Toner, Aidan Publication Date 2015-03-31 I

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Preclinical assessment of the novel compound EL102 in
Title
prostate cancer
Author(s) Toner, Aidan
Publication
2015-03-31
Date
Item record http://hdl.handle.net/10379/5019
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Preclinical Assessment of the Novel
Compound EL102 in Prostate Cancer
Aidan Toner M.Sc. B.Sc.
Thesis submitted to the
National University of Ireland, Galway
for the award of Ph.D. in the
School of Medicine

Supervisors:
Dr. Sharon Glynn Ph.D. M.P.H.
Prof. Frank Sullivan MB MRCPI FFR RCSI
March 2015
Contents Page

Contents i
Declaration vii
Research Outputs viii
List of Figures x
List of Tables xii
List of Abbreviations xiii
Abstract xxi
Acknowledgements xxii
Dedication xxiv
Chapter 1: General Introduction 1
1.1 Prostatic Disease 2
1.1.1 The Human Prostate 2
1.1.2 Prostatitis 3
1.1.3 Benign Prostatic Hyperplasia 4
1.1.4 Prostate Cancer Diagnosis and Incidence 5
1.2 Prostate Cancer Treatment 11
1.2.1 Prostatectomy 11
1.2.2 Radiotherapy 12
1.2.2.1 Brachytherapy 12
1.2.2.2 External Beam Radiation Therapy 13
1.2.3 Hormone Therapy 13
1.2.3.1 Androgen Receptor 14
1.2.3.2 Gonadotropin-Releasing Hormone Agonists
and Antagonists 16
1.2.3.3 Hormone-Responsive Anti-Androgen Therapy 17
1.2.3.4 Castrate-Resistant Anti-Androgen Therapy 18
1.2.3.5 Surgical Anti-Androgen Therapy 20
1.2.4 Prostate Cancer Chemotherapy 20
1.2.4.1 Paclitaxel 20
1.2.4.2 Docetaxel 22
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1.2.4.3 Cabazitaxel 23
1.2.4.4 Mitoxantrone 24
1.2.4.5 Anthracyclines 24
1.2.4.6 Estramustine 25
1.2.5 Chemoresistance in Prostate Cancer 25
1.2.5.1 Multi-Drug Resistance Efflux Protein Pumps 26
1.2.5.2 AR-Mediated Chemoresistance 27
1.2.5.3 Chemoresistance Through Proliferation and
Survival Pathways 28
1.2.5.4 Chemoresistance Conferred by Cancer Stem
Cells 28
1.3 Recent Advances in Prostate Cancer Treatment 29
1.3.1 Sipuleucil-T 29
1.3.2 Radium-223 29
1.3.3 HIF1α Inhibitors 30
1.3.3.1 Heteroaryl and Aromatic HIF1α Inhibitory
Agents 31
1.3.3.2 Steroidal HIF1 Inhibitors 32
1.3.3.3 RNA Antagonists 33
1.3.3.4 mTor Inhibitors 33
1.3.3.5 HSP90 Inhibitors 33
1.3.3.6 Non-Steroidal Anti-Inflammatory Drugs 34
1.3.3.7 Chetomin 34
1.4 Toluidine Sulphonamides 34
1.4.1 ELR510444 30
1.4.2 ELR510552 (EL102) 35
Chapter 2: Materials and Methods
38
2.1 Cell Culture 39
2.1.1 Cell Lines 39
2.1.2 Culture Conditions 40
2.2 Cell Viability and Apoptosis Assays 41
ii
2.2.1 Resazurin-Based Assays 42
2.2.2 Sulforhodamine B-Based Assays 42
2.2.3 Toxicity in Multi-Drug Resistant Cell Lines 43
2.2.4 DEVD Caspase 3/7 Activation Assays 44
2.2.5 Sub-G and Cell Cycle Analysis 45
1
2.2.6 Analysis of PARP Cleavage 46
2.3 Protein Analysis 46
2.3.1 Antibodies 46
2.3.2 Protein Isolation and Quantification 47
2.3.3 Western Blot Analysis 49
2.3.3.1 In-House Gel/Wet Transfer Methodology 49
2.3.3.2 Precast Gel/Dry Transfer Methodology 52
2.3.3.3 Detection of Proteins 52
2.3.4 Cellular Fractionation of Proteins 53
2.3.5 Immunocytochemistry 54
2.3.5.1 Cell Fixation 54
2.3.5.2 Immunostaining 54
2.3.6 HIF1 TransAM ELISA 55
2.4 Genetic Analysis 56
2.4.1 Plasmid Preparation 56
2.4.1.1 Bacterial Transformation 56
2.4.1.2 Miniprep 57
2.4.1.3 Restriction Digest 58
2.4.1.4 Making Glycerol DH5α Stocks 59
2.4.1.5 Maxiprep 59
2.4.2 Transient Transfections 61
2.4.3 Reporter Gene Assays 62
2.4.3.1 Cell Lysis 62
2.4.3.2 CPRG Assay 62
2.4.3.3 Luciferase Assay 63
2.4.4 Total RNA Isolation 63
iii
2.4.5 cDNA Synthesis 64
2.4.6 qPCR 65
2.4.7 Primers 66
2.5 Cell Migration Assays 67
2.6 Tumour Xenograft Models 67
2.7 Preliminary Methodology: Elara Pharmaceuticals
Findings 68
2.7.1 Proteomic Analysis of HIF1α Activity 68
2.7.2. Binding Assays 69
2.7.2.1 Kinase Binding Screen 69
2.7.2.2 In Vitro Pharmacology: Diversity Profile 70
2.8 Statistical Analysis 71
2.9 Suppliers and Distributors 72
Chapter 3: Analysis of the Cytotoxic Profile of
EL102 in A Panel of Prostate Cancer Cell Lines 74
3.1 Introduction 75
3.2 Results 77
3.2.1 Cell Viability Assays and Determination of the
IC Values of EL102 77
50
3.2.2 Tumour Volume Analysis of an In Vivo CWR22
Murine Xenograft Model 79
3.2.3 In Vitro Analysis of the Combined Effect of
EL102 and Docetaxel 83
3.2.4 Cell Death and Induction of Apoptosis in EL102-
Dosed Prostate Cancer Cell Lines 84
3.3 Discussion 90
3.3.1 EL102 Treatment Reduces Prostate Cancer Cell
Viability In Vitro 90
3.3.2 Combined Therapeutic Effect of EL102 and
Docetaxel In Vivo Differs In Vitro 90
3.3.3 EL102-Induced Apoptosis Induction 91

