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

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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

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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

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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

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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

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LIST OF FIGURES

Figure 2.1: Molecular subtypes of breast cancer ...... 7 Figure 2.2: Garcinia hombroniana leaves ...... 13 Figure 2.3: Garcinia hombroniana tree ...... 14 Figure 2.4: Classification of cell death pathway ...... 16 Figure 4.1: Anti-proliferative effect of GH-EOL on MCF-7 cells. The values represent means ± SD of the triplicates. * indicates significant differences (p < 0.05) with respect to the untreated group...... 32 Figure 4.2: Anti-proliferative effect of GH-EOL on MCF-7/TAMR-1 cells. The values represent means ± SD of the triplicates. * indicates significant differences (p < 0.05) with respect to the untreated group...... 33 Figure 4.3: Effect of GH-EOL and tamoxifen on the cellular viability and morphological changes after 24 hours of treatment...... 36

Figure 4.4: The constant IC50 value of GH-EOL on MCF-7 cells ...... 38

Figure 4.5: The constant IC50 value of GH-EOL on MCF-7/TAMR-1 cells ...... 38

Figure 4.6: The constant IC50 value of GH-EOL on MCF-10A cells ...... 39 Figure 4.7: Effect of GH-EOL and tamoxifen on the MCF-7 cell viability within 24 hours of treatment compared with untreated. Pictures were in x4 magnification...... 40 Figure 4.8: Effect of GH-EOL and tamoxifen on the MCF-7/TAMR-1 cell viability within 24 hours of treatment compared with untreated. Pictures were in x4 magnification...... 42 Figure 4.9: Effect of GH-EOL and tamoxifen on the MCF-10A cell viability within 24 hours of treatment compared with untreated. Pictures were in x4 magnification...... 43 Figure 4.10: Percentage distribution of GH-EOL induced apoptotic and necrotic breast cancer cells. MCF-7 and MCF-10A cells were treated with GH-EOL and tamoxifen (20 µg/mL and 15 µM, respectively) while for 24 hours MCF-7/TAMR-1 was treated with GH-EOL and

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tamoxifen (20 µg/mL and 12.5 µM, respectively) for 24 hr. The cells were stained with annexin V-FITC antibody and propidium iodide and analyzed by flow cytometry...... 44 Figure 4.11: The total percentage of apoptotic cells. Data shown are from a representative experiment repeated three times with similar results. There was a significant difference between treated and untreated (p < 0.05)...... 46

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LIST OF ABBREVIATIONS

ATCC American type culture collection

ATP Adenosine triphosphate

BARD1 Breast cancer Associated RING Domain 1

BRCA1 Breast-Cancer Susceptibility Gene 1

DBTRG Human glioblastoma cell line

DMEM/F12 Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12

DMSO Dimethyl sulfoxide

EDTA Ethylenediaminetetraacetic acid

EO Essential oils

EO-L Essential oils leave

ER Estrogen receptor

FBS Fetal bovine serum

FITC Fluorescein isothiocyanate

GH Garcinia hombroniana

GH-EO-L Garcinia hombroniana essential oil leaves hEGF human Epidermal Growth Factor

HER2 human epidermal growth factor receptor 2

IC50 Inhibitory concentration at 50%

LDL Low – density lipoprotein

MCF-10A Human mammary epithelial breast cell line

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MCF-7 A designated nomenclature for a human breast adenocarcinoma cell line

MCF-7/TAMR-1 A designated nomenclature for a human breast adenocarcinoma cell line resistant to tamoxifen

MTT Microtiter tetrazolium oC Celsius

OD Optical density

PBS Phosphate buffered saline

Pen-Strep Penicillin Streptomycin

PI Propidium iodide

PR Progesterone receptor

PS Phosphatidylserine

RNS Reactive nitrogen species

ROS reactive oxygen species

RPMI-1460 Roswell Park Memorial Institute 1640 Medium

SD Standard deviation

SPSS Statistical Package for the Social Sciences

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ABSTRAK

Latar Belakang: Kanser payudara adalah merupakan kanser yang paling biasa menyerangi kaum wanita dan kadar insiden semakin meningkat setiap tahun di seluruh dunia. Tamoksifen ialah ubat kemoterapeutik utama digunakan dalam merawat pesakit kanser payudara. Walaubagaimanapun, komplikasi ginekologi dan rintangan terhadap ubat merupakan antara kesan buruk yang utama terutamanya selepas 10-15 tahun rawatan dengan tamoksifen. Kemudian, kemungkinan berlakunya perulangan kanser payudara yang juga boleh menyumbang kepada penyebab utama kepada kematian yang berpunca daripada kanser tersebut. Garcinia hombroniana (GH) (manggis hutan) ialah sejenis pokok yang telah dilaporkan mempunyai kesan sitotoksik yang baik dalam melawan pertumbuhan pelbagai jenis sel titisan kanser, termasuklah MCF-7. Oleh sebab itu, tujuan kajian ini adalah untuk melanjutkan penyiasatan kesan-kesan anti-proliferasi dan apoptosis minyak pati yang telah diekstrak daripada daun GH terhadap dua jenis sel titisan kanser payudara yang berbeza, iaitu MCF-7 dan MCF-7/TAMR-1.

Kaedah: Minyak pati telah diekstrak daripada daun GH melalui proses penghidrosulingan. Kesan anti-proliferasi minyak pati terhadap sel-sel titisan MCF-7 dan

MCF-7/TAMR-1 ditentukan menggunakan asai MTT dan dinilai melalui pembaca plat mikro. Mekanisme kematian sel ditentukan menggunakan asai pewarna Aneksin V-

FITC/propidium iodida dan diukur secara kuantitatif melalui aliran sitometri. Sel titisan payudara bukan kanser iaitu MCF-10A juga dinilai dalam kedua-dua asai sebagai sel kawalan untuk dibandingkan kepada sel MCF-7 dan MCF-7/TAMR-1.

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Keputusan: Keputusan telah menunjukkan bahawa minyak pati GH mempamerkan kesan anti-proliferasi melawan pertumbuhan kedua-dua jenis sel MCF-7 dan MCF-7/TAMR-1 yang bersandarkan kepada dos dan masa, dengan nilai IC50 masing-masing 35.22 µg/mL and 17.67 µg/mL. Utamanya, minyak pati GH menunjukkan kesan toksik yang rendah terhadap sel titisan payudara bukan kanser, MCF-10A, dengan nilai IC50 76.11 µg/mL.

Tambahan pula, analisis aliran sitometri juga selanjutnya mengesahkan kematian yang diaruh oleh minyak pati GH berlaku melalui mekanisme apoptosis.

Kesimpulan: Kajian ini menyimpulkan bahawa minyak pati GH menunjukkan kesan anti-proliferasi yang kuat terhadap kedua-dua sel kanser payudara manusia iaitu MCF-7 dan MCF-7/TAMR-1. Berdasarkan nilai IC50, MCF-7/TAMR-1 yang mewakili sel titisan payudara yang berfenotip rintangan terhadap tamoksifen, adalah lebih sensitif terhadap rawatan minyak pati GH. Paling utama, minyak pati GH menunjukkan aktiviti sitotoksik yang rendah terhadap sel titisan payudara bukan kanser, MCF-10A. Keputusan-keputusan ini dapat menjelaskan sebahagian kepada tindakan minyak pati yang bersifat selektif terhadap sel-sel titisan kanser payudara tetapi tidak kepada sel normal. Di samping kesan sitotoksik yang selektif, tindakan perencatan minyak pati GH dalam melawan pertumbuhan sel-sel kanser payudara manusia MCF-7 dan MCF-7/TAMR-1 juga diperantarakan melalui apoptosis. Oleh itu, minyak pati GH sangat berpotensi untuk dimajukan sebagai agen anti-kanser yang baru, selektif dan kuat di masa hadapan.

