INVESTIGATION OF ANTIPROLIFERATIVE EFFECTS OF GAMMA- AND DELTA-TOCOTRIENOLS IN HUMAN BREAST CANCER CELL LINES USING GENOMICS AND QUANTITATIVE PROTEOMICS APPROACH

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(1)M. al. ay. a. INVESTIGATION OF ANTIPROLIFERATIVE EFFECTS OF GAMMA- AND DELTA-TOCOTRIENOLS IN HUMAN BREAST CANCER CELL LINES USING GENOMICS AND QUANTITATIVE PROTEOMICS APPROACH. U. ni ve. rs i. ti. PREMDASS RAMDAS. FAKULTI OF MEDICINE DEPARTMENT OF MOLECULAR MEDICINE UNIVERSITY OF MALAYA KUALA LUMPUR 2019.

(2) al. ay. a. INVESTIGATION OF ANTIPROLIFERATIVE EFFECTS OF GAMMA- AND DELTA-TOCOTRIENOLS IN HUMAN BREAST CANCER CELL LINES USING GENOMICS AND QUANTITATIVE PROTEOMICS APPROACH. rs i. ti. M. PREMDASS RAMDAS. U. ni ve. THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY. FACULTY OF MEDICINE DEPARTMENT OF MOLECULAR MEDICINE UNIVERSITY OF MALAYA KUALA LUMPUR 2019.

(3) UNIVERSITY OF MALAYA ORIGINAL LITERARY WORK DECLARATION Name of Candidate: Premdass Ramdas Matric No: MHA120064 Name of Degree: Doctor of Philosophy Title of Project Paper/Research Report/Dissertation/Thesis (“this Work”): INVESTIGATION OF ANTIPROLIFERATIVE EFFECTS OF GAMMA- AND. a. DELTA- TOCOTRIENOLS IN HUMAN BREAST CANCER CELL APPROACH Field of Study: Genomics and Proteomics. al. I do solemnly and sincerely declare that:. ay. LINES USING GENOMICS AND QUANTITATIVE PROTEOMICS. U. ni ve. rs i. ti. M. (1) I am the sole author/writer of this Work; (2) This Work is original; (3) Any use of any work in which copyright exists was done by way of fair dealing and for permitted purposes and any excerpt or extract from, or reference to or reproduction of any copyright work has been disclosed expressly and sufficiently and the title of the Work and its authorship have been acknowledged in this Work; (4) I do not have any actual knowledge nor do I ought reasonably to know that the making of this work constitutes an infringement of any copyright work; (5) I hereby assign all and every rights in the copyright to this Work to the University of Malaya (“UM”), who henceforth shall be owner of the copyright in this Work and that any reproduction or use in any form or by any means whatsoever is prohibited without the written consent of UM having been first had and obtained; (6) I am fully aware that if in the course of making this Work, I have infringed any copyright whether intentionally or otherwise, I may be subject to legal action or any other action as may be determined by UM. Candidate’s Signature. Date:. Subscribed and solemnly declared before, Witness’s Signature. Date:. Name: Designation:. ii.

(4) ABSTRACT The chemoprevention of breast cancer using natural and synthetic compounds to intervene in the early precancerous stages of carcinogenesis before the invasion begins is undertaken as a measure to reduce breast cancer probability for women at high risk. A number of bioactive dietary components are of particular interest in the field of breast cancer. One such compound known as the tocotrienols; a sub-group of Vitamin E family,. a. display anticancer properties and may play a role in cancer prevention. To date, there are. ay. many studies which show that tocotrienols can inhibit the proliferation of human breast cancer cells in vitro. Especially, the γ- and δ- isoforms of tocotrienols have been shown. al. to have a more potent inhibitory effect on numerous cancer cell growth. In this study, the. M. effect of tocotrienol isomers (γ and δ) were used to postulate the mechanism of action of these compounds on DNA methylation process and proteomic footprinting. The use of. ti. DNA methylation profiling and quantitative proteomics provided insights into functions. rs i. and the mechanisms of tocotrienols action in breast cancer. The DNA methylation profiling of known tumour suppressors of breast cancer revealed significant DNA. ni ve. hypomethylation in δ- and γ-tocotrienols-treated MCF-7 and MDA-MB-231 cells. Tocotrienols treatment resulted in methylation changes at the methylated CpG sites of some important tumour suppressor genes such as Hs3st2, Adam23, Cav1 and Cst6. These. U. genes were hypermethylated in untreated cells, but the percentage of methylation was reduced significantly following δ- and γ-tocotrienols treatment which showed the ability of these isomers to potentially reverse the DNA methylation alterations. The data provided new evidence for tocotrienols-mediated DNA methylation alterations as a potential mechanism of breast cancer chemoprevention. The supernatant, cytoplasmic and nuclear protein proteomic profiles of tocotrienols treated MCF-7 and MDA-MB-231 human breast cancer cells were analysed by utilising label-free quantitative proteomic strategy. The key findings of secreted proteomic analysis following tocotrienols iii.

(5) treatment showed downregulation of Cathepsin D expression which is frequently seen over-expressed in various cancer and upregulation of Profilin-1 whose downregulation was reported in various adenocarcinoma. Next, the quantitative label-free proteome profiling of nuclear and cytoplasmic protein of δ- and γ-tocotrienol treated MCF-7 and MDA-MB-231 cells showed the ability of tocotrienols to dysregulate the proteins involved in cell motility, trafficking, metastasis, invasion and proliferation. The cytoplasmic proteome results revealed the ability of tocotrienols to inhibit a group of. a. proteasome proteins such as Psma, Psmb, Psmd and Psme. As the inhibition of. ay. proteasome proteins are known to induce apoptosis in proliferating or transformed cells, the findings from this study identified tocotrienols as a potential proteasome inhibitor. al. that can overcome deficiencies in growth-inhibitory or pro-apoptotic molecules in breast. ti. M. cancer cells.. U. ni ve. rs i. Keywords: Vitamin E, Tocotrienols, Proteomics, Genomics, Breast Cancer.. iv.

(6) ABSTRAK Pencegahan kanser payudara dengan menggunakan kompaun semula jadi dan sintetik untuk melawan tahap awal karsinogenesis sebelum serangan sel kanser bermula amat penting bagi mengurangkan kebarangkalian kanser payudara terutamanya untuk golongan wanita yang berisiko tinggi. Sejumlah komponen diet bioaktif yang mempunyai manfaat bagi pencegahan kanser payudara semakin mendapat perhatian para penyelidik. Salah satu kompaun yang dikenali sebagai tocotrienol; sub-kumpulan. a. keluarga Vitamin E, memaparkan ciri-ciri anti-kanser dan boleh memainkan peranan. ay. penting dalam pencegahan kanser payu dara. Sehingga kini, terdapat pelbagai kajian yang menunjukkan bahawa tocotrienol dapat menghalang percambahan sel-sel kanser. al. payudara manusia. Kesan anti-kanser yang ditunjukkan oleh isofom tocotrienol yang. M. berlainan pada sel-sel kanser payudara manusia didapati berbeza. Isofom γ dan δtocotrienol terutamanya, telah terbukti mempunyai kesan anti-proliferatif yang tinggi. ti. terhadap pertumbuhan sel kanser payu dara. Dalam kajian ini, kaedah metilasi DNA dan. rs i. proteomik kuantitatif telah digunakan untuk mempostulasikan mekanisme tindakan. ni ve. kedua isomer tocotrienol (γ dan δ) terhadap dua jenis sel kanser payu dara iaitu MCF-7 dan MDA-MB-231. Metilasi DNA merupakan suatu proses yang sering dikaitkan dengan tumor kanser payudara. Di dalam kajian ini, hipometilasi DNA yang ketara dapat dilihat pada sel-sel MCF-7 dan MDA-MB-231 yang dirawat dengan δ- dan γ-tocotrienol.. U. Rawatan tocotrienol menunjukkan perubahan metilasi di tapak CpG metilasi beberapa gen-gen penindas tumor penting seperti Hs3st2, Adam23, Cav1 and Cst6. Peratusan metilasi yang tinggi dilihat pada gen-gen ini dalam sel kanser payu dara yang tidak dirawat, manakala peratusan metilasi didapati berkurangkan dengan ketara selepas rawatan δ- dan γ-tocotrienols yang menunjukkan keupayaan isomer ini untuk membalikkan perubahan metilasi DNA. Data kami memberikan penjelasan baru mengenai pengubahsuaian metilasi DNA oleh tocotrienol sebagai mekanisme yang. v.

