APOPTOSIS ACTIVITIES OF NICKEL AND COPPER COMPLEXES FROM THYMOQUINONE AND
DITHIOCARBAMATE ON ORAL CANCER CELL LINES IN VITRO
BY
WASTUTI HIDAYATI SURIYAH
A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy in Pharmaceutical
Sciences (Pharmacology)
Kulliyyah of Pharmacy International Islamic University Malaysia
SEPTEMBER 2018
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ABSTRACT
Oral squamous cell carcinoma (OSCC) has been associated with high morbidity and mortality rate. Metal-based anticancer drugs such as platinum-based agents have been widely used to treat various cancer cells including OSCC. However, their efficiency is limited by the side effects and frequent development of chemoresistant cancer cells. To date, research on metal-based compounds has been extensively continued to develop more promising chemotherapeutic compounds capable to overcome these limitations. Thymoquinone (TQ) has been reported to have numerous biological activities, including anticancer in vitro and in vivo. Similarly, dithiocarbamate (PEDTC) is also known to have in vitro antineoplastic potential against several types of cell lines. Nickel and copper complexes from TQ (NiTQPy, CuTQPy) and PEDTC (NiPEDTC, CuPEDTC) were successfully synthesized and characterized. These metal complexes provide new approach to broaden the spectrum of biological activities their parent ligands. The study is aimed to investigate and determine anticancer potential of the metal complexes derived from TQ and PEDTC. Human OSCC HSC-3 and HSC-4 cell lines were chosen as in vitro model. The cells were exposed to various concentrations of the compound substances and examined by MTT assay for cytotoxicity analysis. Normal human oral fibroblast and human keratinocyte (HACAT) cells were also included in the assay. Zebrafish was used as a model for in vivo toxicity study. The number of apoptotic cells was quantified by flow cytometry and confirmed by Caspase 3/7 assay. Quantitative RT PCR was performed to analyze the mRNA expression of apoptotic-regulator genes. The protein expression was observed by western blot. The MTT assay demonstrated that the metal complexes induced cytotoxicity on HSC-3 and HSC-4 cells. Exposure of the metal complexes at the concentration similar to cancer cells relatively did not affect the normal cells and zebrafish embryo development, except for those treated with copper complexes. Flow cytometry showed the metal complexes increased the number of sub-G1 population, which represent apoptotic cells. The apoptotic activities were supported by Caspase 3/7 analysis. The various degree of apoptosis induction by metal complexes from TQ and PEDTC are associated with the elevation of BAX/BCL-2 ratio in both transcriptional (mRNA) and translational (protein) levels. NiPEDTC and NiTQPy was shown to be the most effective to induce apoptosis in HSC-3 and HSC-4 cells among the metal complexes. To conclude, these models of study are useful to demonstrate apoptosis activities in OSCC. The metal complexes from TQ and PEDTC are suggested to merit further investigation for its potentiation as anticancer agents.
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ملخص البحث
خلايا سرطان الفم الحرشفية (أوسك) مرتبطة بمعدل وفيات عالي. الأدوية المضادة للسرطان القائمة كانت وما زالت تستخدم على نطاق واسع لعلاج الخلايا السرطانية على المعادن مثل معدن البلاتين
ذا العلاج الكيميائي غير مرضية ومع ذلك حتى الآن نتيجة ه خلايا سرطان الفم؛ المختلفة بما في ذلكبسبب الآثار الجانبية والتطور المتكرر للخلايا السرطانية الكيميائية. حتى هذا اليوم الأبحاث المتعلقة بالمركبات القائمة على المعادن مستمرة و على نطاق واسع في تطوير مركبات علاج كيميائي أكثر
نشطة البيولوجية الناتجة من ثيموكينون (تك) هنالك العديد من الدراسات حول الأ فعالية.وديثيوكاربامات (بيدتك) هذان المركبان يعتبران مضادان للجراثيم و للفطريات و كذلك مضادان
تقدم المركبات المعدنية المشتقة من تك و بيدتك yجا جديدا لتوسيع نطاق أنشطتها للسرطان.نيات المركبات المعدنية المستمدة من تك و بيدتك ضد البيولوجية. �دف هذه الدراسة إلى تحديد إمكا
البشرية كنموذج في المختبر. تعرضت 4-و هسك 3-السرطان. تم اختيار خلايا أوسك هسك كيز مختلفة من المركبات و تم فحصها باستخدام فحص م ت ت لتحليل السمية الخلوية.االخلايا لتر
الطبيعية والخلايا الكيراتينية البشرية (هاكات) أيضا في وقد أدرجت الخلايا الليفية الفموية البشريةالفحص. تم استخدام سمكة الزرد كنموذج لدراسة السمية في الجسم الحي. و تم قياس عدد الخلايا
. كما 3/7التي خضعت للموت المبرمج من خلال التدفق الخلوي وأكدت من قبل فحص كاسباس ل عن موت الخلايا ور ن اي لتحديد الجين المسؤ-م استخدم فحص ال رت / ب س ر لتحليل ال
المبرمج. استخمت لطخة وسترن لملاحظة البروتينان المسؤلة. أظهرت اختبارات السمية الخلوية أن ولكن أظهرت تأثير أقل على 4-و هسك 3-المركبات المعدنية خفضت معدل البقاء لخلايا هسك
ر اختبار التدفق الخلوي للمركبات المعدنية زيادة في المرحلة ما الخلايا الطبيعية واجنة سمكة الزرد. أظهو هذا يمثل الخلايا الميتة بطريقة الموت المبرمج. نشاط موت الخلايا المبرمج اكد و دعم 1-قبل ال ج
. ارتبطت درجة مختلفة من موت الخلايا المبرمج من قبل المركبات 3/7بنتائج اختبار الكاسباس ر ن -في كلا ترانسكريبتيونال (م 2-قة من تك و بيدتك مع ارتفاع نسبة باكس / بكلالمعدنية المشت
اي) ومستويات متعدية (البروتين). وقد تبين أن نيبيدتك هي الأكثر فعالية لتحريض موت الخلايا ، نيبيدتك كان الاكثر فعالية، نيتكبي و 4-، في حين أن في خلايا هسك3-المبرمج في خلايا الهسك
في إحداث موت الخلايا المبرمج. وختاما، فإن هذه النماذج من الدراسة كانت فعالة نسبياكوتكبي مفيدة لإثبات أنشطة موت الخلايا المبرمج في خلايا سرطان الفم الحرشفية أوسك.