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ABSTRACT

Background: Breast cancer is the most common cancer affected women and the incidence rate is increasing yearly throughout the world. Tamoxifen is the first-line chemotherapeutic drug used in treating breast cancer patients. However, gynaecological complications and drug resistance are among the major drawback effects of tamoxifen, particularly at 10-15 years of post-treatment. Thereafter, breast cancer recurrence may occur which also contribute to the major causes of breast cancer-related deaths. Garcinia hombroniana (GH) (seashore mangosteen) is a plant that has been reported to possess good cytotoxic effect against the growth of various human cancer cell lines, including the

MCF-7. For this reason, the aim of this study was to further investigate the anti- proliferative and apoptotic effects of the essential oil extracted from the leaves of GH against two different types of breast cancer cell lines, MCF-7 and MCF-7/TAMR-1.

Methods: The essential oil was extracted by hydrodistillation process from the leaves of

GH. The anti-proliferative effects of the essential oil against MCF-7 and MCF-7/TAMR-

1 cancer cell lines were determined using MTT assay and measured by a microplate reader. The mechanism of cell death was determined using Annexin V-FITC/propidium iodide staining assay and quantitatively measured by flow cytometry. The human non- cancerous breast cell line, MCF-10A was also included in both assays as comparative control cells to the MCF-7 and MCF-7/TAMR-1 cells.

Results: The results showed that the GH essential oil exhibited anti-proliferative effect against the growth of both MCF-7 and MCF-7/TAMR-1 cells following dose- and time- dependent manners, with an IC50 of 35.22 µg/mL and 17.67 µg/mL, respectively.

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Importantly, it exhibited low toxicity effect against the non-cancerous human breast cell line, MCF-10A, with an IC50 of 76.11 µg/mL. Additionally, flow cytometric analysis also further confirmed that the cell death induced by GH essential oil occurred via the mechanism of apoptosis.

Conclusions: This study concluded that the essential oil of GH exhibited potent antiproliferative effects against both MCF-7 and MCF-7/TAMR-1 human breast cancer cells. Based on the IC50 values, the MCF-7/TAMR-1, which represents tamoxifen- resistant phenotype breast cancer cell line, was more sensitive towards the GH essential oil treatment. Importantly, GH essential oil demonstrated low cytotoxicity towards the non-cancerous breast cell line, MCF-10A. These findings may at least in part explain to the selectivity of GH essential oil in killing breast cancer cell lines but not in normal counterpart. Besides its selective cytotoxic effect, the growth inhibitory action of GH essential oil against MCF-7 and MCF-7/TAMR-1 human breast cancer cells also mediated by apoptosis. Therefore, GH essential oil could be developed as a new, selective and potent anticancer agent in future.

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CHAPTER 1

INTRODUCTION

1.1 Background of the study

Cancer is defined as a group of disease that is characterized by abnormal cell growth which tends to proliferate in an uncontrollable way and metastasize to distant sites of the body (National Cancer Institute, 2015). GLOBOCAN database indicated that the worldwide cancer morbidity has increased to more than 18 million cases and mortality has increased to more than 9.5 million cases in 2018 (Bray et al., 2018). Breast cancer is now leading cause of cancer incidence among females and it is the second most common cause of death after lung tumour in both males and females (GLOBOCAN, 2018). All over the world, both in developed and developing nations, breast cancer is becoming a major health problem among females. In South-Eastern countries, Malaysian women have the largest mortality rate of breast cancer (Nordin et al., 2018). Although chemotherapies are commonly prescribed for the control of breast cancer, the drugs can also harm normal cells, causing significant adverse effects. Consequently, cancer patients are increasingly seeking out for alternative and complementary medicins such as herbs and medicinal plants (Gomez et al., 2016).

Breast cancer is a disease which has diverse etiologies classified by histological, molecular and phenotypes. Estrogen receptor-positive (ER+) breast cancer is the most frequent reported breast cancer subtypes which depend on the estrogen to support the growth and scattered off the cancer cells (Hon et al., 2016). Tamoxifen and aromatase

1 inhibitors, are examples of the most effective endocrine therapies, to help delay or prevent the growth of ER+ breast cancer by blocking the actions of estrogen. However, resistance to these agents has become a crucial clinical challenge in the management of ER+ breast cancer. The MCF-7/TAMR-1 breast cancer cell line provides a model cell system for studying tamoxifen resistance (Viedma-RodriGuez et al., 2014).

Medicinal plants have been widely used and explored for their good potential as new anticancer agents. Moreover, these plants may exhibit a lesser or minimal level of adverse effects on healthy cells when compared with synthetic drugs (Roy et al., 2017).

Species of genus Garcinia could affect the development and progression of breast cancer.

Specifically, Garcinia species has been demonstrated to attenuate breast cancer progression through its anti-proliferative, anti-metastasic of breast tumour cells, induction of apoptosis and synergistic activity with chemotherapeutic drugs (Li et al., 2017).

Garcinia hombroniana (GH), known as “seashore mangosteen” in Malaysia, is commonly used in traditional Malay medicine to treat different disorders such as abdominal pain and gonorrhoea, however little is known about its toxicological properties (Dyary et al., 2016).

Therefore, the use of GH could be a practical attempt as a potential agent in preventing and treating breast cancer.

1.2 Problem statement

Breast cancer is the most common malignancy in women around the world.

Globally, it is estimated that the ratio of breast cancer is that might occur among women one in four (Bray et al., 2018). Tamoxifen, which is a selective ER regulator, has contributed to the decline in mortality rate among patients with hormone receptor-positive

2 breast cancer. However, development of resistance to tamoxifen has led to disease progression and death (Fagan et al., 2017). Medicinal plants have been regarded as one of the valuable sources of bioactive agents that may contribute to the anticancer activity. This includes Garcinia species, which also produces essential oils. The essential oil can be extracted from different parts of the plant including leaves by various methods. These essential oils contain a mixture of mostly volatile and triterpenoids constituents such as α- copaene, germacrene D and β-caryophllene (Jamila et al., 2015; Tan et al., 2018).

Furthermore, essential oils extracted from Garcinia atroviridis have been demonstrated to induce cytotoxicity against MCF-7 human breast cancer line (Tan et al., 2018).

Therefore, for this study, GH leaf extract was chosen to study its anti-proliferative effect on breast cancer cell lines. Two models of breast cancer cell lines were used in this study, namely MCF-7 and MCF-7/TAMR-1. Besides that, the dose-response effects and the IC50 values of essential oils extracted from leaves of GH were determined and established by using MTT assay. Furthermore, the mode of cell death via apoptosis and/or necrosis was determined by using Annexin-V FITC and propidium iodide staining assay by flow cytometry. The hypothesis of this study was GH essential oils leaves (GH-EOL) may exhibit anti-proliferative effect against both cell lines through the induction of apoptosis.

1.3 Research objectives

For this study, the main objective was to investigate the anti-proliferative effect of

GH-EOL in human breast cancer cells. To achieve this, sub-objectives were constructed and listed as below:

1. to extract essential oils from GH leaves by using hydrodistillation technique.

3

2. to determine the dose-response effects and to establish the IC₅₀ values of GH-EOL

on MCF-7 and MCF-7/TAMR-1 human breast cancer cell lines by using MTT

assay.