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3.3.4 Clinical Potential For EL102 as a
Chemotherapeutic Agent 92
Chapter 4: Exploring the In Vitro Mechanisms of
EL102 in Drug-Resistance, Microtubule Dynamics
and HIF1α Signalling
94
4.1 Introduction 95
4.2 Results 97
4.2.1 EL102 and Microtubule Dynamics In Vitro 97
4.2.2 The Impact of EL102 Treatment on the Growth of
Two Multi-Drug Resistant Cell Models 100
4.2.3 Examining the Inhibitory Effects of EL102 on a
Panel of Kinases 103
4.2.4 EL102 Reduces Cellular HIF1α Protein In Vitro 104
4.2.5 Examining the Effects of EL102-Mediated HIF1α
Deactivation 106
4.3 Discussion 110
4.3.1 EL102 Destabilises Microtubule Structures 110
4.3.2 EL102 Circumvents Two Classic Models of Drug
Resistance In Vitro 112
4.3.3 The Ability of EL102 to Inhibit HIF1α and Certain
Kinases May Have Significant Clinical Implications 113
Chapter 5: Investigating the Potential Role of EL102
as a Disruptor of Androgen Signalling in Prostate
Cancer 115
5.1 Introduction 116
5.2 Results 117
5.2.1 Ligand–Nuclear Receptor Interaction In the
Presence of EL102 117
5.2.2 Determination of the Cytotoxic Profile of EL102
in AR-Positive Cell Line, LNCaP 119
5.2.3 EL102 Decreases AR Protein and AR Gene
Expression In Vitro 120
v
5.2.4 Analysis of the Expression Patterns of AR
Protein Following EL102 Treatment 122
5.2.5 EL102 Disrupts the Binding of AR to Androgen
Response Elements in a Dose-Dependent Manner 124
5.2.6 Investigating the In Vitro Effects of EL102 on
AR-Induced CXCR4 Expression and CXCR4-Mediated
Migration 126
5.3 Discussion 128
5.3.1 EL102 Inhibits the Binding of Androgen and
Progesterone to AR and PR, Respectively, in a Cell-
Free Assay 128
5.3.2 EL102 Inhibits Expression and Activation of AR
In Vitro 129
Chapter 6: Final Discussion
131
6.1 Overview 132
6.2 HIF1α Inhibition and Microtubule Disruption in
Prostate Cancer Treatment 133
6.3 Castration-Resistance and Chemotherapy Resistance
in Prostate Cancer 134
6.4 Experimental Critique 136
6.5 Conclusions 138
Chapter 7: Bibliography 139
Appendices 170
Appendix I – Cell Line Specifications 171
Appendix II – The novel toluidine sulphonamide EL102
shows pre-clinical in vitro and in vivo activity against
prostate cancer and circumvents MDR1 resistance
Toner et al. Br J Cancer (2013) 181
Appendix III – Ambit KinomeScan Results 194
Appendix IV – Cerep Diversity Profile 201