(7) berpotensi untuk kemoterapi kanser payudara. Seterusnya, pendekatan proteomik kuantitatif telah digunakan untuk meneliti profil proteome MCF-7 dan MDA-MB-231 di dalam fraksi supernatan, sitoplasmik dan protein nuklear. Strategi proteomik kuantitatif tidak berlabel telah digunakan untuk menganalisis profil proteome supernatan sel-sel kanser payudara MCF-7 dan MDA-MB-231 yang dirawat dengan tocotrienol. Penemuan utama analisis secretomik selepas rawatan tocotrienol adalah penurunan ekspresi protein Cathepsin D yang dilaporkan mempunyai ekspresi yang tinggi dalam pelbagai jenis. a. kanser manakala ekspresi protein Profilin-1 yang dilaporkan mempunyai ekspresi yang. ay. berkurangan dalam pelbagai adenokarsinoma pula meningkat. Seterusnya, profil proteome kuantitatif tidak berlabel digunakan untuk mengkaji protein nukleus dan. al. sitoplasmik daripada sel-sel MCF-7 dan MDA-MB-231 yang menerima rawatan δ- dan. M. γ-tocotrienol. Hasilnya menunjukkan keupayaan tocotrienol untuk mempengaruhi expresi protein-protein yang terlibat dalam pengedaran, pengangkutan, metastasis,. ti. pencerobohan dan percambahan sel-sel kanser payudara. Tocotrienol juga didapati. rs i. mempengaruhi expresi sekumpulan protein proteasome seperti Psma, Psmb, Psmd dan. ni ve. Psme. Perencatan expresi protein-protein proteasome ini dilaporkan dapat mendorong apoptosis dalam sel-sel kanser. Penemuan dari kajian ini mengenal pasti tocotrienols sebagai penghalang proteasome yang dapat membantu mengatasi kekurangan dalam. U. molekul-molekul pertumbuhan atau pro-apoptosis dalam sel-sel kanser payudara. Kata Kunci: Vitamin E, Tocotrienol, Proteomik, Genomik, Kanser Payu Dara. vi.

(8) ACKNOWLEDGEMENTS My heartiest appreciation goes to my supervisors, Associate Prof. Dr. Puteri Shafinaz Abdul Rahman and Prof. Dr. Ammu Kutty Radhakrishnan for their dedicated efforts, supervision and constant encouragement throughout the research period. I am extremely grateful for their advice, moral support, suggestions and time spent for discussion throughout the completion of this research and thesis.. a. I also wish to convey my thanks to my colleagues and friends, Dr Mangala, Ms. ay. Jeya Seela, Ms Sarmini, Ms Izlina and Ms Ann Nazirah for their assistance throughout. al. the research period.. M. Special thanks extended to Ms. Asmahani Azira Abdu Sani for her moral support, assistance, technical knowledge and help in various ways for my research.. ti. My appreciation also goes to University Malaya, Malaysian Palm Oil Board. rs i. (MPOB), and Ministry of Higher Education for funding and providing financial support. ni ve. for my research project and tuition fees.. I wish to extend my heartfelt thanks and gratitude to my beloved parents Mr.. Ramdas and Madam Gunasikari, my sister Ms Nagadevi, and brothers Mr. Muhammad. U. Sameer and Mr. Muhammad Shazmir for their love and kindness. Last but not least, my deepest gratitude to the Almighty for blessing me with the. strength and knowledge from the beginning of my academic life up to this doctoral level. His benevolence has made me excel and achieve success in all my academic pursuits.. vii.

(9) TABLE OF CONTENTS Abstract ......................................................................................................................... iii Abstrak ............................................................................................................................ v Acknowledgements .....................................................................................................vvii Table of Contents ..................................................................................................... viviii List of Figures ................................................................................................................. x. a. List of Tables ................................................................................................................xvi. ay. List of Symbols and Abbreviations ......................................................................... xviii. al. List of Appendices ........................................................................................................ xx. M. CHAPTER 1: GENERAL BACKGROUND ............................................................... 1 Background ............................................................................................................. 1. 1.2. Objectives................................................................................................................ 4. 1.2. Research Hypotheses .............................................................................................. 5. rs i. ti. 1.1. ni ve. CHAPTER 2: LITERATURE REVIEW ..................................................................... 6 Breast Cancer Statistics ........................................................................................... 6 2.1.1. Breast cancer biology................................................................................. 7. 2.1.2. Risk factor for breast cancer ...................................................................... 8. U. 2.1. 2.1.3. 2.2. Treatment modalities ............................................................................... 10. Vitamin E .............................................................................................................. 13 2.2.1. History of Vitamin E................................................................................ 13. 2.2.2. Chemistry of Tocotrienols ....................................................................... 15. 2.2.3. Emergence of Tocotrienols Research ...................................................... 16. 2.2.4. Tocotrienols and Breast Cancer ............................................................... 17. 2.2.5. Tocotrienols and Other Cancer ................................................................ 18. viii.

(10) 2.3. In Vitro Model of Breast Cancer ........................................................................... 20. 2.4. Epigenetics: DNA Methylation ............................................................................. 21. 2.5. 2.4.1. DNA Methylation in Normal Vs Cancer Biology ................................... 22. 2.4.2. DNA Methylation in Nutrition ................................................................ 24. Quantitative Mass-spectrometry based proteomics .............................................. 25 2.5.1. Label-free Quantitative Proteomics ......................................................... 27. 2.5.2. Label-free quantitative proteomics approach in Breast Cancer Research. Proteomics in Nutrition Research ............................................................ 30. ay. 2.5.3. a. 28. M. Materials................................................................................................................ 32 Cell Lines ................................................................................................. 32. 3.1.2. Cell culture ............................................................................................... 32. 3.1.3. Treatments ............................................................................................... 32. 3.1.4. Chemicals................................................................................................. 32. 3.1.5. LC-MS/MS .............................................................................................. 33. 3.1.6. Methylation Studies ................................................................................. 33. 3.1.7. Consumables ............................................................................................ 33. 3.1.8. General Apparatus ................................................................................... 34. rs i. ti. 3.1.1. U. ni ve. 3.1. al. CHAPTER 3: MATERIALS AND METHODS ........................................................ 32. 3.2. Instrumentation ..................................................................................................... 34. 3.3. Methods ................................................................................................................. 35 3.3.1. Cell lines and cultue conditions ............................................................... 35. 3.3.2. Preparation of treatments ......................................................................... 35. 3.3.3. Cell Viability and Determination of Minimal Inhibitory Concentration . 37. 3.3.4. Adaptation to serum-free media .............................................................. 37. ix.

(11) 3.3.5. DNA methylation studies......................................................................... 38. 3.3.5.1. DNA extraction ................................................................................. 38. 3.3.5.2. Determination of Protein Concentration and Trypsin Digestion ...... 39. 3.3.5.3. Data acquisition and analysis ............................................................ 42. 3.3.6. Label-free quantitative proteomics .......................................................... 42 Secretome analysis ............................................................................ 43. 3.3.6.1a. Protein concentration and digestion .................................................. 43. 3.3.6.1b. Liquid Chromatography and Mass Spectrometry Analysis .............. 44. 3.3.6.1c. Protein Identification and Label-Free Quantification ....................... 45. 3.3.6.2. Cytoplasmic and Nuclear Proteome Analysis ................................... 46. 3.3.6.2a. Extraction of cytoplasmic proteins.................................................... 46. 3.3.6.2b. Extraction of nuclear proteins ........................................................... 47. 3.3.6.2c. Liquid chromatography and mass spectrometry analysis for. M. al. ay. a. 3.3.6.1. Bioinformatics and functional analysis.................................................... 49. ni ve. 3.3.7. Protein identification and label-free quantification ........................... 48. rs i. 3.3.6.2d. ti. cytoplasmic and nuclear fractions ..................................................... 47. 3.3.7.1. DAVID database analysis ................................................................. 49. 3.3.7.2. PANTHER database analysis ............................................................ 50. 3.3.7.3. STRING protein-protein interaction analysis ................................... 50. U. 3.3.8. Statistical Analysis ................................................................................... 51. 3.3.8.1. Cell-proliferation and DNA methylation data................................... 51. 3.3.8.2. Proteomics data ................................................................................. 51. CHAPTER 4: RESULTS ............................................................................................. 53 4.1. Effects of delta- and gamma-tocotrienols on proliferation of human breast cancer cells. .................................................................................................................. 53. x.

