Identification and characterization of a novel Palladacycle, AJ-5, to treat advanced Melanoma and

195 Pages·2014·10.35 MB·English

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The copyright of this thesis vests in the author. No
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quotation from it or information derived from it is to be

published without full acknowledgeement of the source.
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The thesis is to be used for private study or non-
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commercial research purposes only.

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Published by the Universit y of Cape Town (UCT) in terms
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of the non-exclusive license granted to UCT by the author.
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Dedicated to my wife
Mervat

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Declaration
I, Saeb Aliwaini, hereby declare that the work on which this thesis is based is my original work
and that neither the whole work nor any part of it has been, is being, or is to be submitted for
another degree in this or any other University. I empower the University to reproduce for the
purpose of research either the whole or any portion of the contents in any manner whatsoever.
Saeb Aliwaini
February, 2014
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Acknowledgements

Praise be to Allah, the lord of the world, and peace and blessings of Allah be upon the noblest
of the prophets and messengers, Prophet Muhammad.
This study could not be realised without the contribution of a number of individuals.
I wish to express my deepest gratitude and appreciation to my supervisor Sharon, who taught
me how to be a scientist. Thanks Sharon for your enlightening supervision, useful assistance,
continuous and valuable advice. I am also greatly indebted to Prof. Selwyn Mapolie, my co-
supervisor, for his kind assistance, unflagging support and guidance.
Many thanks to my colleagues, the T-box family members, who made the laboratory a
wonderful environment to work in, and provided precious assistance and advice. I would like to
give special thanks to Dr. Jade Peres for standing by me through the toughest times.
My parents, Hussein and Subhia, deserve my deepest gratitude for their continued belief,
support and love.
My father and mother-in-law, Shehada and Subhia, deserve my eternal thanks for their
continuous support; not only during my PhD study, but throughout the time I have known
them.
My dearest wife, Mervat, who loved me always and carried all my responsibilities so that I
could go away to get my PhD, I would not have been able to do this without you and I will never
forget this.
My best children Shahed, Tasneem, Ahmad, Malak and Muhammad, I never forgot you and I
will never leave you again.
This work was kindly supported by the University of Cape Town (UCT), National Research Fund
(NRF) and Muslim Judicial council (MJC).
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Table of Contents

Declaration 3
Acknowledgments 4
Table Contents 5
List of figures and tables 9
Abstract 11
Chapter 1: Literature Review 13
1.1 Introduction 13
1.2 Chemotherapy 14
1.3 Metal based alkylating agents 16
1.3.1 Platinum based compounds 16
1.3.1.1 Mechanism of anti-tumour activity of Platinum based compounds 17
1.3.2 Palladium based compounds 20
- Bidentate nitrogen ligands 21
- Phosphine ligands 23
- Other ligands 24
1.3.2.1 Mechanisms of anti-tumour activity of Palladium based compounds 25
- Induction of DNA damage 25
-Targeting organelles 26
1.4 Cell death 28
1.4.1 Apoptosis 28
1.4.1.1 Intrinsic apoptosis 29
1.4.1.2 Extrinsic apoptosis 31
1.4.2 Autophagy 32
1.4.2.1 Therapeutic modulation of autophagy 38
1.4.3 Cross talk between apoptosis and autophagy 40
1.5 Breast cancer 43
1.6 Melanoma 47
1.7 Aims of this study 50
Chapter 2: Materials and Methods 52
2.1 Cell culture 52
2.2 Mycoplasma test 52
2.3 Treatments 53
2.3.1 Palladium based compounds treatments 53
2.3.2 CDDP treatment 53
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2.3.3 Inhibitors 53
2.4 Cell morphology 54
2.5 Cytotoxicity assays 54
2.6 In Vitro cell migration assay 55
2.7 Mammosphere assay 55
2.8 Flow Cytometry 56
2.8.1 Cell cycle analysis 56
2.8.2 Measurement of apoptosis 57
2.8.3 Analysis of CD44/CD24 marker expression 57
2.9 Fluorescent microscopy 58
2.9.1 Nuclear fragmentation 58
2.9.2 Determination of cytochrome c release into the cytosol 58
2.9.3 Quantification of GFP-LC3 puncta 59
2.10 Electron microscopy 60
2.11 Western blot analysis 61
2.12 Transfection assays 62
2.13 Luciferase assays 64
2.14 Quantitative real-time PCR (qRT-PCR) 65
2.15 Chromatin immunoprecipitation (ChIP) assays 67
2.16 Nude mice study 68
2.16.1 Acute toxicity study 68
2.16.2 Sub- acute toxicity study 69
2.16.3 Anti-tumour activity study 69
2.17 Statistical analysis 70
Chapter 3: Results 71
Introduction 71
3.1 Screening of a group of palladium based compounds in melanoma and breast 73
cancer cell lines
3.2 The palladacycle, AJ-5, exhibits anti-tumour and anti-cancer stem cell activity in 75
breast cancer cells
3.2.1 AJ-5 inhibits cell proliferation and migration of human breast cancer cells 76
3.2.2 AJ-5 has putative anti-cancer stem cell activity 76
3.2.3 AJ-5 induces DNA damage and G1 cell cycle arrest 80
3.2.4 AJ-5 induces intrinsic and extrinsic apoptosis in breast cancer cells 82
3.2.5 AJ-5 induces autophagy and inhibits the mTOR pathway 88
3.2.6 AJ-5 induced cytotoxicity involves autophagy 91
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3.2.7 The p38 MAPK pathway mediates AJ-5 cytotoxicity 95
3.3 AJ-5 inhibits melanoma growth in vitro and in vivo through apoptosis 98
and autophagy
3.3.1 AJ-5 exerts potent anti-proliferative activities against human melanoma cells 98
3.3.2 AJ-5 induces S and G2/M cell cycle arrests 100
3.3.3 AJ-5 induces intrinsic and extrinsic apoptosis in melanoma cells 100
3.3.4 AJ-5 also induces autophagy in melanoma cells 104
3.3.5 AJ-5 regulates proteins involved in the DNA damage response and cell 107
cycle progression through p53 independent manner
3.3.6 AJ-5 induced cytotoxic effect is mediated by the activation of p38 and 111
ERK1/2 MAPK pathways
3.3.7 AJ-5 induced apoptosis and autophagy involves the inhibition 114
of the AKT/mTOR pathway
3.3.8 Inhibition of autophagy decreases AJ-5 induced cell death 118
3.3.9 AJ-5 inhibits melanoma tumour growth in vivo 118
3.4 The role of the developmentally important T-box factor, TBX3, in AJ-5 127
induced cytotoxicity
3.4.1 AJ-5 up-regulates TBX3 post-transcriptionally 127
3.4.2 AJ-5 up-regulates TBX3 in an AKT dependent manner 128
3.4.3 Knocking down TBX3 enhanced AJ-5 induced cytotoxicity 130
3.4.4 Knocking down TBX3 increases apoptosis and autophagy in MCF7 cells 132
3.4.5 TBX3 transcriptionally down regulates PUMA promoter activity 135
3.4.6 The DNA-binding domain of the TBX3 protein is required for repression 135
of PUMA
3.4.7 TBX3 binds the PUMA promoter in vivo 138
Chapter 4: Discussion 141
4.1 AJ-5 is an effective drug against estrogen receptor positive and triple 142
negative breast cancer cells
4.2 AJ-5 is a potent cytotoxic agent in advanced melanoma cell lines 144
4.3 AJ-5 triggers the intrinsic and extrinsic apoptotic pathways 145
4.4 AJ-5 induces autophagic cell death 146
4.5 AJ-5 inhibits the mTOR pathway 147
4.6 AJ-5 cytotoxicity is mediated by the MAPKs 148
4.7 AJ-5 induced apoptosis is p53 independent 149
4.8 Knocking down TBX3 in a TBX3-driven breast cancer cell line sensitises them to 152
AJ-5 treatment
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4.9 Concluding remarks 154
Chapter 5: References 155
Chapter 6: Appendix 185
6.1 Mycoplasma Test 185
6.2 Palladium based compounds 185
6.3 Flow Cytometry 190
6.4 Immunofluorescence 190
6.5 Electron microscopy 190
6.6 Western Blot analysis 190
6.7 Plasmids and DNA constructs 191
6.8 Chromatin immunoprecipitation (ChIP) assay 193