3. to determine the apoptotic or necrotic effects of GH-EOL in MCF-7 and MCF-

7/TAMR-1 human breast cancer cell lines by flow cytometry.

1.4 Research question

In general this study need to investigate the anti-proliferative effect of GH-EOL in human breast cancer cells. Following questions had been developed: Is there an anti- proliferative effect of GH-EOL in tamoxifen resistant human breast cancer cell line (MCF-

7/TAMR-1)? What are the mechanism of GH-EOL in regards to cell death?

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CHAPTER 2

LITERATURE REVIEW

2.1 Cancer

According to the World Health Organization (2018), cancer is defined as a complex disease involving abnormal cells grow uncontrollably and, in some cases, to metastasize

(spread) nearby tissues. Worldwide, cancer is the second dominant cause of death and 9.6 million deaths from cancer were estimated in 2018. Globally, about 1 in 6 deaths is due to cancer. It is also stated that cancers of the males, lung, prostate, colorectal, stomach and liver are the most frequently diagnosed cancers, whereas among females are breast, colorectal, lung, cervix, and thyroid cancers (International Agency for Research on

Cancer, 2018).

The most common or suspected factors increase the risk of developing cancer are advancing age, drinking alcohol, exposure to cancer-causing substances in the environment (carcinogens) such as tobacco smoking and UV radiation from sunlight, chronic inflammation, dietary components or nutrients, hormones such as estrogens, immunosuppressive drugs, infectious agents including viruses, bacteria and parasites, and obesity (National Cancer Institute, 2015). Cancer therapies are being continually developed as increasing knowledge of molecular and tumour biology. There are many therapeutic strategies to prevent and/or cure cancer such as surgery, radiation, chemotherapy, immunotherapy, targeted therapy (drug only acts on cancer cell), and

5 hormone therapy (Zugazagoitia et al., 2016). However, interest is also being shown in alternative treatment using natural products (Shaikh et al., 2016).

2.2 Breast cancer

Breast cancer is the most commonly diagnosed cancer and also the second dominant cause of cancer death among women in the world. In 2018, approximately 2.1 million women diagnosed with breast cancer, accounting for approximately 11.6% of the cancer cases (Bray et al., 2018). In South-East Asia, the incidence rate of breast cancer in

Malaysia is 18 per 100,000 populations in comparison to Singapore and Thailand which at 15 and 11 per 100,000 populations respectively. On the other hand, the median survival time for breast cancer patients diagnosed in stage Ш is 50.77 months in North-East

Peninsular Malaysia (Nordin et al., 2018).

2.3 Breast cancer subtypes

Breast cancer cells often have different types of hormonal receptors including estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER2).

These receptors mediate cell growth signalling. Breast cancer is divided into four molecular subtypes according to these receptors (Figure 2.1). Luminal A breast cancers are ER+, PR+, HER2-, Luminal B breast cancers are known as triple positive, are ER+,

PR+, HER2+, HER2 enriched breast cancers are ER-, PR-, HER2+, and Triple Negative

(basal-like) breast cancers are ER-, PR-, HER2-. Luminal A subtype account for 70 % of all cases, whereas Basal-like, Luminal B, and HER2 make up 15 %, 10 %, and 5 % of all breast cancer cases respectively (Dai et al., 2016).

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Basal HER2 Luminal B Luminal A

15 % 5 % 10 % 70 %

Figure 2.1: Molecular subtypes of breast cancer

2.4 Breast cancer risk factors

Breast cancer is more commonly associated with family history, obesity, sex, ageing, prolonged exposure to estrogen and in postmenopausal women. The family history of breast cancer in a first-degree relative increased the risk for both ER-positive and ER- negative invasive breast cancer, but the level of risk varied by age and more common for women older than age 50 years. Similarly, postmenopausal women who were overweight were at risk of ER-positive and ER-negative cancer (Kerlikowske et al., 2016). The number of breast cancer cases occurred in women is 100 times higher than that in men

(Sun et al., 2017).

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2.5 Treatment of breast cancer

There is a variety of treatment for women diagnosed with breast cancer which include surgery, radiation therapy, chemotherapy, hormonal therapy, and targeted therapies. The most appropriate treatment depends on the stage and type of breast cancer, characteristics of the cancer cells, menopausal status, and the patient’s state of health

(Nounou et al., 2015).

Surgical treatment to remove breast cancer involve two basic types lumpectomy and mastectomy. Lumpectomy, surgically removing the tumour and a small margin of surrounding normal epithelial tissues, but not the entire breast. Mastectomy, surgical removing the all breast includes nipple and areola (American Cancer Society, 2016).

Radiation therapy is often started after lumpectomy to kill any remaining cancer cells in a particular area by damaging DNA via radiation (Balaji et al.,2016).

Chemotherapy is a treatment which uses drugs to enfeeble and damage tumour cells in the body, including cells at the primary site of cancer and any cancer cells that break away and spread throughout the body parts. Chemotherapy is sometimes given before surgery

(neoadjuvant therapy) to shrink larger tumours (Pathak et al., 2018).

Hormone therapy slows or stops the growth of hormone receptor-positive breast cancer cells by blocking hormones from binding to receptors on tumour cells. Tamoxifen is one of the most common hormone therapy medications used to block estrogen receptors in breast cancer cells. It is often given to decrease the size of breast tumour before surgery to remove it, to reduce the risk of breast cancer coming back after surgery, and to treat breast cancer that has already spread. Another examples of hormonal therapies used in the

8 management of metastatic hormone receptor-positive breast cancer are fulvestrant act as an selective estrogen receptor downregulator and Letrozol act as irreversible non-steroidal aromatase inhibitor in postmenopasual women diagnosed with hormone receptor-positive breast cancer (Drãgãnescu and Carmocan, 2017).

Targeted cancer therapies are drugs that block the action of a specific protein

(molecular target) that allows the cancer cells to grow in a rapid or abnormal way without harming normal cells. Hormone replacement therapies, signal transduction inhibitors, modulators of gene expression, apoptosis inducers, angiogenesis inhibitors, immunotherapies, and toxin delivery molecules are types of targeted therapies have been approved for use in cancer treatment (National Cancer Institute, 2019).

2.6 Tamoxifen resistance in breast cancer

Around 2 out of 3 breast cancers are hormone receptor-positive. Estrogen signalling plays a critical role in the proliferation of breast cancer, and therefore reducing the amount of estrogen or blocking its action using endocrine therapy can reduce the risk of early- stage hormone-receptor-positive breast cancers coming back. The selective estrogen receptor modulator (SERM), tamoxifen, is the standard treatment options for estrogen receptor-positive breast cancer patients. Tamoxifen treatment of estrogen receptor (ER)- positive breast cancer reduces the annual breast cancer death rate by 31%. However, about half of patients with advanced ER-positive disease immediately fail to respond to tamoxifen and approximately 40% will acquire the resistance during the treatment

(Hultsch et al., 2018). Acquired resistance to hormone therapy remains a major challenge for women with estrogen receptor-positive metastatic breast cancers. Multiple

9 mechanisms responsible for endocrine resistance may include deregulation of various components of the ER pathway itself, alterations in cell cycle and cell survival signalling molecules, and the activation of alternative signalling pathways that can provide tumours with alternative proliferative and survival stimuli (Hayes and Lewis-wambi, 2015). For instance, breast cancer cells resist to tamoxifen treatment by overactivation the phosphatidylinositol-3-kinase (PI3k)/Akt and the mammalian target of rapamycin

(mTOR) intracellular signalling pathway through cross-talk between the estrogen receptor and this growth factor signalling pathway. In consequence, reducing apoptosis and allowing the proliferation of cancer (Won et al., 2016). Another example, tamoxifen- resistant breast cancer cells are resistant to genotoxicity mechanism of tamoxifen (DNA damaging through oxidative stress) by a mutation in tumour suppressor genes BARD1 and BRCA1 (gens to protect normal cells from conversion to cancer cells). This will lead to loss or reduction in gens functions and then normal cells progress to cancer cells (Zhu et al., 2018). Due to severe side effects and multidrug resistance, these treatment approaches become increasingly ineffective. However, turning to complementary treatment approach can be a big solution for this situation, as it is evident that compounds derived from the natural source have a great deal of anticancer activity (Mitra and Dash,

2018).