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APPROVAL PAGE
The thesis of Wastuti Hidayati Suriyah has been approved by the following:
_____________________________ Abdul Razak Kasmuri
Supervisor
_____________________________ Fiona How Ni Foong
Co-supervisor
_____________________________ Muhammad Taher Internal Examiner
_____________________________ Iekhsan Othman
External Examiner
_____________________________ Amin Malik Shah bin Abdul Majid
External Examiner
_____________________________ Kamarul Ariffin Khalid
Chairman
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DECLARATION
I hereby declare that this thesis is the result of my own investigations, except where
otherwise stated. I also declare that it has not been previously or concurrently
submitted as a whole for any other degrees at IIUM or other institutions.
Wastuti Hidayati Suriyah
Signature ........................................................... Date .........................................
vi
INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA
DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH
APOPTOSIS ACTIVITIES OF NICKEL AND COPPER COMPLEXES FROM THYMOQUINONE AND
DITHIOCARBAMATE ON ORAL CANCER CELL LINES IN VITRO
I declare that the copyright holders of this thesis are jointly owned by the student and IIUM.
Copyright © 2018 Wastuti Hidayati Suriyah and International Islamic University Malaysia. All
rights reserved.
No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below
1. Any material contained in or derived from this unpublished research may be used by others in their writing with due acknowledgement.
2. IIUM or its library will have the right to make and transmit copies
(print or electronic) for institutional and academic purposes.
3. The IIUM library will have the right to make, store in a retrieved system and supply copies of this unpublished research if requested by other universities and research libraries.
By signing this form, I acknowledged that I have read and understand the IIUM Intellectual Property Right and Commercialization policy. Affirmed by Wastuti Hidayati Suriyah ……..…………………….. ……………………….. Signature Date
vii
‘Allah is the Light of the heavens and the earth.
The parable of His Light is as a niche within which is a lamp, the lamp is within glass,
the glass as if it were a pearly star lit from a blessed olive tree, neither of the east nor of the west,
whose oil would almost glow even if untouched by fire. Light upon light..
Allah guides to His Light whom He wills. And Allah sets forth parables for mandkind, and Allah is Knowing of all things’
(Quran, 24:35)
To my parents, my family, and my teachers, who enlighten my world with love and knowledge
from the light which Allah places in our heart and to Allah we shall return..
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ACKNOWLEDGEMENTS
All praises to Allah (SWT) the Most Beneficent and the Most Merciful for giving me knowledge, strength and courage to complete the thesis. May Allah’s peace and blessings be upon our beloved prophet Muhammad (SAW) and his family. This study would have never been possible without the support, understanding and care of many people to whom I owe my sincere thanks and appreciation. May Allah the Almighty bless and reward them all abundantly.
My deepest appreciation and gratitude to my supervisor, Associate Professor Dr. Abdul Razak Kasmuri for all his generosity in teaching and guidance for establishment of this study. The motivations and knowledge he has shared with me would not be forgotten. I am deeply indebted to my co-supervisor, Assistant Professor Dr. Fiona How Ni Foong for her encouragement, valuable guidance and support throughout the study period. Without their excellent supervisions, this work might not have been reality.
I also wish to thank and give my appreciation to Assoc. Prof. Dr. Muhammad
Taher, Assoc. Prof Dr. Alfi Khatib, and Sr. Nurulwahida Saad for their kind help and guidance. My sincere thanks to all the helpful laboratory staff in Kulliyyah of Pharmacy and Kulliyyah of Science, especially to Sr. Hazan Haryanti, Br Walid, Sr. Muiezzah, Sr. Maliza Azrain, Sr. Noor Hafiah and Sr. Sriviowarti Noerdin, for their cooperation in laboratory technical assistance.