(12) 4.2. DNA methylation profiles of tumour suppressor genes in human breast cancer cells treated with tocotrienols ........................................................................................ 57. 4.3. Quantification of secreted proteins in culture supernatant of MCF-7 and MDAMB-231 cells treated with tocotrienols ................................................................. 64. 4.4. Label-free mass spectrometry quantification of nuclear and cytoplasmic proteins isolated from MCF-7 and MDA-MB-231 cells following treatment with tocotrienols ............................................................................................................ 70. 4.5.1. a. Functional Bioinformatics Analysis...................................................................... 95 Functional annotation and pathway enrichment of differentially expressed. ay. 4.5. protein datasets ........................................................................................ 95 Analysis using the PANTHER software ........................................... 95. 4.5.1.2. Analysis using the DAVID software............................................... 107. M. Protein- protein interaction analysis using STRING ............................. 113. ti. 4.5.2. al. 4.5.1.1. rs i. CHAPTER 5: DISCUSSION ..................................................................................... 129 Effects of tocotrienols on the proliferation of human breast cancer cells ........... 129. 5.2. Effects of tocotrienols on the CpG promoter methylation of tumour suppressor. ni ve. 5.1. genes in human breast cancer cells ..................................................................... 131 Effects of tocotrienols on the secretome of human breast cancer cells .............. 137 5.3.1. MDA-MB-231 cells ............................................................................... 137. 5.3.1. MCF-7 cells ........................................................................................... 140. U. 5.3. 5.4. 5.5. Effect of tocotrienols on the cytoplasmic proteome in breast cancer cells ......... 142 5.4.1. MDA-MB-231 cells ............................................................................... 142. 5.4.2. MCF-7 cells ........................................................................................... 151. Effect of tocotrienols on the cytoplasmic proteome in breast cancer cells ......... 154 5.5.1. MDA-MB-231 cells ............................................................................... 154. xi.

(13) 5.5.2. MCF-7 cells ........................................................................................... 154. CHAPTER 6: CONCLUSION .................................................................................. 159 6.1. General Conclusion ............................................................................................. 159. 6.2. Limitations of the study ...................................................................................... 160. 6.3. Future Studies ..................................................................................................... 161. REFERENCES ........................................................................................................... 162. a. LIST OF PUBLICATIONS AND PAPERS PRESENTED .................................... 217. U. ni ve. rs i. ti. M. al. ay. APPENDIX ................................................................................................................. 219. xii.

(14) LIST OF FIGURES Figure 2.1: Number of new cases in Malaysia, year 2018, females, all ages. .................. 7 Figure 2.2: A) Tocotrienol has three unsaturated, three double bonds on the isoprenoid side chain while B) tocopherol's side-chain is saturated (phytyl side chain) ................ 14 Figure 2.3: Chemical structures of isoforms of tocotrienols ......................................... 16 Figure 2.4: The methylation of cytosine nucleotides occurs mainly on the 5th carbon. 22. a. Figure 2.5: Cytosines 5′ to guanosines (CpG) islands in promoter region of tumour suppressor gene are methylated resulting in hypermethylation and gene silencing. ...... 23. ay. Figure 3.1: Overview of the study approach .................................................................. 36. al. Figure 4.1: Comparing the effects of tocotrienols on the proliferation of MCF-7 human breast cancer cells in the presence (10% FBS) or absence of serum .............................. 54. M. Figure 4.2: Comparing the effects of tocotrienols on the proliferation of MDA-MB-231 human breast cancer cells in the presence (10% FBS) or absence of serum .................. 55. ti. Figure 4.3: Heat map comparing methylation status of genes in the genomic DNA of MDA-MB-231 and MCF-7 cells (untreated and treated with tocotrienols) ................... 58. ni ve. rs i. Figure 4.4: DNA methylation status of tumour suppressor genes that were significantly altered in MDA-MB-231 human breast cancer cells when treated with IC50 concentrations of tocotrienols......................................................................................... 59 Figure 4.5: DNA methylation status of tumour suppressor genes that were significantly altered in MCF-7 human breast cancer cells when treated with IC50 concentrations of tocotrienols .................................................................................................................... 60. U. Figure 4.6: Venn diagrams showing differentially methylated genes following treatment with tocotrienols in MDA-MB-231 and MCF-7 human breast cancer cells .................. 63 Figure 4.7: Venn diagrams of differentially expressed proteins following tocotrienols treatment with number of unique elements in union and intersections between different breast cancer cell lines and tocotrienol isoforms ............................................................ 69 Figure 4.8: Venn diagrams of differentially expressed cytoplasmic and nuclear proteins of δ- and γ-T3 isoforms treated MDA-MB-231 cells ..................................................... 88 Figure 4.9: Venn diagrams of differentially expressed cytoplasmic and nuclear proteins of δ- and γ-T3 isoforms treated MCF-7 cells ................................................................. 91. xiii.

(15) Figure 4.10: Gene ontology showing biological processes, molecular functions, cellular components, protein class and pathways for differentially methylated genes in MDAMB-231 cells in response to treatment with tocotrienols ............................................... 96 Figure 4.11: Gene ontology showing biological processes, molecular functions, cellular components, protein class and pathways for differentially methylated genes in MCF-7 cells in response to treatment with tocotrienols ............................................................. 97 Figure 4.12: Gene ontology showing biological processes, molecular functions, cellular components, protein class and pathways for differentially regulated proteins secreted by MDA-MB-231 cells in response to treatment with tocotrienols .................................... 99. ay. a. Figure 4.13: Gene ontology plotted showing biological processes, molecular functions, cellular components, protein class and pathways for differentially regulated proteins secreted by MCF-7 cells in response to treatment with tocotrienols ............................ 100. al. Figure 4.14: Gene ontology showing biological processes, molecular functions, cellular components, protein class and pathways for differentially regulated cytoplasmic proteins produced by MDA-MB-231 cells in response to treatment with tocotrienols ............. 102. ti. M. Figure 4.15: Gene ontology showing biological processes, molecular functions, cellular components, protein class and pathways for differentially regulated cytoplasmic proteins produced by MCF-7 cells in response to treatment with tocotrienols .......................... 103. rs i. Figure 4.16: Gene ontology showing biological processes, molecular functions, cellular components, protein class and pathways for differentially regulated nuclear proteins produced by MDA-MB-231 cells in response to treatment with tocotrienols ............. 105. ni ve. Figure 4.17: Gene ontology showing biological processes, molecular functions, cellular components, protein class and pathways for differentially regulated nuclear proteins produced by MCF-7 cells in response to treatment with tocotrienols .......................... 106. U. Figure 4.18: Protein-protein interaction network for differentially expressed secreted proteins of MDA-MB-231 cells in response to tocotrienols treatment ........................ 114 Figure 4.19: Protein-protein interaction network for differentially expressed secreted proteins of MCF-7 cells in response to tocotrienols treatment..................................... 116 Figure 4.20: Protein-protein interaction network for differentially expressed cytoplasmic proteins of MDA-MB-231 cells in response to γT3 treatment .................................... 118 Figure 4.21: Protein-protein interaction network for differentially expressed cytoplasmic proteins of MDA-MB-231 cells in response to δT3 treatment ..................................... 119 Figure 4.22: Protein-protein interaction network for differentially expressed cytoplasmic proteins of MCF-7 cells in response to γT3 treatment ................................................. 121. xiv.

(16) Figure 4.23: Protein-protein interaction network for differentially expressed cytoplasmic proteins of MCF-7 cells in response to δT3 treatment ................................................. 122 Figure 4.24: Protein-protein interaction network for differentially expressed nuclear proteins of MDA-MB-231 cells in response to γT3 treatment ..................................... 124 Figure 4.25: Protein-protein interaction network for differentially expressed nuclear proteins of MDA-MB-231 cells in response to δT3 treatment ..................................... 125 Figure 4.26: Protein-protein interaction network for differentially expressed nuclear proteins of MCF-7 cells in response to γT3 treatment ................................................. 127. ay. a. Figure 4.27: Protein-protein interaction network for differentially expressed nuclear proteins of MCF-7 cells in response to δT3 treatment ................................................. 128 Figure 5.1: Profilin 1 (Pfn1) binds to actin and affects the structure of the cytoskeleton where in upregulated conditions, profilin 1 prevents the polymerisation of actin ....... 138. M. al. Figure 5.2: The downregulated proteasomes proteins formed the most prominent network based on the protein–protein interaction for differentially expressed cytoplasmic proteins in response to γT3 in MDA-MB-231 cells ................................................................... 144. ti. Figure 5.3: The KEGG proteasome pathway shows involvement of downregulated cytoplasmic proteins in response to γT3 in MDA-MB-231 cells ................................. 146. ni ve. rs i. Figure 5.4: The differentially expressed transport proteins formed the most prominent network based on the protein–protein interaction for differentially expressed cytoplasmic proteins in response to δT3 in MDA-MB-231 cells ..................................................... 148. U. Figure 5.5: The binding of cytochrome c (Cytc) to Apaf-1 triggers the activation of caspase-9, which then accelerates apoptosis by activating other caspases .................. 151. xv.