2.7 Natural products derived from the plant as an alternative treatment for breast cancer

Natural plants have been used to prevent and treatment of breast cancer. Huge number of natural compounds from plants are identified and showed very promising anti- cancer properties with less toxic side effects compared to current treatments such as

10 chemotherapy. Anticancer properties of natural, synthetic or biological and chemical agents to reverse, suppress or prevent carcinogenic progression. Natural compounds reduced the aggressiveness of breast cancer through various mechanisms of action, such as downregulating ER-α expression and activity, inhibiting proliferation, migration, metastasis and angiogenesis of breast tumour cells, inducing apoptosis, cell cycle arrest and sensitizing breast tumour cells to radiotherapy and chemotherapy (Mitra and Dash,

2018).

The plant kingdom produces naturally occurring secondary metabolites which are being attracted to the interest of scientific and research to investigated and designed cancer therapeutics from natural compounds especially from phytochemicals (Greenwell and

Rahman, 2015). Many dietary natural products could affect the development and progression of breast cancer, such as mangosteen (Garcinia mangostana L.) known as

“queen of fruits”, Pomegranate (Punica granatum L.), Mango (Mangifera indica L.), and

Jujube (Ziziphus jujube) were used to prevent and treatment of breast cancer in South-East

Asia (Li et al., 2017). Substantial experimental studies on the effectiveness of Garcinia hombroniana extract indicated that this plant is an anti-cancer agent (Dyary et al., 2016;

Jamila et al., 2015).

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2.8 Taxonomy of Garcinia hombroniana plant

Table 2.1: Taxonomy hierarchy of Garcinia hombroniana plant

Kingdom Plantae

Phylum Tracheophyta

Class Magnoliopsida

Order Malpighiales

Family Clusiaceae

Genus Garcinia L.

Species Garcinia hombroniana Pierr

Adapted from (GBIF Backbone Taxonomy)

2.9 Garcinia hombroniana

Garcinia is the major genus in the family Clusiaceae with over 400 species found throughout the tropics of Africa and Asia and about 50 species found in the lowland and mountains of Peninsular Malaysia (Tan et al., 2018). The genus Garcinia is reported to possess pharmacological activities such as antimicrobial, anti-inflammatory, anticancer, hepatoprotective and anti-HIV activities. Some studies have reported anti‐cholinesterase activity of the plant on the nervous system disorders (Jamila et al., 2017). Traditionally, numerous parts of Garcinia plant (fruits, leaves, flowers, stem and bark) have been utilized to treat various ailments such as abdominal pain, leucorrhoea, gonorrhoea, diarrhoea, dysentery, wound infection, suppuration, and chronic ulcer in Malaysia,

Thailand, Indonesia, Sri Lanka, Philippines and China (Jamila et al., 2015).

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Figure 2.2: Garcinia hombroniana leaves

Adapted from (plantsystematics.org)

Garcinia hombroniana plant belongs to family Clusiaceae. It is a small or medium evergreen tropical tree which produces globose fruit in a bright red colour. The plant is native to the tropical rainforests of Southeast Asian countries such as Vietnam, Cambodia,

Malaysia and Thailand (Chew and Lim, 2018). It is known as “Manggis hutan” (seashore mangosteen) in Malaysia or “Waa” in Thailand. In Malaysia, it is found in the longshore area, from the lowland forests near the sea to the lower mountain forests and the highlands.

13

Figure 2.3: Garcinia hombroniana tree

Adapted from (toptropicals.com)

In Asian and West Africa, this plant had been used for its medicinal purpose. The plant roots are used to make a herbal decoction for women as an anti-infective agent after childbirth and the leaves are used to relieve itching. The previous studies show that twigs, stem bark, pericarp, and leaves Garcinia hombroniana to contain alkaloids, flavonoids, phenols, saponins, tannins, xanthones, benzophenones, and terpenoids. Garcinia hombroniana leaves aqueous extract shows little toxicity on the vital organs such as the liver, kidneys, heart, and spleen in laboratory animals (Dyary et al., 2016). Furthermore,

Garcinia hombroniana leaves aqueous extract has numerous therapeutic effects such as anti-diabetic properties due to the à-glucosidase inhibitor agent, antioxidant and

14 lipoxygenase inhibitor activity, and potential antitrypanosomal activity (Marlin et al.,

2017; Triadisti et al., 2017; Dyary et al., 2015). In addition to that, the methanol extract of the twigs of Garcinia hombroniana has strong antioxidant activity on human low- density lipoprotein (LDL) and antiplatelet aggregation activities, and antibacterial activity against methicillin-resistant Staphylococcus aureus and S. aureus (Saputri and Jantan,

2012; Klaiklay et al., 2013). Naturally, bioflavonoids from the bark of Garcinia hombroniana display significant antioxidant and antibacterial activities, and good dual inhibition on both acetylcholinesterase and butyryl cholinesterase (Jamila et al., 2014;

Jamila et al.,2015). Garcinia hombroniana bark extract was also reported to possess good cytotoxic effect against human breast cancer (MCF7) and human glioblastoma (DBTRG) cell lines which might be due to the phenolic compounds (Jamila et al., 2014; Jamila et al.,2017).

2.10 Cell death

Cell death is a very well-organized fundamental activity that is equally complex in regulation as cell division and differentiation. Balance homeostasis in the body is a crucial factor in avoiding the growth of cancer cells. Thus, activation of the cell death mechanism is vital in maintaining the homeostasis of proliferative of health and normal cells (Green and Lambi, 2015). Cell death occurs when the cells are confronted with a process which is reversible at first before it becomes irreversible. Loss of plasma membrane integrity, cell fragmentation with its nucleus into apoptotic bodies, and engulfment its fragments by adjacent cells are the molecular or morphological criteria to define dead cell in vivo. Cell death, due to the presence of its genetic regulation, can be divided into programmed (or active) ones, such as apoptosis and autophagy, and not programmed (or nonactive) such

15 as necrosis (Galluzzi and Vitale, 2018). Figure 2.4 shows a summary of the classification of the cell death pathway.

Figure 2.4: Classification of cell death pathway

Adapted from (Danial and Hockenbery, 2018)

2.11 Apoptosis

Apoptosis is programmed and an active form of cell death in which a highly specific and orderly set of biochemical changes underlie the unique morphologic changes and the ultimate disposition of the dying cell and its contents. It is distinguished by cell shrinkage, nuclear fragmentation, plasma membrane blebbing and finally by the separation of the cellular components into apoptotic bodies. These apoptotic bodies are removed by phagocytes which attracted by the “eat me” signal from the exposure of

16 phosphatidylserine on the plasma membrane. Apoptosis induction can be activated by two different pathways, the intrinsic and extrinsic pathways. The extrinsic pathway is mediated by death receptors, while the intrinsic or mitochondrial pathway is triggered by the release of apoptogenic proteins, such as cytochrome c, which activated caspase proteins that are the main effector molecules that induce this process (Hassan et al., 2014).