Sincere thanks go to my special teammate, Sr. Hanisuhana Hamidon, Sr.
Rahima, Sr. Sama Shaban, Sr. Syafinaz, Sr. Lailinajmie Hussain and Sr. Murni for the stimulating discussions and their warm supports during this research journey. Not to forget to all my friends in the postgraduate studies and all those who contributed in this research, I feel very much grateful for your assistance.
Special thanks go to my beloved mother Maryati, my dear husband,
Solachuddin and my sweet children, Ayako Aziza, Ar-Razi Ryoshi, and Shafa Syahida for their prayers, patience, unconditional love and enc ouragement throughout this study.
Last but not least, thanks are also due to the RACE 14-015-0021 and IIUM
Research EDW B 0902-212 grants for their financial support to develop my scientific discoveries.
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TABLE OF CONTENTS
Abstract ………………………………………………………………………….……………………………… ii Abstract in Arabic ……………………………………………………………………...……………………iii Approval Page …………..…………………………………………………….………………………………iv Declaration ……………………………………………………………………….……………………………..v Copyright Page ………………………………………………………………….…………………………….vi Dedication ………………………………………………………………………….……………………………vii Acknowledgements ………………………………………………………….………………………………viii List of Tables …………………………………………………………………….…………………………….xv List of Figures ………………………………………………………………….…………………………….. xvi List of Equations ……………………………………………………….…………………………………… xviii List of Abbreviations ……………………………………………………….………………………………xix List of Symbols …………………………………………………………….…………………………………xxiii CHAPTER ONE: INTRODUCTION .................................................................. 1
1.1 Background of the Study ........................................................................ 1 1.2 Research Hypothesis ............................................................................... 7 1.3 Objectives ............................................................................................... 7
1.3.1 Specific Objectives ....................................................................... 7 1.4 Flowchart of Study ................................................................................. 8
CHAPTER TWO: LITERATURE REVIEW ..................................................... 9
2.1 Head and Neck Cancer: An Overview ................................................... 9 2.1.1 Epidemiology, Etiology and Risk Factors of Head and
Neck Cancer: Oral Squamous Cell Carcinoma ............................ 10 2.1.2 Diagnosis, Prognosis and Treatments of Head and Neck
Cancer ........................................................................................... 13 2.2 Molecular Basis of HNSCC Carcinogenesis .......................................... 16
2.2.1 Multistep Process of Carcinogenesis ............................................ 16 2.2.2 Genetics of Cancer ........................................................................ 17
2.2.2.1 The Oncogenes ................................................................. 18 2.2.2.2 The Tumor suppressor genes and proteins ....................... 20 2.2.2.3 Stability Genes .................................................................. 23
2.2.3 Regulation of Apoptosis ............................................................... 24 2.3 Chemotherapy Drugs for Head and Neck Cancer .................................. 28
2.3.1 Cancer Drugs Resistance .............................................................. 31 2.4 The Medicinal Approaches in Metal Complexes: A Strategy for
Cancer Therapy ...................................................................................... 34 2.5 Metal Ions and Metal-Based Complexes Properties ............................... 35
2.5.1 Non Platinum Metal Complexes as Anti-Cancer Drugs ............... 35 2.5.2 Copper, Nickel, and Its Complexes .............................................. 35
2.6 The Biological Active Ligands and Its Properties .................................. 38 2.6.1 Thymoquinone .............................................................................. 38 2.6.2 Dithiocarbamate ............................................................................ 39
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2.7 In vitro and in vivo Anticancer Model of Study: Toxicity and Screening ................................................................................................ 40 2.7.1 Oral Squamous Carcinoma Cell lines (HSC-3 and HSC-
4): An in vitro Model of Study ..................................................... 41 2.7.2 Zebrafish: An in vivo Model of Study .......................................... 43
CHAPTER THREE: SYNTHESIS AND CHARACTERIZATION OF METAL THYMOQUINONE COMPLEXES AND METAL DITHIOCARBAMATE COMPLEXES .............................................................. 45
3.1 Introduction ............................................................................................ 45 3.2 Objective ................................................................................................. 46 3.3 Materials and Methods ........................................................................... 46
3.3.1 Materials, Chemicals and Equipment ........................................... 46 3.3.1.1 Materials .......................................................................... 46 3.3.1.2 Chemicals ........................................................................ 46 3.3.1.3 Equipments ...................................................................... 47
3.3.2 Synthesis of Metal Complexes from Thymoquinone (TQ) .......... 47 3.3.3 Synthesis of Metal Complexes from Potassium Phenethyl
Dithiocarbamates (PEDTC) .......................................................... 48 3.3.4 Melting Point Determination ........................................................ 48 3.3.5 Fourier Transform Infrared (FTIR) Spectroscopy ........................ 48 3.3.6 Atomic Absorption Spectroscopy (AAS) ..................................... 49 3.3.7 CHNS Elemental Analyses ........................................................... 49 3.3.8 Gas Chromatography-Mass Spectrometry (GC-MS) .................... 49
3.4 Results .................................................................................................... 50 3.4.1 Nickel Complex (NiTQPy) and Copper Complex
(CuTQPy) from Thymoquinone (TQ) .......................................... 50 3.4.1.1 Fourier Transform Infrared (FTIR) Spectroscopy of
NiTQPy and CuTQPy ................................................................... 51 3.4.1.2 Gas Chromatography-Mass Spectrometry (GC-MS) of
NiTQPy and CuTQPy ................................................................... 52 3.4.2 Nickel Complex (NiPEDTC) and Copper Complex
(CuPEDTC) from Potassium Phenethyl Dithiocarbamate (PEDTC) ....................................................................................... 53