(17) LIST OF TABLES Table 2.1: Most common adverse effects of cancer therapy .......................................... 12 Table 2.2: The contents of tocopherols and tocotrienols (mg/100 g of oil) in some common edible oils......................................................................................................... 15 Table 3.1: Instruments used in this study ....................................................................... 34 Table 3.2: Digestion reactions for DNA methylation assay ........................................... 40 Table 3.3: Reaction mix set up for quantitative PCR .................................................... 41. a. Table 3.4: Cycling conditions used for the qPCR step ................................................... 41. ay. Table 4.1: The half maximal inhibitory concentration (IC50) values of delta- and gammatocotrienols on human breast cancer cells ...................................................................... 56. al. Table 4.2: Methylation state of genes in response to tocotrienols treatment in MDA-MB231 and MCF-7 human breast cancer cells ................................................................... 61. M. Table 4.3: Differentially expressed secreted proteins in MDA-MB-231 cells treated with tocotrienols compared with untreated control ................................................................ 66. ti. Table 4.4: Differentially expressed secreted proteins in MCF-7 cells treated with tocotrienols compared with untreated control ................................................................ 67. rs i. Table 4.5: Differentially expressed cytoplasmic proteins (P<0.05) in tocotrienol-treated MDA-MB-231 cells compared with untreated control .................................................. 71. ni ve. Table 4.6: Differentially expressed nuclear proteins (P<0.05) in tocotrienol-treated MDA-MB-231 cells compared with untreated control .................................................. 76 Table 4.7: Differentially expressed cytoplasmic proteins (P<0.05) in tocotrienol-treated MCF-7 cells compared with untreated control ............................................................... 81. U. Table 4.8: Differentially expressed nuclear proteins (P<0.05) in tocotrienol-treated MCF7 cells compared with untreated control ......................................................................... 84 Table 4.9: List of the unique proteins in union and intersections between different types of protein data sets of MDA-MB-231 cells cytoplasmic and nuclear protein ................ 89 Table 4.10: List of the unique proteins in union and intersections between different types of protein data sets of MCF-7 cells cytoplasmic and nuclear protein ............................ 93 Table 4.11: List of genes that matched with (A) cancer disease database and (B) with significant functional categories by DAVID analysis .................................................. 108 Table 4.12: List of secreted proteins in tocotrienol-treated (A) MDA-MB-231 and (B) MCF-7 cells matched with functional categories by DAVID analysis ....................... 109. xvi.

(18) Table 4.13: List of cytoplasmic proteins secreted by tocotrienol-treated (A) MDA-MB231 and (B) MCF-7 cells matched with functional categories by DAVID analysis ... 111. U. ni ve. rs i. ti. M. al. ay. a. Table 4.14: List of nuclear proteins secreted by tocotrienol-treated (A) MDA-MB-231 and (B) MCF-7 cells matched with functional categories by DAVID analysis ........... 112. xvii.

(19) LIST OF SYMBOLS AND ABBREVIATIONS :. automated gain control. BCA. :. bicinchoninic acid. CID. :. collision induced dissociation. CV. :. coefficient of variance. Da. :. Dalton. T3. :. delta-tocotrienols. DEC. :. dependent enzyme control. DMEM. :. Dulbecco’s modified Eagle’s medium. DMSO. :. dimethylsulphoxide. ESI. :. electrospray ionisation. FBS. ::. foetal bovine serum. FDR. :. false discovery rate. T3. :. gamma-tocotrienols. ay. al. M. ti. :. [Glu-1]-fibrinopeptide. :. gonadotropin-releasing hormone. ni ve. GnRH. rs i. GFP. a. AGC. :. high-energy collision dissociation. KEGG. :. Kyoto Encyclopaedia of Genes and Genomes. LC. :. liquid chromatography. M. :. methylated. Ms. :. methylation sensitive. Md. :. methylation dependent. MCF-7. :. Michigan Cancer Foundation-7. MDA-MB-231. :. M.D. Anderson Metastasis Breast cancer 231. MS. :. mass spectrometry. MSE. :. mass spectrometry elevated. U. HCD. xviii.

(20) :. normalised collision energy. PANTHER. :. Protein ANalysis THrough Evolutionary Relationships. PFN1. :. Profilin-1. PLGS. :. ProteinLynx Global Server. QToF. :. quadrupole time-of-flight. SEC. :. sensitive enzyme control. SFM. :. serum-free medium. STRING. :. Search Tool for the Retrieval of Interacting Genes/Proteins. T3. :. tocotrienols. UM. :. unmethylated. UPLC. :. ultra-performance liquid chromatography. WHO. :. World Health Organisation. WST. :. Water-soluble tetrazolium. U. ni ve. rs i. ti. M. al. ay. a. NCE. xix.

(21) LIST OF APPENDICES. U. ni ve. rs i. ti. M. al. ay. a. APPENDIX A: EpiTect methyl II complete PCR array (94) human breast cancer gene panel ............................................................................................................................. 219. xx.

(22) CHAPTER 1 INTRODUCTION 1.1. BACKGROUND Breast cancer is the second most common type of cancer in the world after lung. cancer. It accounts for 11.6% of all cancer incidences in humans (Bray et al., 2018) and it is the fifth most common cause of cancer deaths (World Health Organization, 2014).. a. In addition, breast cancer is the most common female malignancy in Malaysia with. ay. 32.7% of new cases reported in 2018 (Organization, 2018) and worldwide (Bray et al., 2018). There is a marked geographical difference in the worldwide incidence of breast. al. cancer, with a higher incidence in developed countries compared to developing countries. M. (Ghoncheh, Pournamdar, & Salehiniya, 2016). In Asian countries, breast cancer is the most commonly diagnosed cancer among women and on an average; about one in nine. ti. women in Malaysia are at risk, compared to one in eight in Europe and the United States.. rs i. In 2008, 1.38 million cases were diagnosed worldwide and an estimated 207,900 new cases with 9,840 deaths for 2010. Studies in the US estimated that 1 in 8 women are at. ni ve. risk of breast cancer in a lifetime. In Malaysia, 2018 statistics states that 37 per 100,000 are at risk of breast cancer, with 7593 reported cases diagnosed that year. The mortality rate of breast cancer in Malaysia is 11.82% and 2894 have reported dead in 2018; it is. U. the most common cause of cancer death in women for both developed and developing countries with 268 000 deaths (Yip, Pathy, & Teo, 2014). The estimated death of breast cancer triumphs the deaths from lung cancer with 189,000 to 188,000. Due to the favorable survival rates of breast cancer, it is now ranked the 5th most common cause of death from cancers worldwide (Globocan, 2012) One of the common treatment approaches to breast cancer is surgery to remove the tumour foci and/or mastectomy. Surgery is generally followed by chemotherapy and 1.

(23) radiotherapy, which has several side-effects. For many years now, scientists have understood that the onset of breast cancer is a gradual and stepwise process. Therefore, chemoprevention of breast cancer, the attempt to use natural or bioactive compounds to intervene in the early pre-cancerous stages of carcinogenesis before invasion begins, is undertaken as a measure to reduce breast cancer risk for women at high risk of developing this disease. Owing to this, the treatment for breast cancer has changed to a new perspective and researchers are finding evidence to prove that natural compounds exhibit. a. anti-cancer activity with increased health benefits and reduced or no side-effects as. ay. compared to other chemoprevention drugs. In the more recent years, chemoprevention researches are focused on finding substances or components of natural sources that can. al. prevent or inhibit carcinogenesis. Targeted cancer therapies using natural bioactive. M. compounds are also used to target specific characteristics of cancer cells, such as a gene or protein that allows the cancer cells to grow in a rapid or abnormal way. One such. rs i. ti. natural bioactive compound is tocotrienols (T3) which is used in this study. Tocotrienols are a member of the vitamin E family. Vitamin E is a generic term. ni ve. referring to an entire class of compounds that is further divided into two major subgroups known as tocopherols and T3. Both tocopherols and T3 exist naturally in four isoforms, namely alpha (α), beta (β), δ (delta) and gamma (γ) (Schneider, 2005).. U. Tocotrienols and its derivatives are currently gaining a lot of attention as there are many evidence in the literature which show that they are effective chemopreventive natural compounds that can be used for prevention and/or treatment of breast cancer (T. C. Hsieh, Elangovan, & Wu, 2010). In the literature, there are several studies that report on the anti-proliferative (Parajuli, Tiwari, & Sylvester, 2015) and anti-cancer (T. C. Hsieh et al., 2010) effects of T3. The anti-proliferative effects of the four T3 isoforms on human breast cancer cells appear to be different. For instance, γT3 and δT3 were reported to exert a more potent anti-proliferative effects on these cancer cells compared to αT3 or 2.