2.12 Necrosis

Necrosis has been considered as an accidental or not programmed and passive of cell death, and the endpoint commonly associated with very severe toxic damage.

Necrotic cell death is characterized by an increase in cell volume, swelling of organelles, plasma membrane rupture and provoking an inflammatory response. The morphological features associated with necrosis include cellular energy depletion, damage to membrane lipids, and loss of function of homeostatic ion pumps/channels. It is considered as a toxic process because these necrotic cells are reported to release harmful chemicals that can induce damage to other cells. Necrosis is caused by factors external to the cell, such as lytic viral infection, physical trauma, complement-mediated lysis, depletion of ATP, loss of ionic homeostasis, and excessive ROS/RNS (Manning and Zuzel, 2010).

2.13 Essential oils and their bio-activities

The essential oil is a mixture of concentrated hydrophobic liquid containing volatile compounds synthesized by medicinal and aromatic plants as secondary metabolites. There are obtained from various parts of the plant such as flowers, fruits, leaves, twigs, and barks

(Kumar et al., 2018). There are a variety of methods for obtaining essential oils from the plant. Steam distillation method was found to be one of the promising techniques for

17 extraction of essential oil from plants as reputable distiller will preserve the original quantities of the plant. The distillation was conducted in a Clevenger apparatus in which boiling, condensing and decantation were done (Rassem et al., 2016). Essential oils show a broad range of bioactivities, especially antimicrobial activity, and have long been utilized for treating various human ailments and diseases. Depending on type and concentration, essential oils possess antimutagenic, antiproliferative, antioxidant, and detoxifying capabilities acting on different routes in the cancer cell as well as cancer preventative potentialities. The cytotoxic activity of essential oils is mostly due to the presence of phenols, aldehyde and alcohol (Blowman et al., 2018).

18

CHAPTER 3

METHODOLOGY

3.1 Materials and chemicals

3.1.1 Chemicals and reagents

All chemicals and reagents used in this study are listed in Table 3.1

Table 3.1: List of chemicals and reagents

Chemicals and reagents Supplier

Dimethyl Sulphoxide (DMSO) Fisher Scientific, UK

Roswell Park Memorial Institute medium (RPMI- Gibco, USA

1640)

Phosphate Buffered Saline (PBS) Gibco, USA

Penicillin-Streptomycin (Pen-strep) antibiotics Gibco, USA

solution

Dulbecco's Modified Eagle Medium/Nutrient Gibco, USA Mixture F-12 (DMEM/F-12), no phenol red Fetal Bovine Serum (FBS) Gibco, USA

Trypsin-EDTA Gibco, USA

5-diphenyltetrazolium bromide dye (MTT) Calbiochem, Germany

Trypan blue Gibco, USA

Accutase Millipore, USA

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Garcinia hombroniana leaves Penang Botanical Garden

MCF-7 cell line American Type Culture Collection (ATCC), Virginia, USA MCF-7/TAMR-1 cell line American Type Culture Collection (ATCC), Virginia, USA MCF10A cell line American Type Culture Collection (ATCC), Virginia, USA Annexin V FITC Roche, Germany

Propidium Iodide (PI) dye Roche, Germany

Tamoxifen citrate salt Nacalai tesque, Japan

3.1.2 Consumables

All consumables used in this study are listed in Table 3.2

Table 3.2: List of consumables

Consumables Supplier

Cryogenic vials and Cryoboxes Nalgene, USA

Micropipette tips (10µl, 200µl, 1000µl) Labcon, Germany

Serological pipettes (2ml, 5ml, 10ml) Nunc, Denmark

Centrifuge tubes (15ml, 50ml) Nest Biotechnology Co., Ltd., China

Tissue culture flasks (25 cm², 75 cm²) Becton Dickinson, USA

Syringes (10ml) Terumo Corporation, Philippines

Syringes membrane filter (0.22 µm) BD Plastipak, Spain

96-well microtiter plates Essen Bioscience, USA

Aluminium foils Reynolds Consumer Products Inc., USA

20

Coverslips Deckglaser, Germany

Counting chamber Becton Dickinson, USA

3.1.3 Laboratory equipment

All of the laboratory equipment used in this study are listed in Table 3.3

Table 3.3: List of laboratory equipment

Name and brand Supplier

Airstream Class II Biological Safety ESCO, Singapore

Cabinet

Centrifuge Hettich Zentrifugen, Germany

Incubator ThermoScientific, USA

Water bath Memmert, Germany

Inverted microscope OLYMPUS, USA

Hemocytometer LD-Laboroptik Ltd., UK

Microplate reader Biotek, UK

Flow cytometer Becton Dickinson, USA

Clevenger-type apparatus Custom Made

3.2 Extraction of essential oils by hydrodistillation technique

The leaves of Garcinia hombroniana were collected at Penang Botanical Garden.

The voucher specimen (USM 11748). The leaves of Garcinia hombroniana were washed with distilled water and cut into small pieces prior to hydrodistillation. The hydrodistillation was carried out for 5 hours using a Clevenger-type apparatus. The

21

extracts were carefully concentrated using gentle steam of nitrogen gas at room temperature, yielding pale yellow oils. The essential oils isolated from the leaves were kept at 4 ⁰c until analysis. A stock concentration 10 000 µg/ml was prepared by dissolving the oils in dimethyl sulphoxide (DMSO) and kept at -20 ⁰c until use.

3.3 Cell culture

3.3.1 Human breast adenocarcinoma cell lines

Two types of adenocarcinoma cell lines were used throughout this study. MCF-7 is an ER-positive cell line and MCF-7/TAMR-1 is an ER-positive tamoxifen resistance cell line, originated from human breast adenocarcinoma with epithelial morphology. MCF-

10A is an ER-alpha negative normal breast cell line. These cells were obtained from

American Type Culture Collection (ATCC, USA). These cells were grown in suitable condition and the medium was changed every 2 days. All cells handling and medium preparation were carried out using aseptic technique in class II safety cabinet.

3.3.2 Reagents for cell culture work

3.3.2.1 Medium

MCF-7 was cultured and maintained in RPMI-1640 growth medium (Gibco, USA).

MCF-7/TAMR-1 and MCF-10A were cultured and maintained in Dulbecco's Modified

Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) (Gibco, USA). Both medium stored

22

at 4 ⁰C. The reagents were thawed before used and filtered-sterilised with 0.22 µm disposable filter unit.

3.3.2.2 Heat-inactivated Fetal Bovine Serum (FBS)

A bottle of FBS (100 ml) (Gibco, USA) was stored at -20 ⁰C. The serum was filtered-sterilised with 0.22 µm disposable filter units and aliquoted into sterile 50 ml tubes and kept at -20 ⁰C. The FBS was thawed in a water bath at 37 ⁰C prior to use.

3.3.2.3 Phosphate-Buffered Saline (PBS)

Ten-time Phosphate-Buffered Saline (Gibco, USA) was filtered-sterilised with 0.22

µm disposable filter units and aliquoted into sterile 50 ml tube with distilled added to produce 1x working concentration. The PBS was stored at room temperature.

3.3.2.4 Penicillin-Streptomycin (Pen-strep) antibiotics solution

A bottle of ready-made containing Penicillin-Streptomycin (Pen-strep) solution

(Gibco, USA) was filtered-sterilised with 0.22 µm disposable filter units and aliquoted into sterile 15 ml tubes and kept at -20 ⁰C. The antibiotic was thawed in a water bath at 37

⁰C prior to use.