3.4.2.1 Fourier Transform Infrared (FTIR) Spectroscopy of NiPEDTC and CuPEDTC ............................................................ 54
3.4 Discussion and Conclusion ..................................................................... 55 CHAPTER FOUR: IN VITRO CYTOTOXICITY AND IN VIVO TOXICITY STUDIES OF NICKEL AND COPPER COMPLEXES DERIVED FROM THYMOQUINONE AND DITHIOCARBAMATE ON HSC-3 AND HSC-4 CELL LINES .......................................................................................................... 57
4.1 Introduction ............................................................................................ 57 4.2 Objective ................................................................................................. 60
4.2.1 Specific Objectives ....................................................................... 60 4.3 Materials and Methods ........................................................................... 60
4.3.1 Materials, Chemicals, and Equipment for Cytotoxicity Study ............................................................................................. 60 4.3.1.1 Materials .......................................................................... 60
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4.3.1.2 Chemicals ........................................................................ 61 4.3.1.3 Equipments ...................................................................... 61 4.3.1.4 Reagent for Assay ............................................................ 62
4.3.2 General Procedures for Cell Culture ............................................. 62 4.3.2.1 Cell Thawing ................................................................... 62 4.3.2.2 Cell Subculturing and Maintaining ................................. 63 4.3.2.3 Cell Freezing ................................................................... 63 4.3.2.4 Cell Counting ................................................................... 63
4.3.3 Sample Preparation ....................................................................... 64 4.3.4 Observation of HSC-3 and HSC-4 Cells Growth ......................... 64 4.3.5 The Cytotoxic Study of Metal TQ Complexes and Metal
PEDTC Complexes on HSC-3 and HSC-4 Cells Using MTT Assay ................................................................................... 64
4.3.6 The Cytotoxic Study of Metal TQ Complexes and Metal PEDTC Complexes on HOF and HACAT Cells Using MTT Assay ................................................................................... 65
4.3.7 Materials, Chemicals, and Equipment for Zebrafish Toxicity Study .............................................................................. 65 4.3.7.1 Materials and Chemicals .................................................. 65 4.3.7.2 Equipments ....................................................................... 66
4.3.8 General Procedures for Zebrafish Toxicity Study ........................ 66 4.3.8.1 Test Compounds ............................................................... 66
4.3.9 Mortality Scoring of Zebrafish Toxicity Study ............................ 67 4.3.10 The Zebrafish Embryo Observation After Treatment ................. 67
4.4 Statistical Analysis ................................................................................. 67 4.5 Results .................................................................................................... 68
4.5.1 The Normal Growth Curves of HSC-3 and HSC-4 Cells ............. 68 4.5.2 Metal TQ Complexes and Metal PEDTC Complexes
Reduced the Viability of HSC-3 Cell Line ................................... 68 4.5.3 Metal TQ Complexes and Metal PEDTC Complexes
Reduced the Viability of HSC-4 Cell Line ................................... 70 4.5.4 The Effect of Metal TQ Complexes and Metal PEDTC
Complexes on The Viability of HOF and HACAT Cell Lines ............................................................................................. 72
4.5.5 The Toxicity Effects (LC50 values) of TQ Metal Complexes and PEDTC Metal Complexes on Zebrafish Embryo ......................................................................................... 73
4.6 Discussion and Conclusion ..................................................................... 77 CHAPTER FIVE: APOPTOSIS ANALYSIS OF NICKEL AND COPPER COMPLEXES DERIVED FROM THYMOQUINONE AND DITHIOCARBAMATE ON HSC-3 AND HSC-4 CELL LINES ...................... 81
5.1 Introduction ............................................................................................ 81 5.2 Objectives ............................................................................................... 84
5.2.1 Specific Objectives ....................................................................... 84 5.3 Materials and Methods ........................................................................... 84
5.3.1 Materials, Chemicals, and Equipment .......................................... 84 5.3.1.1 Materials ........................................................................... 84 5.3.1.2 Chemicals ......................................................................... 84
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5.3.1.3 Equipments ....................................................................... 84 5.3.1.4 Reagent for Assay ............................................................. 85
5.3.2 Sample Preparation ....................................................................... 85 5.3.3 Methods ......................................................................................... 85
5.3.3.1 Flowcytometry/Cell Cycle Analysis ................................ 85 5.3.3.2 Caspase 3/7 Assay ........................................................... 86
5.4 Sample Analysis ..................................................................................... 86 5.5 Results .................................................................................................... 87
5.5.1 Metal TQ and PEDTC Complexes Induced Apoptosis in HSC-3 and HSC-4 Cell Lines (Flow cytometry): ........................ 87 5.5.1.1 Cell Cycle Analysis on HSC-3 cells Induced by
Metal TQ and PEDTC Complexes in 24 and 48 h .......... 87 5.5.1.2 Cell Cycle Analysis on HSC-4 cells Induced by
Metal TQ and PEDTC Complexes in 24 and 48 h .......... 90 5.5.2 Metal TQ and Dithiocarbamates Complexes Activate
Caspase 3/7 in HSC-3 and HSC-4 Cell Lines .............................. 93 5.6 Discussion and Conclusion ..................................................................... 94
CHAPTER SIX: STUDY ON THE EXPRESSION OF BAX/BCL-2 GENE AND PROTEIN ACTIVITY OF METAL DITHIOCARBAMATES COMPLEXES AND METAL THYMOQUINONE COMPLEXES IN HSC-3 AND HSC-4 CELL LINES .......................................................................................................... 98