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List of Figures and Tables
Figures Page

Chapter 1: Literature Review
1.1 Regulation of caspase dependent apoptotic pathways 30
1.2 Molecular mechanism of autophagy 33
1.3 Crosstalk between apoptosis and autophagy 41
Chapter 3: Results
3.1 The cytotoxic effects of a panel of palladium based compounds on breast cancer 74
and melanoma cell lines
3.2 AJ-5 induces cytotoxicity and inhibits migration of breast cancer cells 77
3.3 AJ-5 displays activity against cancer stem-like cells from the MCF7 cell line 79
3.4 AJ-5 induces sub-G1 peak and G1 cell cycle arrest 81
3.5 AJ-5 activates the DNA damage response 83
3.6 AJ-5 induces apoptosis in breast cancer cells 84
3.7 AJ-5 induced apoptosis involves both intrinsic and extrinsic apoptosis 86
3.8 AJ-5 induces cytochrome c release from the mitochondria to the cytoplasm 87
3.9 AJ-5 induces vacuoles in breast cancer cells 89
3.10 AJ-5 induces autophagy in breast cancer cells. 90
3.11 AJ-5 induces autophagy flux in breast cancer cells 92
3.12 AJ-5 inhibits the mTOR pathway and activates concurrent apoptosis and 93
autophagy
3.13 Inhibition of autophagy decreases the cytotoxic effect of AJ-5 in MCF7 and 94
MDA-MB-231 cells
3.14 Inhibition of autophagy reduces AJ-5 induced apoptosis 96
3.15 p38 MAPK mediates AJ-5 induced autophagy and apoptosis in breast cancer 97
cells
3.16 AJ-5 induces a potent cytotoxic effect on melanoma cell lines 99
3.17 AJ-5 induces sub-G1 peak and S and G2/M cell cycle arrest 101
3.18 AJ-5 induces apoptosis in melanoma cells 102
3.19 AJ-5 induces molecular markers of apoptosis in melanoma cells 103
3.20 AJ-5 induces high levels of vacuolization in melanoma cells 105
3.21 AJ-5 induces autophagosomes formation in melanoma cells 106
3.22 AJ-5 induces continuous autophagy flux in ME1402 and WM1158 cells 108
3.23 Confirmation of AJ-5- induced autophagy flux 109
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