(24) βT3 (Shun et al., 2004). Exposure to T3 could activate the apoptotic pathways in types of tumour cells, including breast cancer (Shun et al., 2004), colorectal cancer (Wada et al., 2017) , lung cancer (Lim, Loh, Ting, Bradshaw, & Zeenathul, 2014a) and prostate cancer (Luk et al., 2011; Yap et al., 2008). In addition, several studies have shown that high dietary intake of T3 can suppress carcinogen-induced mammary tumourigenesis in experimental animals (Hafid, Radhakrishnan, & Nesaretnam, 2010).. a. To date, most of the molecular mechanisms exerted by T3 on tumour cells and. ay. its biosynthesis pathway(s) are still unclear. Owing to this, considerable effort has been expanded in investigating the effects of T3 and its individual fractions in cancer. al. prevention. The advent of newer cutting-edge technologies now available such as. M. genomics and proteomics has paved the way for scientists to start making headway into elucidating these mysteries as well as to scrutinise the suitability of developing T3 as a. ti. potential candidate for prevention or treatment of breast cancer. Research on T3 offers. rs i. a promising discovery as its underlying molecular mechanism has still not been clearly understood. Furthermore, the existing literature on T3 in the area of epigenetics and. ni ve. proteomics is limited. In addition, to date there is no literature on the DNA methylation process of epigenetics and MS/MS quantitative proteomic profiling of T3 in human breast cancer cells. Therefore, there is a need to elucidate these scientific evidences in. U. order to understand the mechanism(s), pathway(s) affected and biological properties of T3 at the molecular level. This research thesis comprehensively reports the novel molecular pathways and mechanisms mediated by T3 in human breast cancer cells. The data generated from this study could support several important mechanisms that partially explain the mode of actions of T3 as well as uncover some newer molecular pathways and mechanisms. The information on differentially expressed protein profiling and DNA methylation genes induced by T3 uncovered in this thesis work could lead to novel findings and understanding that could further assist future chemo-preventive measures. 3.

(25) The unique feature of this study is the effort taken to understand proteome alteration using (i) nuclear proteins; (ii) cytoplasmic proteins and (iii) proteins recovered from cell culture media. The proteomic signature identified in these proteins following the treatment with T3 isoforms can efficiently pave the way for greater scientific discovery besides uncovering many novel proteins, which were not previously reported. These results could also provide some insight on the protein–protein interacting networks. a. affected by T3.. ay. Another important aspect explored in this study is on the DNA methylation profiling of 84 tumour suppressor genes, frequently reported to be methylated in breast. al. cancer. One known advantage of using T3 as chemotherapeutic agents is their selectivity. M. for cancer cells [10]. This could be largely due to the reduced antioxidant defences expressed by these malignant cells when compared to non-malignant cells (Sznarkowska,. ti. Kostecka, Meller, & Bielawski, 2016) or may involve many other unknown factors. The. rs i. selectivity of T3 for cancer cells may be dependent on the ability of the former to specifically target and downregulate proteins, which are abnormally upregulated in. ni ve. carcinogenic but not in normal cells. As such, identification of more potent molecular targets of T3 and its isoform may provide novel insights into the anticancer mechanisms. U. of T3 and potential therapeutic strategies for breast cancer. 1.2. RESEARCH OBJECTIVES. The overall objective of this study is to elucidate the molecular mechanisms underlying differentially expressed proteins and changes in DNA methylation pattern induced in human breast cancer cells treated with T3 isoforms (γT3 and δT3).. 4.

(26) The specific objectives of this study are as follows: i). To investigate the time and dose dependent effects of T3 isoforms (γT3 and δT3) on MCF-7 and MDA-MB-231 human breast cancer cell lines. ii). To study the nuclear, cytoplasmic and supernatant protein expression profiling in MCF-7 and MDA-MB-231 human breast cancer cells following treatment with T3 isoforms (γT3 and δT3) To investigate the DNA methylation profiles of tumour suppressor genes. a. iii). ay. modulated by the T3 isoforms (γT3 and δT3) in MCF-7 and MDA-MB-231 human breast cancer cells.. To identify aberrant pathways and molecular mechanisms modulated by T3. al. iv). M. isoforms (γT3 and δT3) via the integration of findings from proteomics and DNA. RESEARCH HYPOTHESIS. rs i. 1.3. ti. methylation.. ni ve. The study is based on four research hypotheses (HA), which are listed below:. HA1:. Tocotrienols (γT3 and δT3) will inhibit proliferation of MCF-7 and MDAMB-231 human breast cancer cells in a dose- and time-dependent manner. U. HA2:. HA3:. Tocotrienols (γT3 and δT3) will modulate oncoproteins related to breast cancer in the secretome and proteome of MCF-7 and MDA-MB-231 human breast cancer cells Pathway profiling on the effects of tocotrienols (γT3 and δT3) on the proteome of human breast cancer cells (MCF-7 and MDA-MB-231) will uncover novel pathways and mechanisms. HA4:. Tocotrienols (γT3 and δT3) will reverse the DNA methylation of tumour suppressor genes in MCF-7 and MDA-MB-231human breast cancer cells. 5.

(27) CHAPTER 2 LITERATURE REVIEW 2.1. BREAST CANCER STATISTICS. Breast cancer (BC) is the most common malignancy in women around the world. According to GLOBOCAN 2018, breast cancer is the most common cancer in women;. a. accounting for 11.6% of all cancers (Bray et al., 2018). In 2018, about 2.1 million newly. ay. diagnosed female breast cancer cases were reported worldwide, which accounts for about 1 in 4 cancer cases among women (Bray et al., 2018). The same authors reported that. al. there were 626,679 (6.6%) cases of deaths due to breast cancer worldwide (Bray et al.,. M. 2018). Hence, the incidence of breast cancer in women worldwide was reported to be 24.2% while the mortality rate was estimated to be 15%. (Bray et al., 2018). A similar. ti. trend was observed in the United States of America (USA) where, the incidence rate of. rs i. cancer was reported to have increased over last decade. In 2018, there was a total of 1,735,350 new cancer cases and 609,640 cancer-related deaths in the USA (Siegel,. ni ve. Miller, & Jemal, 2019). These global breast cancer data confirm that this malignancy is a leading concern across the globe and pose serious threats to the well-being of women worldwide. In America, Africa and Asia, there is a rise in the incidences of breast cancer. U. mainly could be due to adverse lifestyle changes which increase the risk for this disease. The mortality of breast cancer in these regions is increasing, while lack of proper diagnosis and therapy being the main reason (Torre et al., 2015).. Breast cancer is also a leading cause of death among women in Malaysia. The overall percentage of breast cancer patients in Malaysia is 31% compared to other types of cancer (Organization, 2018). Nearly 3,500 new breast cancer cases are diagnosed every year. In Malaysia, the number of new cases of female breast cancer in 2018 was 6.

(28) reported to be 7,593 (32.7%) followed by colorectal cancer at 2,795 (12%) (Figure 2.1). About 1 in 20 women in Malaysia are at risk of developing breast cancer. Breast cancer is the number one female malignancy in Malaysia and in 2018 alone, there were 2,894 (11.82%) deaths due to this cancer (Organization, 2018). Current data show that the number of breast cancer patients is steadily growing in Malaysia and as such, there is a. Figure 2.1:. rs i. ti. M. al. ay. a. need to identify effective strategies to reduce the incidence and mortality.. Number of new cases in Malaysia, year 2018, females, all ages.. ni ve. (Adapted from Globocan, Malaysia,2018). 2.1.1. Breast cancer biology. U. Tumour of the breast that started from ductal hyper-proliferation can develop into a benign tumour that may further progress to become metastatic carcinomas following stimulation by various factors such as lifestyle, carcinogens and heredity. Initially, breast cancer was categorised as oestrogen-receptor (ER)-positive (such as MCF-7 and T47D cells) or ER-negative (such as MDA-MB-231, MDA-MB-468 and MDA-MB-453) breast cancers. With the discovery of few other important biomarkers, breast cancer was further categorised into molecular subtypes, such as luminal A and luminal B, human. 7.