6.1 Introduction ............................................................................................ 98 6.2 Objective ................................................................................................. 101
6.2.1 Specific Objectives ....................................................................... 101 6.3 Materials And Methods .......................................................................... 101
6.3.1 Materials, Chemicals, and Equipment .......................................... 101 6.3.1.1 Materials ........................................................................... 101 6.3.1.2 Chemicals ......................................................................... 102 6.3.1.3 Equipments ....................................................................... 103 6.3.1.4 Reagents for Assay ........................................................... 103
6.3.2 Cell Culture and Sample Preparation for qRT-PCR Analysis ........................................................................................ 103
6.3.3 Total RNA Extraction ................................................................... 104 6.3.3.1 Phase Separation ............................................................... 104 6.3.3.2 RNA Precipitation ............................................................ 104 6.3.3.3 RNA Wash and Re-dissolve ............................................. 105 6.3.3.4 Spectrophotometric Analysis ............................................ 105
6.3.4 Quantitative Real-Time RT-PCR Analysis ................................... 105 6.3.4.1 Reverse-Transcription PCR .............................................. 105 6.3.4.2 Quantitative Real-Time RT-PCR of the Genes ................ 106
6.3.5 Protein Lysate and Sample Preparation for Western Blotting ......................................................................................... 108 6.3.5.1 Preparation of Lysate from Cell Culture .......................... 108 6.3.5.2 Determination of Protein Concentration .......................... 109 6.3.5.3 Preparation of Sample for Loading into Gel .................... 109
6.3.6 Protein Separation by SDS PAGE Electrophoresis ...................... 109 6.3.7 Transferring the Protein and Staining (Western Blot) .................. 110 6.3.8 Protein Detection .......................................................................... 110
xiii
6.4 Statistical Analysis ................................................................................. 111 6.5 Results .................................................................................................... 112
6.5.1 The mRNA Expressions of BAX and BCL-2 in HSC-3 Cells Line ...................................................................................... 112
6.5.2 The mRNA Expressions of BAX and BCL-2 in HSC-4 Cells Line ...................................................................................... 114
6.5.3 The Protein Expressions of BAX and BCL-2 in HSC-3 Cell line ....................................................................................... 116 6.5.4 The Protein Expressions of BAX and BCL-2 in HSC-4 Cell line ....................................................................................... 118
6.6 Discussion and Conclusion ..................................................................... 119 CHAPTER SEVEN: GENERAL DISCUSSION AND CONCLUSION .......... 122
7.1 Discussion .............................................................................................. 122 7.2 Conclusions ............................................................................................ 129
REFERENCES:……………………………………………………………..........130 APPENDIX I: LIST OF PUBLICATION/PRESENTATIONS ........................ 150 APPENDIX II: CERTIFICATE OF PRESENTATION 1 ................................ 156 APPENDIX III: CERTIFICATE OF PRESENTATION 2 ............................... 157 APPENDIX IV: CERTIFICATE OF PRESENTATION 3 ............................... 158 APPENDIX V: CERTIFICATE OF PRESENTATION 4 ................................. 159 APPENDIX VI: ARTICLE ................................................................................... 160 APPENDIX VII: IR SPECTRA OF PEDTC AND THE METAL COMPLEXES ........................................................................................................ 161 APPENDIX VIII: GC-MS OF TQ ....................................................................... 162 APPENDIX IX: GC-MS OF NITQPY ................................................................. 163 APPENDIX X: GC-MS OF CUTQPY ................................................................. 164 APPENDIX XI: STANDARD PROTEIN CONCENTRATION CURVES ..... 165 APPENDIX XII: VALIDATION OF GAPDH QRT-PCR IN HSC-4 CELL LINE ........................................................................................................................ 166 APPENDIX XIII: VALIDATION OF GAPDH QRT-PCR IN HSC-3 CELL LINE ............................................................................................................ 167 APPENDIX XIV: VALIDATION OF 18SRNA QRT-PCR IN HSC-4 CELL LINE ............................................................................................................ 168 APPENDIX XV: VALIDATION OF 18SRNA QRT-PCR IN HSC-3 CELL LINE ........................................................................................................................ 169 APPENDIX XVI: VALIDATION OF BAX QRT-PCR IN HSC-4 CELL LINE ........................................................................................................................ 170 APPENDIX XVII: VALIDATION OF BAX QRT-PCR IN HSC-3 CELL LINE ........................................................................................................................ 171 APPENDIX XVIII: VALIDATION OF BCL-2 QRT-PCR IN HSC-4 CELL LINE ............................................................................................................ 172 APPENDIX XIX: VALIDATION OF BCL-2 QRT-PCR IN HSC-3 CELL LINE ........................................................................................................................ 173 APPENDIX XX: THE HOF AND HACAT GROWTH KINETICS AND THE IC50 VALUES ................................................................................................ 174
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APPENDIX XXI: IN VIVO TOXICITY TEST ON ZEBRAFISH EMBRYO................................................................................................................ 175 APPENDIX XXII: IMMUNOBLOTTING OF b-ACTIN AND a-TUBULIN ............................................................................................................... 176 APPENDIX XXIII: PONCEAU STAINING ...................................................... 177
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LIST OF TABLES
Table No. Page No.
