted in pancreatic cancer vii EBM-2 Endothelium basal medium -2 ECM Extracellular matrix EDTA Ethylenediaminetetraacetic acid EGFR Epidermal growth factor receptor FAP Fibroblast activation protein FGF Fibroblast growth factor FOLFIRINOX Folinic acid, 5FU, irinotecan and oxaliplatin GCAO 4-(N-(S-cysteinylglycylacetyl)amino)phenylarsonous acid GD Growth delay GDEPT Gene-directed enzyme prodrug therapy GFP Green fluorescent protein GSAO 4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid GSNO S-nitrosoglutathione GST Glutathione transferase hEGF Human epidermal growth factor hENT1 Human equilibrative nucleoside transporter 1 HER2 Human epidermal growth factor receptor-2 hFGF-B Human fibroblast growth factor HMEC Human dermal microvascular endothelial cells HNE 4-hydroxynoneal HPLC High performance liquid chromatography HUVEC Human umbilical vein endothelial cells IGF-1 Insulin-like growth factor-1 viii IMDM Iscove's modified dulbecco's media KRAS Kirsten rat sarcoma viral oncogene homologue LTC4 Leukotriene C4 MAPK Mitogen-activated protein kinase MMP Matrix metalloproteinase MPTP Mitochondrial permeability transition pore MRP Multi-drug resistance proteins MTT 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide N/A Not applicable NHGs N-hydroxyguanidines NIEHS National Institute of Environmental Health Sciences NP No processing N-PSC Normal human pancreatic stellate cells NSCLC Non-small cell lung cancer P Processing PAO Phenylarsonous acid PCNA Proliferating cell nuclear antigen PDAC Pancreatic ductal adenocarcinoma PDGE Prostaglandin E PDGF Platelet-derived growth factor PI Propidium iodide PI3K Phosphatidylinositol 3-kinase PSA Prostate specific antigen ix PSC Pancreatic stellate cells PSMA Prostate specific membrane antigen R3-IGF-1 Recombinant insulin-like growth factor RPMI Roswell park memorial institute SD Standard deviation SDF-1 Stromal cell-derived factor 1 SE Standard error SERCA Sarcoendoplasmic reticulum calcium transport ATPase T Treated TA-PSC Tumour associated human pancreatic stellate cell TGDi Tumour growth delay index TGF-h1 Transforming growth factor h1 TGFβ Transforming growth factor β TVQT Tumour volume quadrupling time uPA Urokinase-type plasminogen activator VDEPT Virus-directed enzyme prodrug therapy VEGF Vascular endothelial growth factor α-SMA Alpha-smooth muscle actin γGT γ-glutamyl transferase x Table of contents Declarations ..................................................................................................................................