(29) epidermal growth factor receptor 2 (HER2)-positive breast cancers (Perou & BørresenDale, 2011; Reis-Filho & Pusztai, 2011). There is yet another type of breast cancer, which is known as the basal-like breast cancer which does not express ER, progesterone receptor (PR) or HER-2 and is also known as triple-negative cancer (Hudis & Gianni, 2011). Macrophages and stromal influences in the tumour microenvironment play. a. crucial roles in the initiation and progression of breast cancers. Studies have shown that. ay. mammary gland of a rat can become neoplastic only when the stroma is exposed to carcinogen, i.e. not the extracellular matrix (ECM) or epithelium (Maffini, 2004; Soto,. al. 2017). Macrophages can generate an inflammatory microenvironment, which in turn. M. induces angiogenesis, enabling cancer cells to evade surveillance by the host’s immune system (Diversity & Tumor, 2010; Dumars et al., 2016). In general, cancer is often. ti. thought to be caused by genetic abnormalities that includes mutations in oncogenes or. rs i. tumour suppressor genes (Hanahan & Weinberg, 2011; Vogelstein & Kinzler, 2004). However, in reality carcinogenesis involves multi-processes, which includes both genetic. ni ve. and epigenetic changes (Lustberg & Ramaswamy, 2011). 2.1.2. Risk factors for breast cancer. U. There are some key risk factors known to contribute to the onset of breast cancer in woman. One of the major risk factor of breast cancer is aging, where the incidence of breast cancer has been strongly associated with increasing age in woman. For instance, in the United States of America (USA), more than 90% of the mortality rate due to breast cancer were observed in woman over the age of 60 (Siegel, Miller, & Jemal, 2017). Family history is a major risk factor in most cases of breast cancer, where the risk was reported to be two-fold higher in woman whose siblings or close relatives were diagnosed. 8.

(30) with breast cancer (Brewer, Jones, Schoemaker, Ashworth, & Swerdlow, 2017). In addition, there a large body of literature describing the association of breast cancer with family history of first-degree female relatives (Anderson, Bladström, Olsson, & Möller, 2000; Colditz, Kaphingst, Hankinson, & Rosner, 2012; Figueiredo et al., 2007; Hemminki, Granström, & Czene, 2002; Parazzini, La Vecchia, Negri, Franceschi, & Bocciolone, 1992; Peto, Easton, Matthews, Ford, & Swerdlow, 1996). Many of the inherited familial breast cancer was reported to be associated with mutations in BRCA1. ay. a. and BRCA2 genes.. Other important risk factors of breast cancer are related to reproductive factors. al. such as early menarche, late menopause, later age pregnancy and low parity (Horn,. M. Åsvold, Opdahl, Tretli, & Vatten, 2013). These factors are strongly associated with ER status (Dall & Britt, 2017). Endogenous oestrogen produced by the ovary of a pre-. ti. menopausal woman as well as exogenous oestrogen obtained from contraceptives pills. rs i. and hormone replacement therapy (HRT) increases the risk of breast cancer (Endogenous. ni ve. Hormones and Breast Cancer Collaborative Group et al., 2013). Lifestyle factors such as diet, smoking, obesity, lack of physical activities,. consumption of alcohol are also important risk factors of breast cancer and major lifestyle. U. changes have been shown to be very effective in the prevention of breast cancer (Katzke, Kaaks, & Kühn, 2015; Y.-S. Sun et al., 2017). Smoking at a very young age (Gaudet et al., 2016; Kispert & McHowat, 2017) and consumption of alcohol pose a high risks for breast cancer. Analysis of data from 20 studies concluded that consumption of alcohol is associated with increased levels of oestrogen, which is a risk factor for ER-positive and -negative breast cancers (Jung et al., 2016).. 9.

(31) The World Health Organisation (WHO) reported that between 30-50% of cancer cases are preventable (“WHO Cancer prevention,” 2017). Prevention of cancer offer long-term cost-effective strategy to control this disease. The emerging studies on cancer prevention and nutrition show that consumption of vitamins, antioxidants, and mineral as part of one’s diet can help to prevent cancer (Katzke et al., 2015). It is becoming more apparent that breast cancer can be prevented by reducing the risk factors and adopting a. Treatment modalities. ay. 2.1.3. a. more effective lifestyle strategy.. al. Treatment responses vary in different breast tumour sub-types and this make breast cancers very difficult to be controlled and/or treated (S. Moulder & Hortobagyi, 2008).. M. In addition, drug resistance as well as the side-effects of current treatment modalities. ti. further weaken the effectiveness of these treatment approaches (Cazzaniga & Bonanni,. rs i. 2012; DeSantis, Ma, Bryan, & Jemal, 2014). Current treatment modalities used at an early stage of breast cancer without metastasis has a higher chance to be cured. After. ni ve. diagnosis, the best therapeutic option for a breast cancer patient is decided by a team of specialists. Surgery and removal of tumour mass may not always be the best option for. U. every patient.. The treatment options for a breast cancer patient can vary from one patient to. another depending on its’ tumour biology and sub-types (e.g. triple negative or HER2positive). Surgery is still the mainstay of treatment in early breast cancer (McLaughlin, 2013). Cancer treatments modalities such as chemotherapy, radiotherapy, hormonal therapy and surgery often cause major side-effects in the patients (Cleeland et al., 2012). Some of the side-effects associated with breast cancer treatment can affect the quality of. 10.

(32) life of these patients, which can affect every part of their bodies. Furthermore, triplenegative breast cancers do not respond to hormonal therapy (Reddy, 2011). Traditional chemotherapy and radiotherapy can also kill normal healthy cells along with the cancer cells, which causes severe systemic syndrome such as nausea, pain and lethargy (Dantzer, Meagher, & Cleeland, 2012). Targeted anti-cancer therapy using tyrosine kinase inhibitors (TKI) is used to interfere with aberrant signal transduction of. a. cancer cells, which inhibits the growth of these cells. This approach is reported have. ay. lesser side-effects due to its target-specific inhibition (Segota, 2014). However, some of the TKI used in targeted therapy often interfere with signals of normal cells, which is. M. Vincent, 2010; Sankhala et al., 2009).. al. required for its growth and normal functions (Shepard D. & Garcia J., 2009; Fakih &. ti. In most cases, the level of toxicity, especially organ-specific toxicity of drugs. rs i. used in cancer therapy is not known until it is tested in a large subset of cancer patients. Besides the specific adverse effects as described in Table 2.1 (Cleeland et al., 2012),. ni ve. most of these drugs used in cancer therapy were also reported to cause secondary tumour incidences. These situations urge the researchers to find an alternative strategy which can. U. be used to effectively prevent and treat breast cancer while having negligible side-effects.. 11.

(33) Most common adverse effects of cancer therapy. ni ve. rs i. ti. M. al. ay. a. Table 2.1:. In recent days, treatment for breast cancer has changed to a new perspective and. researchers are finding evidence to prove that natural compounds can also exhibit anti-. U. cancer activity with increased health benefits and reduced or no side-effects as compared to other chemoprevention drugs. Chemoprevention researches in recent days are focused on finding substances or components of natural sources that can prevent or inhibit carcinogenesis. Targeted cancer therapies using natural bioactive compounds are also used to target specific characteristics of cancer cells, such as a gene or protein that allows the cancer cells to grow in a rapid or abnormal way. One such compound is tocotrienols (T3) and its derivatives, which currently gaining a lot of attention as there are many evidences that show that these compounds could prove to be very useful 12.

(34) chemopreventive natural compounds that can be used for prevention or treatment for breast cancer. 2.2 2.2.1. VITAMIN E History of Vitamin E. Vitamin E was first discovered by Herbert Evans in 1922 at the University of California,. a. USA. In the same year, a paper claiming vitamin E to be 'substance X', (for it was an. ay. unknown substance); was essential for maintaining fertility in rats was published (Evans & Bishop, 1922). Almost 14 years later, Evan and his team reported the isolation of the. al. first of many isoforms of vitamin E i.e. alpha-tocopherol (αToc) from wheat germ oil. M. (Evans, H. M., Emerson, O. H. & Emerson, 1936). This discovery was confirmed by Fernholz in 1937 (Fernholz, 1937). In the same year, the antioxidant activity of vitamin. ti. E emerged (Olcott & Emerson, 1937). There were several authors who reported on the. rs i. high antioxidant activities of vitamin E (αToc) on lipid peroxidation in the 1980s (Burton, Cheeseman, Doba, Ingold, & Slater, 1983; Cadenas, Ginsberg, Rabe, & Sies, 1984; Cruz,. ni ve. Wimberley, Johansen, & Friis-Hansen, 1983; Ohki, Takamura, & Nozawa, 1984; Tappel & Dillard, 1981). The synergistic interaction between αToc and ascorbate was found to be very effective in preventing lipid peroxidation (Doba, Burton, & Ingold, 1985;. U. Mashiko et al., 2015; K. Sato, Niki, & Shimasaki, 1990). Thirty-seven years following the discovery of αToc, a new family of vitamin E i.e. tocotrienol (T3) was discovered by Bunyan et al. (Bunyan, McHale, Green, & Marcinkiewicz, 1961). They reported that tocotrienols differed from tocoperols as T3 had an isoprenoid side chain while αToc had phytyl side chain (Figure 2.1). A paper on the isolation of T3 from latex from Havea brasiliensis (rubber tree), was published in Nature in 1965 (Dunphy, Whittle, Pennock, & Morton, 1965). However, T3 did not get 13.