2.1 TNM classification stage of HNSCC 14
2.2 The clinical characteristics of patient of oral squamous cell carcinomas 42
3.1 The physical and analytical data of metal TQ complexes 50
3.2 Selected functional group of metal TQ complexes 52
3.3 The physical data of metal PEDTC complexes 53
3.4 Selected functional group of metal PEDTC complexes 55
4.1 The IC50 of samples on HSC-3 cells 70
4.2 The IC50 of samples on HSC-4 cells 70
4.3 Concentration of TQ and its metal ccomplexes, PEDTC and its metal complexes, and CDDP exposed to HOF and HACAT cells 73
4.4 The 50% of lethal concentration (LC50) values 74
6.1 Compositions for the reverse transcription reaction 106
6.2 Primers list for PCR 106
6.3 Compositions for PCR 106
6.4 The qRT-PCR reaction under optimal amplification conditions 107
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LIST OF FIGURES
Figure No. Page No.
1.1 Research flowchart 8
2.1 Molecular model of HNSCC 17
2.2 The various p53 tumor suppressor functions 21
2.3 The morphological features of apoptosis 25
2.4 The electron microscopy of necrotic and apoptotic cells 25
2.5 Apoptosis pathways 26
2.6 Mechanisms of drug resistance in human cancer cells 31
2.7 Picture of Nigella sativa, the seeds and thymoquinone chemical structure 38
2.8 Dithiocarbamate core structure 39
2.9 Adult zebrafish (Danio rerio) female (upper) and male (lower) 43
3.1 FTIR spectra of TQ and its metal complexes (NiTQPy and CuTQPy), Ni(II) acetate, Co(II) acetate, and Pyridin as the solvent 51
3.2 Structures of NiPEDTC and CuPEDTC 54
4.1 Microscopic visualization of OSCC, HSC-3 and HSC-4 (upper) and normal/non-canceruos cell lines, HOF and HACAT (lower) at a magnification of X40 58
4.2 Growth kinetics of HSC-3 and HSC-4 cell lines 68
4.3 The HSC-3 cell viability after exposed by the compounds in various concentrations for 24 and 48 h 69
4.4 The HSC-4 cell viability after exposed by the compounds in various concentrations for 24 and 48 h 71
4.5 The cell viability of HOF (A) and HACAT (B) after induced by the compounds for 24 and 48 h 72
4.6 Control (DMSO) zebrafish embryo 74
4.7 The toxicity effect of zebrafish embryos (48, 72, 96, 120 hpf) after exposed by the compounds in various concentrations 75
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4.8 Morphology of zebrafish embryo (72 hpf) after exposed by the sample with certain concentration respectively 76
5.1 The DNA content-frequency histograms of untreated (Control) and compounds-treated cells (24 and 48 h) in HSC-3 cells 88
5.2 Cell cycle profile of HSC-3 cells 89
5.3 The DNA content-frequency histograms of untreated (Control) and compounds-treated cells (24 and 48 h) in HSC-4 cells 91
5.4 Cell cycle profile of HSC-4 cells 92
5.5 Caspase 3/7 activities in HSC-3 cells 93
5.6 Caspase 3/7 activities in HSC-4 cells 94
6.1 The relative expression of BAX levels in HSC-3 cells at 12 and 24 h in control, treated-control and samples-treated groups 112
6.2 The relative expression of BCL-2 levels in HSC-3 cells at 12 and 24 h in control, treated-control and samples-treated groups 112
6.3 The ratio of BAX/BCL-2 in HSC-3 cells at 12 and 24 h in control, treated-control and samples-treated groups 113
6.4 The relative expression of BAX levels in HSC-4 cells at 12 and 24 h in control, treated-control and samples-treated groups 114
6.5 The relative expression of BCL-2 levels in HSC-4 cells at 12 and 24 h in control, treated-control and samples-treated groups 114
6.6 The ratio of BAX/BCL-2 in HSC-4 cells at 12 and 24 h in control, treated-control and samples-treated groups 115
6.7 The protein expressions of BAX and BCL-2 in HSC-3 cells at 24 h in control, treated-control and samples-treated groups (A) 116
6.8 The protein expressions of BAX and BCL-2 in HSC-4 cells at 24 h in control, treated-control and samples-treated groups (A) 118
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LIST OF EQUATIONS Equation No. Page No.