3.3.2.5 Complete growth medium

A complete growth medium was used in this study to culture and maintain the cell lines. The medium for MCF-7 cell line consist of RPMI-1640 solution supplemented with

23

10 % (v/v) FBS and 1 % (v/v) of Pen Strep. The 50 ml medium was prepared by adding 5 ml FBS, 50 µl Pen Strep and 45 ml RPMI-1640 media. The medium for MCF-7/TAMR-

1 cell line consists of DMEM/F12 without phenol red solution supplemented with FBS,

Bovine insulin and Tamoxifen. The 50 ml medium was prepared by adding 0.5 ml FBS,

15 µl Bovine insulin, 5 µl Tamoxifen and 49.5 ml DMEM/F12 without phenol red media.

The medium for MCF-10A cell line consists of DMEM/F12 with phenol red solution supplemented with horse serum, insulin, hEGF enzyme, hydrocortisone, and Pen Strep.

The 50 ml medium was prepared by adding 47.5 ml DMEM/F12 with phenol red solution,

2.5 ml horse serum, 125µl insulin, 50µl hEGF enzyme, 500µl hydrocortisone, and 50µl

Pen Strep. All prepared media were kept at 4 ⁰C and thawed before used.

3.3.2.6 Trypsin (0.25%, w/v)/ EDTA (0.03%, w/v) solution

Ready-made trypsin of 0.25%, EDTA 0.03% was filter-sterilised, aliquoted into sterile 15 ml tubes and stored at -20 ⁰C until use. The trypsin was thawed in 37 ⁰C prior to use.

3.3.2.7 Cryogenic Medium

The cryogenic medium for MCF-7 cell line contained 95% RPMI complete medium and 5% DMSO. The 10 ml cryogenic medium was prepared by adding filtered-sterilised

9.5 ml RPMI complete media and 0.5 ml DMSO. The cryogenic medium for MCF-

7/TAMR-1 and MCF-10A cell line contained 95% DMEM/F12 complete medium and 5%

DMSO. The 10 ml cryogenic medium was prepared by adding filtered-sterilised 9.5 ml 24

DMEM/F12 complete media and 0.5 ml DMSO. Both cryogenic mediums were prepared fresh and kept cold prior to use.

3.3.3 Culture procedures and conditions

All tissue culture procedures were carried out in a sterile condition (Airstream Class

II Biological Safety Cabinet) using aseptic techniques to avoid any contamination. Cells were cultured in growth medium and maintained in a humidified incubator at 37 ⁰C in an atmosphere in a 5 % CO2 and 95 % air.

3.3.4 Thawing of cells from frozen storage

The frozen cells were retrieved from the -80 ⁰C storage and thawed in a water bath at 37 ⁰C for 2 minutes. The entire frozen cells were gently pipetted into a 15 ml centrifuge tube containing 2 ml pre-warmed complete growth medium. The content centrifuged at

1000 rpm for 5 minutes to remove DMSO for MCF-7 cell and at 300 g for 4 minutes to remove DMSO for MCF-7/TAMR-1 and MCF-10A cells. The supernatant was aspirated and the cell pellet was then resuspended in 1-2 ml of complete growth medium. The cell suspension was then slowly pipetted into a 25 cm² tissue culture flask containing 4-5 ml culture media. The cell morphology was observed using an inverted phase-contrast microscope to check for cell viability. The culture flask was incubated in a humidified atmosphere containing 5 % CO2 at 37 ⁰C. The growth medium was replaced every 2-3 days.

25

3.3.5 Sub-culturing of cells

The cells were sub-cultured into a new flask when they reached about 70 – 80 % confluency in the flask to avoid nutritional deficiency and microbial contaminants. The old medium from the flask was discarded and cells were rinsed with 2 ml PBS. After that, the PBS was discarded and 1 ml trypsin-EDTA solution was added into the flask. The flask was left for about 2 minutes in a CO2 incubator for allowing adherent cells to detach from the surface of the flask. The flask was gently tapped to ensure complete cells detachment and the process was followed by observing the flask using the inverted microscope. Then, 1 ml of fresh culture medium was added into the flask to inactivate the trypsin activity. The medium (containing the cells) was aspirated and transferred into a sterile 15 ml centrifuge tube. The tube was centrifuged at 1000 rpm for 5 minutes for

MCF-7 cell and at 300 g for 4 minutes for MCF-7/TAMR-1 and MCF-10A cells. The supernatant was then removed and the cell pellet was re-suspended in 1 ml growth medium. The mixture containing cells was aspirated and dispensed at a 1:4 dilution

(normally into 4-5 flasks). The medium volume in each new 75 cm² flasks was made up to 6-10 ml by adding fresh growth medium and the cultures were incubated in a humidified atmosphere containing 5% CO2 at 37 ⁰C.

3.3.6 Cell counting for seeding

Approximately 10 µl of the cell suspension was diluted with 90 µl of trypan blue and 10 µl of the diluted cell suspension was pipetted onto a haemocytometer. The total number of viable cells was counted using Equation 1:

Number of viable cells/ml = (A × Dilution Factor) / 4 × 104

A = Total of unstained cell in the calculated grid

26

3.3 Assessment of anti-proliferative effect by MTT assay

The cells (MCF-10A, MCF-7, and MCF-7/TAMR-1) were seeded at a density of (1 x 104, 2 x 104, and 5 x 104) cells per well respectively in triplicates and incubated overnight at 37 ⁰C with 5% CO2. Cells were treated with essential oil isolated from the leaves of Garcinia hombroniana. The concentrations of GH-EOL used in this study were

(0, 1.56, 3.125, 6.25, 12.5, 25. 50, and 100) µg/ml. Tamoxifen at 30µm concentration was used as a positive control in this study. The cells were incubated at time points 24h, 48h, and 72h respectively. At each time points, 10 µl (5mg/ml) of MTT reagent (Calbiochem,

Germany) was added to each well and is wrapped in aluminium foil to prevent light access to the solution and is incubated for 4 hours at 37 ⁰C. After 4 hours the plate is taken out from the incubator and the medium containing MTT solution was discarded from each well and 100 µl of DMSO was added. The absorbance at 570 nm was measured by a microplate reader. The dose-response effect and the half minimal inhibitory concentration

(IC₅₀) values of Garcinia hombroniana essential oils were determined from the graph to get the treatment concentration that caused 50% cell death. The cell viability (%) was calculated as follows:

Percentage cell viability = (Mean O.D. treatment – Mean O.D. blank) / (Mean O.D. untreated cell – Mean O.D. blank) × 100 %

Where O. D. is the optical density and blank is media.

3.4 Cell optimization

To determine the optimal IC50 for GH-EOL and tamoxifen for assessment on the mechanism of apoptosis and/or necrosis by flow cytometry, cells were grown in 75-cm2 tissue culture flasks to obtain stock cultures. Confluent cells were then harvested using

27

trypsin/EDTA solution and pelleted as described in section 3.3.5. The cells were then re- suspended in assay medium and counted using a haemocytometer as described in section

3.3.6. The cells suspension was then adjusted to a concentrations 5 x 105 cells/mL for

MCF-7 and MCF-10A cells and 1 x 106 cells/mL and seeded in 5 mL growth medium in

2 o 25-cm tissue culture flasks and allowed to adhere for overnight in 37 C, 5 % CO2 incubator. Cells were treated with GH-EOL and tamoxifen after 70 – 80% confluency with the following concentrations as shown in table 3.4.

Table 3.4: Cells optimization with different concentrations and treatment media volumes of GH-EOL and tamoxifen.