(35) much attention from the researchers until its ability to lower blood cholesterol (Qureshi et al., 1991) and its potent anti-cancer (Guthrie, Gapor, Chambers, & Carroll, 1997; K.. Figure 2.2:. M. al. ay. a. Nesaretnam et al., 1992) activities emerged in the 80s and 90s.. A) Tocotrienol has three unsaturated, three double bonds on the isoprenoid side chain while B) tocopherol's side-chain is saturated. ti. (phytyl side chain). rs i. (Adapted from PubChem Substance Database (“PubChem,” accessed. ni ve. 10th May 2019). The rice bran oil is a by-product of industrial rice milling and is an important. natural source of T3 especially the γT3 (Sohail, Rakha, Butt, Iqbal, & Rashid, 2017).. U. However, the content of other T3 isoforms is very low in the rice bran oil. Cereals such as oat (Peterson, 1995), rye (Michalska et al., 2007) and barley (Falk, Krahnstöver, van der Kooij, Schlensog, & Krupinska, 2004) were also reported to contain T3, but only very small amounts. Annato bean (Bixa orellana, family: Bixaceae) is another important source of T3, which contains almost 100% tocotrienols, including the desmethyl T3 and is completely free from tocopherols. The annato bean extract, which is used as dietary supplement contains two isoforms of T3, namely T3 (10%) and T3 (90%) (Frega,. 14.

(36) Mozzon, & Bocci, 1998). The main sources of vitamin E (Toc and T3) are shown in Table 2.2. Table 2.2:. The contents of tocopherols and tocotrienols (mg/100 g of oil) in some. rs i. ti. M. al. ay. a. common edible oils. ni ve. (Adapted from Ghosh et al., 2008) 2.2.2. Chemistry of Tocotrienols. U. The vitamin E family is composed of both tocotrienols (T3) and tocopherols (Toc). Both classes of vitamin E share certain structural features including a common polar chromanol head (Figure 2.1), which is a structure that consists of two rings, one phenolic and one heterocyclic that are fused as well as a phytyl or isoprenoid chain at the C-2 position. Tocotrienols differ from tocopherols only by their side chains (Figure 2.1). Both have substituted methyl groups on the chromanol ring at an identical position. Tocotrienols have unsaturated isoprenoid side-chain with double bonds in the 3’, 7’ and 11’ positions, whilst Toc have saturated phytyl carbon chain. Although Toc exist only 15.

(37) as free chromanols in nature, T3 can also occur naturally in the esterified forms (Schneider, 2005). Owing to the presence of the unsaturated bonds in the isoprenoid side chains, the T3 are attributed with a unique conformation. The names of the four T3 isoforms (, ,  and ) are derived from the substituted methyl groups on the chromanol. Figure 2.3:. M. al. ay. a. ring (Figure 2.3).. Chemical structures of isoforms of tocotrienols (Adapted from Shahidi. Emergence of Tocotrienols Research. ni ve. 2.2.3. rs i. ti. & De Camargo, 2016). The second isomer of Toc, i.e. δToc was isolated from soybean oil in 1947 (Stern & Robeson, 1947). The existence of another class of vitamin E i.e. T3 was further reported in 1964 (Pennock, Hemming, & Kerr, 1964). and two-years after this, δT3 was. U. discovered and isolated from plant sources (Whittle, Dunphy, & Pennock, 1966). The biological significance of T3 was not appreciated until T3 from barley (Hordeum vulgare L.) was first identified as an important compound that can interfere with human cholesterol-genesis by Qureshi and co-workers in 1986 (Qureshi, Burger, Peterson, & Elson, 1986). This was the first report to demonstrate the beneficial effects of T3 in human health. During the 1990s, the biological properties of Toc and T3 began to be more clearly delineated, especially for γToc and δToc as well as γT3 and δT3. A number. 16.

(38) of health-related biological properties of Toc and T3 have been identified, including its anti-cholesterolemic (Chan, 1998; Parker, Pearce, Clark, Gordon, & Wright, 1993), antihypertensive (Cheng et al., 2017), antioxidant (Nor Azman et al., 2018; Yoshida, Niki, & Noguchi, 2003), immunomodulatory (Abdul Hafid, Chakravarthi, Nesaretnam, & Radhakrishnan, 2013; Hafid et al., 2010) and more importantly anti-cancer (K. Nesaretnam et al., 1992; Kalanithi Nesaretnam et al., 2004) properties. In the following. Tocotrienols and Breast Cancer. ay. 2.2.4. a. decade, evidence on the anti-cancer activities of T3 started to appear and accumulate.. al. Scientific evidence on the anti-cancer potential of T3 can be traced back to 1986, whereby in this year Sylvester and co-workers (1986) demonstrated the ability of dietary. M. palm oil to reduce progression of mammalian tumour caused by carcinogens (Sylvester,. ti. Russell, Ip, & Ip, 1986). This evidence was further confirmed by another in vitro study,. rs i. which reported on the anti-proliferative effects of TRF isolated from palm oil on mammalian tumour cells (McIntyre et al., 2000; Shah, Gapor, & Sylvester, 2003). Initial. ni ve. evidence of the anti-cancer properties of T3 was also reported in 1989 in a rodent experimental model, where it was shown that supplementation of female rats with TRF from palm oil can prevent chemically-induced mammary tumourigenesis (K. Nesaretnam. U. et al., 1992). Later, T3 were shown to inhibit proliferation of oestrogen receptor (ER)positive (MCF-7) and ER-negative (MDA-MB-435) cultured human breast cancer cell lines, either on its’ own or in combination with tamoxifen (Guthrie et al., 1997). The relative anti-proliferative effects of T3 isoforms against MCF-7 human breast cancer cells were reported as γT3 > δ-3 > αT3> (Ramdas et al, 2010). In addition, T3 are also reported to inhibit the proliferation and growth of murine breast cancer cells (Abdul Hafid et al., 2013; Bachawal, Wali, & Sylvester, 2010; Parajuli et al., 2015) and supress the growth of murine breast tumour in experimental models (Abdul Hafid et al., 2013).. 17.

(39) Ever since these results were reported decades ago, many other important evidence had emerged, which further confirmed the powerful anti-tumorigenic effects of T3 on various cancer cell lines such as prostate (Yap et al., 2008), lung (Phutthaphadoong, Yodkeeree, Chaiyasut, & Limtrakul, 2012) and liver (Abdul Rahman Sazli et al., 2015). There are also a few studies that have reported the ability of individual T3 isoforms, especially T3 and T3 to inhibit the proliferation and growth of various cancer cells (Constantinou, Charalambous, & Kanakis, 2019; Ding, Peng, Deng, Fan, &. a. Huang, 2017; Ramdas et al., 2011; K. Tang et al., 2019; Thomsen, Andersen, Steffensen,. ay. Adimi, & Jakobsen, 2019; Yap et al., 2008). Besides its antioxidant and anti-. al. inflammatory effects, these T3 isomers were also found to mediate the intracellular signalling of cancer cells via various mechanisms such as promoting apoptosis (Sakai,. M. Okabe, Tachibana, & Yamada, 2006; Yamasaki et al., 2014), anti-angiogenesis (Yang Li et al., 2011; Weng-Yew, Selvaduray, Ming, & Nesaretnam, 2009), antimetastatic (Abdul. ti. Hafid et al., 2013; Husain et al., 2017) and other important cellular mechanisms. Tocotrienols and Other Cancers. ni ve. 2.2.5. rs i. (Montagnani Marelli et al., 2019).. Research over the last decade had revealed the potential benefits of T3 on various types. U. of cancers (Abraham, Kattoor, Saldeen, & Mehta, 2019). Beside breast cancer, T3 were reported to exert anti-cancer effects of on colorectal cancer cells. For instance, previous study had shown that δT3 inhibited the proliferation of colon cancer cells via downregulation of Wnt-1, β-catenin and the Cyclin D1 pathways (Ahmed, Alawin, & Sylvester, 2016; J.-S. Zhang et al., 2015).. Gamma-T3 was reported to inhibit. proliferation of HT-29 colon cancer cells via downregulation of Bcl-2 and upregulation of Caspase (W.-L. Xu et al., 2009). It was also reported that γT3 inhibited the growth of human gastric cancer cells via the deactivation of the NF-κB pathways, using both cell18.