4.1 Total number of viable cells/mL = (average viable cell count per square) x dilution factor x 104 64
6.1 Expression fold change = 2–ΔΔCt
ΔCt = (a target gene) – (a reference gene). ΔΔCt = ΔCt (a target sample) – ΔCt (a reference sample) 107
xix
LIST OF ABBREVIATIONS
A A AAS ABL ABC
Adenine Ampere Atomic Absorption Spectroscopy Abelson murine leukemia viral oncogene ATP binding cassette
AIDS Acquired immunodeficiency syndrome AIM Adipogenesis-inducing medium AJCC AKT
American Joint Committee on Cancer Murine thymoma viral oncogene homolog
ANOVA APAF-1 ATP
Analysis of Variance Apoptotic protease activating factor-1 Adenosine Tri Phosphate
BAK BAX BCL-2 BCL-XL BCRP BER BH3 BLAST BME
BCL-2 homologous antagonist/killer BCL-2-associated X B-cell lymphoma 2 B-cell lymphoma-extra large Breast cancer resistance protein Base excition repair BCL-2 homology domain 3 Basic Local Alignment Search Tool β Mercaptoethanol
BQ BSA
Betel quid Bovine Serum Albumin
C Caspases
Cytosine Cysteine aspartic acid proteoases
CDDP cis-diamminedichloridoplatinum(II) CDC CDC25A CDK CDKN2A CDKN2B
Centers for Disease Control Cell division cycle 25A Cyclin dependent kinase Cyclin-dependent kinase inhibitor 2A Cyclin dependent kinase inhibitors 2B
cDNA C3H8O CHCl3
Complementary Deoxyribonucleic Acid Isopropanol Chloroform
CHNS CICD
Carbon, Hydrogen, Nitrogen, Sulphur Caspase-independent cell death
cm CO2 CT Cu CytC
Centimeter Carbondioxide Computed Tomography Copper Cytochrome C
DEPC DeSigN
Diethylpyrocarbonate Differentially Expressed Gene Signatures-Inhibitors
xx
DEVD Aspartyl-L-Glutamyl-L-Valyl-L-Aspartic Acid DISC Death Inducing Signalling Complex dH2O double Distilled Water DMEM Dulbecco’s Modified Eagle Medium DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic Acid dNTP deoxynucleoside Triphosphate DTCs DTT ECL
Dithiocarbamates Dithiothreitol Electro Chemiluminescence
Eds./ed. EDTA e.g. EGF EGFR EGTA ELlSA EMT erbB
Editions/edition Ethylene diamine tetraacetic acid (exempli gratia); for example Epidermal growth factor Epidermal growth factor receptor Ethylene glycol-bis(β-aminoethyl ether Enzyme-Linked Immunosorbent Assay Epithelial-mesenchymal transition Erythroblastosis oncogene B
ERK Extracellular signal et al EtOH FADD
(et alia); and others Ethanol Fas-associated death domain
FasR FBS FET Fig
Fas Receptor Foetal Bovine Serum Fish Embryo Toxicity Figure
FTIR Fourier transformed infrared g g G GC-MS GLOBOCAN
gram gravity Guanine Gas Chromatography-Mass Spectrometry Global Cancer Incidence, Mortality and Prevalence
h hour/s HAART HACAT H2O
Highly active antiretroviral therapy Cultured Human Keratinocyte Cell Water
HEPES HER
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Human epidermal growth factor receptor
HHV-8 hNEIL1/2
Human herpesvirus type 8 Human DNA Glycosylase Nei-like 1/2
HNSCC HOF Hoggi hpf HPV HR HRP IBD
Head and Neck Squamous Cell Carcinoma Human Oral Fibroblast human 8-oxoguanine DNA glycosylase hours post fertilization Human papillomavirus Homologous recombination Horseradish Peroxidase Inflammatory bowel disease
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ICRACU Integrated Centre for Research Animal eraC and Use
IGF insulin-like growth factor IR Infrared KCl Potassium Chloride kBR Potassium bromide kDa kilodalton LCK MAPK
Lymphocyte-specific protein tyrosine kinase Mitogen-activated protein kinase
MeOH Methanol min minute MIX mg
Methylisobutylxanthine miligram
mL MLH1
milliliter Mut-L homologin 1
Mm MMPs
Millimolar Matrix metalloproteinases
MMR MOS MRI
Mismatch repair Moloney murine sarcoma oncogene Magnetic Resonance Imaging
mRNA MRP MSH2 MT mTOR MTS2
messenger Ribonucleic Acid Multidrug-resistance-associated protein Mut-S homologin 2 Metallothionein Mammalian target of rapamycin multiple tumor suppressors 2
MTT 3-(4,5-dimethylthiazol-2-y)2,5-diphenyltetrazolium bromide
Na3VO4 Sodium Orthovanadate NaCl Sodium Chloride NaF Sodium Fluoride NaHCO3 NaN3
Sodium Bicarbonate Sodium Azide
NaOH NER NHEJ