Treatments GH-EOL Tamoxifen

Conc. Treatment Cell Conc. Treatment Cell (µg/mL) media viability (µM) media viability Cell lines volume (%) volume (%) (mL) (mL) 36 5, 6, 7 0 35, 5 0 30, 25 MCF-7 20 5 50 20 5 25

15 5 50

10 5 100

10 5 100 15 6 0

15 5 100 20 6 0

20 5 50 15 5 0 MCF-7/TAMR-1 20 5.5 40 20 5 0

20 6 0 12.5 5 50 25 6 0

MCF-10A 35, 20 5, 6 100 15 5 100

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3.5 Treatment cells for apoptosis

Cells were grown in 25-cm2 culture flasks at optimum cell density for 24 hours. At

70 – 80 % confluency, the growth medium was changed to assay medium and cells were treated with GH-EOL (IC50) and tamoxifen for 24 hours. Following incubation, the cells were then observed under a microscope to evaluate the cell morphology before they were subjected to apoptosis detection by flow cytometry as described below.

3.6 Assessment on the mechanism of apoptosis and/or necrosis by flow cytometry

Apoptosis was detected using Annexin V-FITC and propidium iodide (PI) dye. The phosphatidylserine (PS) on the surface of the apoptotic cell membrane was detected using

Annexin V-FITC which emitted green fluorescence whereas PI staining enters the plasma membrane of the necrotic dead cell which emitted red fluorescence.

For flow cytometric analysis, cells (MCF-10A, MCF-7, and MCF-7/TAMR-1) were seeded at a density of 5 x 105 cells/ml, 5 x 105 cells/ml, and 1 x 106 cells/ml in 5 ml medium

o respectively and allowed to adhere for overnight in 37 C, 5 % CO2 incubator. Cells were treated with essential oils for 24, 48, and 72 hrs. At each incubation period, both floating cells were collected in 15 ml centrifuge tube and adherent cells were detached as by using

Accutase (Millipore, USA). Both floating and adherent cells were pelleted by centrifugation at 300 x g for 2 minutes. The supernatant was then removed and 1 ml PBS was added to wash the cells. The cells were centrifuged 300 x g for 2 minutes and PBS was discarded. This step was repeated twice. The cell pellet was re-suspended in 1 ml of

1x binding buffer. Then, the concentration of cells was adjusted to 1 x 106 cells/ml and

29

transferred out 100 µl of the mixture into the Falcon round bottom tube. Then, 5 µl of the two-stain kit was added in each flow tube and incubated at room temperature with no light for 10 minutes. Following incubation, 400 µl binding buffer was added to each flow tube before the cells were analysed using flow cytometry.

3.7 Statistical analysis

All data were presented as a mean ± standard deviation (SD) of three independent experiments and statistically significant (p < 0.05) was determined using Independent

Student T-test and the SPSS software Statistic version 22.0.

30

CHAPTER 4

RESULTS

4.1 Anti-proliferative effect of GH-EOL and morphological changes

4.1.1 Anti-proliferative studies

Anti-proliferative studies were done on the cell lines and the viability of the cells was determined by the absorbance of 3-(4, 5-dimethyl thiazolyl-2)-2, 5- diphenyltetrazolium bromide (MTT) assay and the results are shown in Figure 4.1, Figure

4.2, and Figure 4.3. The results of the treated cells were recorded at three-time points which were 24 hours, 48 hours, and 72 hours. The lowest concentration used is 1.56µg/mL and the highest is 100µg/mL.

Figure 4.1 demonstrates the effect of GH-EOL on the proliferation of MCF-7 cells.

As compared to untreated cells, GH-EOL has significantly (p < 0.05) inhibited the growth proliferation of MCF-7 cells at most of the concentrations except 3.125 µg/mL, following

24 hours. While at 48 hours, the proliferation of cells was significantly (p < 0.05) at all concentrations of GH-EOL. A significant (p < 0.05) inhibition was observed at concentrations ranging from 6.25 µg/mL to 100 µg/mL at 72 hours incubation, indicating that there was no significant difference on the cell proliferation at concentrations 1.56

µg/mL and 3.125 µg/mL. Among other concentrations, GH-EOL at the concentrations 50

µg/mL and 100 µg/mL has induced greatest inhibition effects (p < 0.05) on MCF-7 cells at all incubation times with the lowest percentage at 48 hours.

31

Figure 4.1: Anti-proliferative effect of GH-EOL on MCF-7 cells. The values represent means ± SD of the triplicates. * indicates significant differences (p < 0.05) with respect to the untreated group.

Figure 4.2 shows the effect of GH-EOL on the proliferation of MCF-7/TAMR-1 cells. As compared to untreated cells, the GH-EOL have significantly (p < 0.05) inhibited the growth proliferation of MCF-7/TAMR-1 cells from the concentration of 6.25 µg/mL to 100 µg/mL at all incubations. At 3.125 µg/mL, the growth inhibition was only observed significantly (p < 0.05) at 48 hours. The lowest percentage of cell viability following the treatment of GH-EOL was significantly (p < 0.05) observed with approximately 28% at concentrations 25 µg/mL, 50 µg/mL, and 100 µg/mL.

32

Figure 4.2: Anti-proliferative effect of GH-EOL on MCF-7/TAMR-1 cells. The values represent means ± SD of the triplicates. * indicates significant differences (p < 0.05) with respect to the untreated group.

Figure 4.3 demonstrates that the effect of GH-EOL on the proliferation of MCF-

10A cells. From the graph, GH-EOL display significant (p < 0.05) inhibition on MCF-

10A cells at concentrations ranging from 25 µg/ml to 100 µg/mL following 24 hours incubation. However, there was a significant (p < 0.05) induction of the cell proliferation at a concentration of 1.56 µg/mL at 24 hours with cell viability of approximately 105%.

While at 48 hours, the proliferation of cells was significantly (p < 0.05) inhibited at concentrations ranging from 6.25 µg/ml to 100 µg/mL. A significant (p < 0.05) inhibition was observed at most of the concentrations except at a concentration of 1.56 µg/mL following 72 hours incubation where there was which no significant differences compared to untreated cells. The concentration that inhibited the proliferation of cells to the significant (p < 0.05) lowest percentage of cell viability was 100 µg/mL with 24% at 48 hours.

33

Figure 4.3: Anti-proliferative effect of GH-EOL on MCF-10A cells. The values represent means ± SD of the triplicates. * indicates significant differences (p < 0.05) with respect to the untreated group.

4.1.2 Effect of GH-EOL on treated cell line morphology

MCF-7, a breast carcinoma cell line, MCF-7/TAMR-1, a breast carcinoma tamoxifen resistance cell line, and MCF-10A, an epithelial breast cell line was incubated with different concentrations (1.56, 3.125, 6.25, 12.5, 25, 50, and 100) µg/mL of GH-EOL at three different time points (24, 48, and 72) hours. Direct observation through inverted light microscope had shown that the GH-EOL treated cells were reduced in number and there were few morphological changes that can be distinguished from an untreated group of cells.

Figure 4.4 showed the images of untreated cell lines (a) MCF-7, (b) MCF-7/TAMR-

1, and (c) MCF-10A morphological characteristics of cell lines after 24 hours seeding

34

a

b

c

Figure 4.4: Morphological characteristics of (a) MCF-7 (b) MCF-7/TAMR-1 (c) MCF- 10A cells one day after culture under magnification of 4x

35

Figure 4.5 showed (a) the image of MCF-7 cells with 50 µg/mL GH-EOL for 24 hours of treatment with some morphological changes such as cell elongation, (b) the image of MCF-7 cells with (30 µM) tamoxifen for 24 hours of treatment with some morphological changes such as vascularization, (c) the image of MCF-7/TAMR-1 cells with 50 µg/mL GH-EOL for 24 hours of treatment with some morphological changes such as rounding of cell, (d) the image of MCF-7/TAMR-1 cells with (30 µM) tamoxifen for

24 hours of treatment with some morphological changes such cell shrinkage.

a b

c d

Figure 4.3: Effect of GH-EOL and tamoxifen on the cellular viability and morphological changes after 24 hours of treatment.