(40) based and experimental model approaches (Manu et al., 2012). Another study reported on the ability of γT3 to inhibit the invasion and migration of gastric cancer cells (Y. H. Zhang et al., 2018). Tocotrienols were also shown to be effective against lung cancer, where a study showed that all T3 isoforms induced apoptosis in lung cancer cells by activating the caspase-8 apoptotic pathway (Lim et al., 2014a). The ability of δ-T3 in inducing. a. cytotoxicity to the glioblastoma cells SF-295 in vitro were also reported (de Mesquita et. ay. al., 2011). In addition, all T3 isoforms were also reported to induce apoptosis in glioblastoma cells via activation of the caspase-8 pathway and by disrupting the integrity. al. of the mitochondrial membrane (Lim et al., 2014a). Yap and co-workers (2008) reported. M. that T3 are novel compounds that had high potential to be used for the prevention and treatment of prostate cancer (Yap et al., 2008). The authors showed that T3 enhanced. ti. apoptosis and autophagy in human prostate cancer cells. Using a murine cancer model,. rs i. it was reported that treatment with γT3 significantly reduced growth of tumour in prostate (Barve et al., 2010), colorectal cancer (Prasad, Gupta, Tyagi, & Aggarwal, 2016a) and. ni ve. liver (Siveen et al., 2014). The ability γT3 to inhibit proliferation of pancreatic cancer cells was reported to take place via inhibition of the NF-κB pathway (Hodul et al., 2013). Other cancer cells that were shown to be inhibited by T3 include skin cancer cells where. U. δT3 was reported to inhibit proliferation of human melanoma cells and triggered endoplasmic reticulum stress-mediated apoptosis (Montagnani Marelli et al., 2016) while γT3 induced apoptosis in human T cell lymphoma (Wilankar et al., 2011) and H2452 (A. Sato et al., 2016)cells through inactivation of the Ras oncogene pathways. All these past and recent evidence collectively support the powerful anti-cancer potential of T3 on various cancer types.. 19.

(41) 2.3. In Vitro Model of Breast Cancer. The MCF-7 cell line is the most commonly used xenograft model to study experimental models of human breast cancer. This cell line was established from a pleural effusion at Michigan Cancer Foundation in 1973 (A. V. Lee, Oesterreich, & Davidson, 2015; Soule, Vazquez, Long, Albert, & Brennan, 1973) This cell line represents an early stage breast cancer and due to its’ ER-positive status, it is dependent on oestrogen for its growth in. a. vitro. The MCF-7 cells are non-invasive although it originated from a metastases of. ay. advanced stage tumour mass and this has shown in animal model (Aliaga et al., 2004). These cells express the wild-type p53 (Okumura et al., 2002). Hence, the xenograft. al. model of MCF-7 is a very useful model for testing anti-oestrogen therapies such as. M. tamoxifen for identification of drugs resistance mechanisms (Kao et al., 2009).. ti. The MDA-MB-231 cell line was isolated at MD Anderson from a pleural. rs i. effusion of a patient with invasive ductal carcinoma (Cailleau, Young, Olivé, & Reeves, 1974). This cell line is characterised by ER-negative, progesterone receptor (PR)-. ni ve. negative and E-cadherin negative status and also expresses mutated p53. The MDA-MB231 cells are characterised by the presence of human epidermal growth factor receptor 2 (HER2) (Chavez, Garimella, & Lipkowitz, 2010). These cells represent a good model. U. of triple-negative breast cancer (Hanahan, Wagner, & Palmiter, 2007; Y. Kang et al., 2003) and are the most commonly used as cell model to represent the late-stage breast cancer (Amaro et al., 2016). The MDA-MB-231 cells are invasive in vitro and when implanted orthotopically, produce xenografts that spontaneously metastasise to lymph nodes (Yoneda, Williams, Hiraga, Niewolna, & Nishimura, 2001). Variants of MDAMB-231 cells with unique metastatic properties have been selected from the parental cell line.. 20.

(42) 2.4. Epigenetics: DNA Methylation. Epigenetic refers to the changes, which takes place inside the gene without causing a change the DNA sequences. There are two types of epigenetic mechanisms, which is interconnected such as DNA methylation and covalent modification of the histones. The DNA methylation and histone modification are the two important mechanisms, which regulate many biological process such as genomic imprinting, X-chromosome. a. inactivation and the changes in the developmental genes (Du, Johnson, Jacobsen, &. ay. Patel, 2015). Dysregulation of this mechanisms give rise to the onset of chronic disease such as cancer (Taberlay & Jones, 2011). Although the epigenetic changes do not change. al. the DNA sequence as seen in mutation, it significantly affects the transcription process. M. and changes the gene expression profiles (Newell-Price, Clark, & King, 2000). There are many studies, which showed that hypermethylation of the promoter region of tumour. ti. suppressor genes can cause the onset and development of cancer (Böck et al., 2018;. rs i. Martínez-Calle et al., 2019; Ng & Yu, 2015). Methylation of DNA is an important epigenetic mechanism, which occurs due to addition of a methyl (CH3) group to the. ni ve. DNA, and this action changes the function of the genes. However, unlike the genetic changes, epigenetic mechanisms are reversible (Kanwal & Gupta, 2012) and this had paved ways for many researchers to find potent inhibitors, which can be used as a strategy. U. to stop or reverse some of these processes. During DNA methylation process, a methyl group is covalently added to the 5-. carbon ring of the base cytosine, which produces 5-methylcytosine which is known as the "fifth base" DNA in the CpG islands (Carroll, 2017; Eriksson, Lennartsson, & Lehmann, 2015) (Figure 2.4). Addition of the methyl group(s) to the promoter region inhibits transcription of the affected gene. Most of the DNA methylation occurs at the cytosine-guanine rich (CpG) islands in the tumour suppressor genes, and this greatly. 21.

(43) influence the expression of these genes (Deaton & Bird, 2011). There is growing literature that supports the role of epigenetics, especially the DNA methylation mechanism to help unravel how some of the bioactive natural compounds affect this. ni ve. rs i. ti. M. al. ay. a. mechanism.. Figure 2.4:. The methylation of cytosine nucleotides occurs mainly on the 5th carbon. (Adapted from Carroll, 2017). DNA Methylation in Normal Vs Cancer Biology. U. 2.4.1. In normal cells, there are three main targets repressed by the DNA methylation process. The first target is the general parental gene imprinting, which are differentially expressed from the parental chromosomes. They are the main key regulators of the embryogenesis and adult life. In many cases, the inactive allele is marked with DNA methylation, and the mono-allelic expression is lost in the absence of methylation (Feinberg, 2007). Secondly, the transposons and the other repeated sequences, which make up a large. 22.

(44) fraction of the mammalian genome (Walsh & Bestor, 1999). Thirdly, some genes are methylated in a tissue-specific manner (Illingworth et al., 2008). Tumour cells are often noted to exhibit abnormal DNA methylation patterns whereby a number of tumour suppressor genes were found to be methylated and inactive (Esteller, 2007). These abnormal DNA methylation patterns are the initial causal event and onset of cellular transformation, which initiates the very first step of tumorigenesis. a. (Feinberg, Ohlsson, & Henikoff, 2006). In normal cells, the promoter region of the. ay. genes/tumour supressor genes are rarely methylated and have a regular expression. However, in cancer cells, cytosines 5′ to guanosines (CpG) islands in promoter region of. al. tumour suppressor gene are methylated resulting in hypermethylation and gene silencing. M. (M. Yang & Park, 2012) (Figure 2.5). Demethylating agents are able to re-establish the expression of the tumour suppressor genes silenced by the DNA methylation event and. ti. as such, have great potential to be used as therapeutic targets for many types of cancer. U. ni ve. rs i. such as breast and leukaemia (Smith, Otterson, & Plass, 2007).. Figure 2.5:. Cytosines 5′ to guanosines (CpG) islands in promoter region of tumour. suppressor. gene. are. methylated. resulting. in. hypermethylation and gene silencing. (Adapted from Yang & Park, 2012). 23.