Sodium Hydroxide Nucleotide excision repair Nonhomologous end-joining
Ni NIST nm No. NP40 OECD OPC
Nickel National Institute of Standards and Technology nanometer Number/Numbers Nonyl phenoxypolyethoxylethanol Organisation for Economic Co-operation and Development Oropharingeal cancer
OSCC PAHs
Oral Squamous Cell Carcinoma Polycyclic aromatic hydrocarbons
PBS Phosphate Buffer Saline PCR PDGF
Polymerase Chain Reaction Platelet-derived growth factor
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PDK PEDTC PET
PI-3K–dependent kinase Potassium phenethyl dithiocarbamate Positron Emission Tomography
pH PI
Potential of Hydrogen Propidium iodide
PI3K PIM1 PMSF PRAD1 PVDF qRT-PCR Ras RAF1 Rb
Phosphatidylinositol 3-kinase Proviral integration site 1 Phenylmethylsulfonyl fluoride Parathyroid Adenomatosis 1 Polyvinylidene fluoride Quantitative Real Time-Reverse Transcription PCR Rat sarcoma Raf-1 Proto-Oncogene, Serine/Threonine Kinase Retinoblastoma
RNA RNA-seq ROS RPM
Ribonucleid Acid Sequence-based RNA Reactive oxygen species Revolutions per minute
RT-PCR Reverse Transcription Polymerase Chain Reaction SDBS SDS-PAGE Sec
Spectral Database for Organic Compounds Sodium dodecyl sulfate polyacrylamide gel electrophoresis second
SEM SH3GL2 SILAC SOP
Standard Error of Means SH3 Domain Containing GRB2 Like 2, Endophilin A1 Stable Isotope Labeling with Amino Acids in Cell Culture Standard Operational Procedure
Src SREBP STAT
Sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog Sterol Regulatory Element Signal transducer and activator transcription
T TBS T TGFs
Thymine Tris-buffered Saline with Tween 20 Transforming growth factors
TNF TNM TRAIL tRNA TS TSNAs TSGs
Tumor Necrosis Factor Tumor, Nodes, Metastases TNF-related apoptosis-inducing ligand Transfer Ribonucleic Acid Thymidylate synthase Tobacco-specific nitrosamines Tumor suppressor genes
TQ UV/VIS V
Thymoquinone Ultraviolet–visible spectroscopy Volt
WHO ZFET
World Health Organization Zebrafish Embryo Toxicity
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LIST OF SYMBOLS a β cm-1
oC ΔCt µL µg
Alpha Beta Wavenumber Celcius degree Delta cycle threshold Microlitre Microgram
P
the probability of obtaining the result
*
statistical significance denotation
m/z
mass-to-charge ratio
n
sample sizes
IC50
LC50
® ™
half maximal inhibitory concentration median lethal concentration registered trademark trademark
1
CHAPTER ONE
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Oral squamous cell carcinoma (OSCC) is the most common type of head and neck
squamous cell carcinoma (HNSCC), which arises in oral cavity. OSCC causes high
morbidity and mortality, with total of 145,300 deaths per year worldwide and
estimated of new cancer is 300,400 cases. Overall, the OSCC mortality was common
in males than females with ratio of 2:1. There are geographical variations in the
incidence of oral cancer across the world, with the highest incidence rate was found in
South and Southeast Asia, which is accounted more than 100,000 cases (Vigneswaran
& Williams, 2014; Ferlay et al., 2015). Tobacco use, alcohol consumption and human
papillomavirus (HPV) as either individually or combination, are some of the risk
factors that complicate the increase of the occurrence of the squamous cell carcinoma
(Döbróssy, 2005).
The OSCC development is a multistep and progressive in nature. It starts with
uncontrolled proliferation of epithelial cells such as basal cell hyperplasia, dysplasia,
carcinoma in situ and ends up with advanced OSCC (Lehrbach et al., 2003). The early
malignant lesion is usually in the form of an erytholeukoplastic lesion that is often
asymptomatic (Bagan et al., 2010). OSCC is considered as a cancer with a poor
prognosis, for only 50–63% of the five year survival rate is reported. Almost 2/3 of
the cases of oral cancers were detected at the late stage of disease (Güneri & Epstein,
2014).
The main principle of the treatments for patients with OSCC is either
radiotherapy or radical surgery, which is often combined with adjuvant chemotherapy