To investigate synergism in activity from combination of platinum compounds and that from combination between phytochemicals in ovarian tumour models

By

ACKNOWLEDGEMENT

I would take this opportunity to thank my research supervisor, family and friends for their support and guidance without which this research would not have been possible.

DECLARATION

I, [type your full first names and surname here], declare that the contents of this dissertation/thesis represent my own unaided work, and that the dissertation/thesis has not previously been submitted for academic examination towards any qualification. Furthermore, it represents my own opinions and not necessarily those of the University.

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TABLE OF CONTENTS

ACKNOWLEDGEMENTII

DECLARATIONIII

CHAPTER 2: MATERIALS AND METHODS1

2.1 Introduction1

2.2 Synthesis of the compounds1

2. 3 Syntheses of mononuclear compounds3

2.4 Syntheses of trinuclear compounds8

2.5 Characterization of compounds14

2.6 Preparation standard solution for A.A.S.15

2.7 Calculating percentage yield of the compound22

2.8 Molar conductivity22

2.9 Spectral studies22

2.10 Biological activity24

2.11 Phosphate buffered saline (PBS)27

2.12 HEPES28

2.13 Cells recovery from liquid nitrogen28

2.14 Cell subculture29

2.15 Storage of the cell lines31

2.16 MTT-Based cytotoxicity assay31

CHAPTER 3: RESULTS34

3.1 Characterization of compounds34

3.2 Characterization of mononuclear complexes34

3.3 Elemental compositions of TH5, TH6 and TH736

3.4 Characterization of the trinuclear compounds37

3.5 Elemental compositions of TH1, TH8 and TH1439

3.6 Spectral studies39

3.7 Nuclear magnetic resonance spectra (1H NMR) of TH5, TH6, TH7, TH1, TH8 and TH1444

3.8 Molar conductivity45

3.9 Cytotoxicity46

3.10 Activity summary50

3.11 Cell uptake and DNA binding51

3.12 DNA binding54

3.13 Gel electrophoresis56

3.14 Interaction between the compounds and ssDNA56

3.15 Interaction with pBR322 plasmid DNA58

3.16 BamH1 Digestion61

3.17 Interaction with nucleobases (HPLC)64

3.18 Binding ratio65

CHAPTER 4: DISCUSSION67

4.1 Ovarian cancer and ovarian surface epithelium67

4.2 Oncogenic signalling pathways in ovarian cancer68

4.3 GnRH73

4.4 Gonadotrophins79

4.5 Activin and inhibin85

CHAPTER 5: CONCLUSION88

REFERENCES93

CHAPTER 2: MATERIALS AND METHODS

2.1 Introduction

As stated earlier, in this project a number of mononuclear trans-planaraminepalladium(II) complexes and trinuclear complexes containing two planaramine ligands bonded to the central metal ions have been prepared, characterized, and investigated for their antitumour activity against a number of ovarian cancer cell lines. The cell uptake and nature of binding of the compounds with DNA have also been determined. This chapter describes all the experimental methods related to the study. The method of preparation of cisplatin has also been included, as the compound has been used as a reference as applied to studies on activity, cell uptake and binding with DNA.

2.2 Synthesis of the compounds

2.2.1 Materials used

2-hydroxy pyridine, 3-hydroxy pyridine, 4-hydroxy pyridine were obtained from Sigma Chemical Company St. Louise USA, transplatin and cisplatin, were obtained from Sigma-Aldrich Pty Ltd, NSW, Australia and used as received without further purification. N,N-dimethylformamide [DMF] [C3H7NO], dimethyl sulfoxide (DMSO), 1,6- diaminohexane dihydrochloride, tetramethylene diamine dihydrochloride, sodium hydroxide, silver nitrate (AgNO3); potassium chloride (KCl) and potassium iodide (KI) were obtained from BDH Chemicals, Australia Pty. Ltd., concentrated hydrochloric acid (HCl), potassiumtetrachloroplatinate(II) (K2[PtCl4]), potassium tetrachloropalladate(II) ([K2[PdCl4]), ethanol, methanol, acetone, diethyl ether were purchased from APS chemicals, Australia, concentrated ammonia solution, triethyl amine, dichloromethane, 28% ammoni

2.2.2 Preparation of cisplatin and transplatin

The preparation of cisplatin (and transplatin) was first described by Peyrone in 1845. The method is based on the action of aqueous ammonia on potassium tetrachloroplatinate(II). Although the method was later modified by Kauffman and Cowan (Kauffman and ...