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ALAA TAHA YASIR AL KANAN Hj.Pdf MECHANISMS OF ANTI-PROLIFERTATIVE EFFECT OF GARCINIA HOMBRONIANA ESSENTIAL OILS LEAVES IN MCF-7 AND MCF-7/TAMR-1 HUMAN BREAST CANCER CELL LINES BY ALAA TAHA YASIR AL KANAN DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE (HEALTH TOXICOLOGY) ADVANCED MEDICAL AND DENTAL INSTITUTE UNIVERSITI SAINS MALAYSIA 2019 DECLARATION I hereby declare that this research was sent to universiti sains malaysia (USM) for the degree of Master of Science in Health Toxicology. It has not been sent to other Universities. With that, this research can be used for the consultation and can be photocopied as reference. Sincerely, ------------------------- ALAA TAHA YASIR AL KANAN (P-IPM0060/18) ACKNOWLEDGEMENT In the name of Allah, the Most Beneficient, the Most Merciful. Praise to Allah, who has given me the opportunity, strength, knowledge and ability to complete this work and my Master study. Firstly, I would like to thank and show my deepest appreciation to my supervisor Dr Nik Nur Syazni Nik Mohd Kamal for her unflagging enthusiasm, valuable guidance and constant encouragement throughout the tenure of my study in IPPT, USM. Her valuable knowledge and her logical way of thinking have been of great value for me. I would also like to express gratitude to my co-supervisor Dr Tan Wen Nee, who help me with the essential oil extraction as the first step of my laboratory work and I also appreciate the support from PhD candidates, Nurul Izzati and Musthahimah Muhamad who always assist me in this research. My acknowledgement would be incomplete without my deepest thanks to the pillar of my strength, my wife and the blessings of my mother who helped me at every stage of my personal and academic life and longed to see this achievement come true. Lastly, I would also express lots of thanks to anyone who directly and indirectly involves in finishing this project. Thank you. Alaa Taha Yasir Alkanan P-IPM0060/18 Health toxicology 2018/2019 i TABLE OF CONTENTS ACKNOWLEDGEMENT .......................................................................................... i TABLE OF CONTENTS .......................................................................................... ii LIST OF TABLES ..................................................................................................... v LIST OF FIGURES .................................................................................................. vi LIST OF ABBREVIATIONS ................................................................................ viii ABSTRAK .................................................................................................................. x ABSTRACT .............................................................................................................. xii CHAPTER 1 INTRODUCTION .............................................................................. 1 1.1 Background of the study .................................................................................. 1 1.2 Problem statement ............................................................................................ 2 1.3 Research objectives .......................................................................................... 3 1.4 Research question ............................................................................................. 4 CHAPTER 2 LITERATURE REVIEW .................................................................. 5 2.1 Cancer .............................................................................................................. 5 2.2 Breast cancer .................................................................................................... 6 2.3 Breast cancer subtypes ..................................................................................... 6 2.4 Breast cancer risk factors ................................................................................. 7 2.5 Treatment of breast cancer ............................................................................... 8 2.6 Tamoxifen resistance in breast cancer ............................................................. 9 2.7 Natural products derived from the plant as an alternative treatment for breast cancer ............................................................................................................. 10 2.8 Taxonomy of Garcinia hombroniana plant ................................................... 12 ii 2.9 Garcinia hombroniana ................................................................................... 12 2.10 Cell death ....................................................................................................... 15 2.11 Apoptosis ........................................................................................................ 16 2.12 Necrosis .......................................................................................................... 17 2.13 Essential oils and their bio-activities .............................................................. 17 CHAPTER 3 METHODOLOGY ........................................................................... 19 3.1 Materials and chemicals ................................................................................. 19 3.1.1 Chemicals and reagents ............................................................................. 19 3.1.2 Consumables ............................................................................................. 20 3.1.3 Laboratory equipment ............................................................................... 21 3.2 Extraction of essential oils by hydrodistillation technique ............................ 21 3.3 Cell culture ..................................................................................................... 22 3.3.1 Human breast adenocarcinoma cell lines .................................................. 22 3.3.2 Reagents for cell culture work .................................................................. 22 3.3.3 Culture procedures and conditions ............................................................ 25 3.3.4 Thawing of cells from frozen storage ....................................................... 25 3.3.5 Sub-culturing of cells ................................................................................ 26 3.3.6 Cell counting for seeding ........................................................................... 26 3.3 Assessment of anti-proliferative effect by MTT assay .................................. 27 3.4 Cell optimization ............................................................................................ 27 3.5 Treatment cells for apoptosis ......................................................................... 29 3.6 Assessment on the mechanism of apoptosis and/or necrosis by flow cytometry ........................................................................................................ 29 3.7 Statistical analysis .......................................................................................... 30 CHAPTER 4 RESULTS .......................................................................................... 31 4.1 Anti-proliferative effect of GH-EOL and morphological changes ................ 31 iii 4.1.1 Anti-proliferative studies ........................................................................... 31 4.1.2 Effect of GH-EOL on treated cell line morphology .................................. 34 4.2 Determination of the constant IC50 values for GH-EOL on MCF-7, MCF- 7/TAMR-1, and MCF-10A cells .................................................................... 37 4.3 Cells optimization .......................................................................................... 39 4.4 Apoptosis detection ........................................................................................ 43 4.4.1 Mode of cell death induced by GH-EOL in breast cancer cell lines ......... 45 4.4.2 Mode of cell death induced by GH-EOL in MCF-10A cell line .............. 45 CHAPTER 5 DISCUSSION .................................................................................... 47 CHAPTER 6 CONCLUSION ................................................................................. 50 6.1 Conclusion ...................................................................................................... 50 6.2 Recommendation ............................................................................................ 50 REFERENCES ......................................................................................................... 52 iv LIST OF TABLES Table 2.1: Taxonomy hierarchy of Garcinia hombroniana plant ............................. 12 Table 3.1: List of chemicals and reagents ................................................................. 19 Table 3.2: List of consumables ................................................................................. 20 Table 3.3: List of laboratory equipment .................................................................... 21 Table 3.4: Cells optimization with different concentrations and treatment media volumes of GH-EOL and tamoxifen. ................................................. 28 Table 4.1: The IC50 values of GH-EOL determined using MCF-7, MCF-7/TAMR- 1, and MCF-10A cells ........................................................................ 37 v LIST OF FIGURES Figure 2.1: Molecular subtypes of breast cancer ........................................................ 7 Figure 2.2: Garcinia hombroniana
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