vi
Declaration:

I, Aidan Toner, certify that this thesis is my own work, with the exception of
instances wherein I have presented collaborative data and have clearly
identified and credited the collaborating party by name and/or appropriate
citation. I also, certify that I have not previously obtained a degree in this
University or elsewhere else on the basis of any of this work.

Signed:_____________________________ Date: _____________

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Research Outputs:
Peer-Reviewed Research Publication:
The novel toluidine sulphonamide EL102 shows pre-clinical in vitro and in vivo
activity against prostate cancer and circumvents MDR1 resistance. Toner et al.
Br J Cancer. 2013 Oct 15; 109 (8):2131-41.
Oral Presentations:
EL102, A Novel Chemotherapeutic Candidate for the Treatment of Prostate
Cancer, Inhibits HIF1α Activity, AR Expression and Microtubule Stability. NUIG
College of Medicine, Nursing and Health Science Postgraduate Research Day,
May 28, 2014.
Introducing novel toluidine sulfonamide EL102: A potential chemotherapeutic
agent in prostate cancer. (Abstract published Journal of Clinical Urology.
September 2013 vol. 6 no. 5 p338). Irish Society of Urology Annual Meeting
2013 Sept. 20-21.
Introducing novel toluidine sulphonamide EL102, a potential chemotherapeutic
in prostate cancer. (Abstract published 2013 BJU International, Vol.111,
Supplement 3, p61.) British Association of Urological Surgeons (BAUS),
Manchester, U.K., June 17th – 20th 2013.
Abstracts and Poster Presentations:
Further preclinical assessment of compound EL102 in the treatment of prostate
cancer. Journal of Clinical Oncology, 2014 ASCO Annual Meeting Abstracts.Vol
32, No 15_suppl (May 20 Supplement), 2014.
Characterisation of a New Class of Prostate Cancer Chemotherapeutics. Irish
Association for Cancer Research (IACR) Annual Meeting, Galway Bay Hotel,
Galway. Feb 27 & 28, 2014.
Preclinical analysis of EL102, a novel compound proposed for the treatment of
prostate cancer. 3rd Annual Academic Screening Workshop Sheraton Baltimore
North Hotel, MD. Sept 17 & 18, 2013.
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