36

4.2 Determination of the constant IC50 values for GH-EOL on MCF-7, MCF-

7/TAMR-1, and MCF-10A cells

The IC50 values obtained following GH-EOL treatment of MCF-7 breast cancer cell line, MCF-7/TAMR-1 resistance breast cancer cell line, and MCF-10A epithelial breast cells (as listed in Table 4.1) were plotted against the duration of treatment as shown in figures 4.6, 4.7, and 4.8 to obtain a constant IC50 values for treatment of each cell line for subsequent experiments. Treatment with GH-EOL showed a time-dependent cytotoxic activity in each cell line tested. The constant IC50 values of GH-EOL in MCF-7, MCF-

7/TAMR-1, and MCF-10A cells were 35.22 µg/mL, 17.67 µg/mL, and 76.11 µg/mL, respectively.

Table 4.1: The IC50 values of GH-EOL determined using MCF-7, MCF-7/TAMR-1, and MCF-10A cells

Time – points The average IC50 value of GH-EOL (µg/mL)

(hours) MCF-7 MCF-7/TAMR-1 MCF-10A

24 35.67 16.67 67.33

48 32.33 16 73.33

72 37.67 20.33 87.67

37

Figure 4.4: The constant IC50 value of GH-EOL on MCF-7 cells

Figure 4.5: The constant IC50 value of GH-EOL on MCF-7/TAMR-1 cells

38

Figure 4.6: The constant IC50 value of GH-EOL on MCF-10A cells

4.3 Cells optimization

MCF-7, a breast carcinoma cell line, MCF-7/TAMR-1, a breast carcinoma tamoxifen resistance cell line, and MCF-10A, an epithelial breast cell line were treated with different concentrations in different volumes below and above the IC50 value for GH-

EOL and tamoxifen for 24 hours (Table 3.4). Following incubation, cells were then observed under a phase-contrast inverted microscope to evaluate the percentage of anti- proliferation by comparison between attached (live) cells and floating (dead) cells. Then select the concentration which inhibited 50% of cells proliferation as IC50 value before they were subjected to apoptosis detection by flow cytometry.

Figure 4.9 showed the images of MCF-7 cells after 24 hours (a) untreated in 5 mL,

6 mL, and 7 mL, cells were adherent with clear cell to cell boundaries. Images of MCF-7 cells after 24 hours treatment with (b) GH-EOL 36 µg/mL in 5 mL, 6 mL, and 7 mL with

39

100 % inhibition (floating cells), (c) GH-EOL 20 µg/mL in 5 mL with 50 % inhibition

(floating and adherent cells), (d) tamoxifen 35 µM, 30 µM, 25 µM, and 20 µM in 5 mL with 75 % - 100 % inhibition, (e) tamoxifen 15 µM in 5 mL with 50 % inhibition, and (f) tamoxifen 10 µM in 5 mL with 0 % inhibition. Treatments showed various degree of cell inhibition percentage and the optimum IC50 is 20 µg/mL in 5 mL and 15 µM in 5 mL for

GH-EOL and tamoxifen respectively which were used for apoptosis detection by flow cytometry.

a b

c d

e f

Figure 4.7: Effect of GH-EOL and tamoxifen on the MCF-7 cell viability within 24 hours of treatment compared with untreated. Pictures were in x4 magnification.

40

Figure 4.10 showed the images of MCF-7/TAMR-1 cells after 24 hours (a) untreated in 5 mL, and 6 mL, cells were adherent with clear cell to cell bounderies. Images of MCF-7/TAMR-1 cells after 24 hours treatment with (b) GH-EOL 10 µg/mL, and 15

µg/mL in 5 mL with 0 % inhibition (adherent cells), (c) GH-EOL 20 µg/mL in 5 mL with

50 % inhibition (floating and adherent cells), (d) GH-EOL 20 µg/mL, and 25 µg/mL in

5.5 mL, and 6 mL with 60 % - 100 % inhibition (floating cells), (e) tamoxifen 12.5 µM in

5 mL with 50 % inhibition, (f) tamoxifen 15 µM, and 20 µM in 5 mL, and 6 mL with 80

% - 100 % inhibition. Treatments showed various degree of cell inhibition percentage and the optimum IC50 is 20 µg/mL in 5 mL, and 12.5 µM in 5 mL for GH-EOL and tamoxifen respectively which were used for apoptosis detection by flow cytometry.

41

a b

c d

e f

Figure 4.8: Effect of GH-EOL and tamoxifen on the MCF-7/TAMR-1 cell viability within 24 hours of treatment compared with untreated. Pictures were in x4 magnification.

Figure 4.11 showed the images of MCF-10A cells after 24 hours (a) seeding in 5 mL with 70 – 80 % confluency, (b) treated with GH-EOL 36 µg/mL in 5 mL with 0 % inhibition, (c) treated with GH-EO-L 20 µg/mL in 5 mL with 0 % inhibition, and (d) treated with tamoxifen 15 µM in 5 mL with 0 % inhibition.

42

a b

c d

Figure 4.9: Effect of GH-EOL and tamoxifen on the MCF-10A cell viability within 24 hours of treatment compared with untreated. Pictures were in x4 magnification.

4.4 Apoptosis detection

The inhibitory effect was further affirmed with apoptosis analysis with flow cytometry. The result showed that the tested cells for the apoptotic rate had shown significant differences after incubation of 24 hours. Figure 4.12 summarized the percentage of the apoptotic cells which induced by GH-EOL and tamoxifen on treated cells compared with untreated cells obtained after data analysis from three independent experiments. For each quadrant, lower left indicated the viable cells, lower right indicated the early stage of the apoptotic cell, upper right indicated the late stage of apoptotic cells, and upper left indicated the necrotic cells.

43

Untreated GH-EOL Positive Tamoxifen

0.41% 4.7% 0.7% 10.6% 0.9% 18.3% 0.2% 3.92% MCF

-

7 92.8% 2.1% 56.6% 31.76% 42.1% 38.6% 92.53% 3.35%

MCF

1.28% 6.31% 9.06% 24.33% 11.58% 24.19% 0.9% 5.87%

-

7/TAMR

89.39% 3.02% 50.19% 16.42% 54.67% 9.29% 90.1% 3.13%

-

1

PropidiumIodide

MCF 0.3% 3.4% 0.2% 4.2% 0.5% 4.3% 0.3% 1.87%

-

10A

95.4% 0.9% 94.5% 1.0% 94.4% 0.9% 97.04% 0.79%

Annexin-V-FITC 24 hours

Figure 4.10: Percentage distribution of GH-EOL induced apoptotic and necrotic breast cancer cells. MCF-7 and MCF-10A cells were treated with GH-EOL and tamoxifen (20 µg/mL and 15 µM, respectively) while for 24 hours MCF-7/TAMR-1 was treated with GH-EOL and tamoxifen (20 µg/mL and 12.5 µM, respectively) for 24 hr. The cells were stained with annexin V-FITC antibody and propidium iodide and analyzed by flow cytometry.

44

4.4.1 Mode of cell death induced by GH-EOL in breast cancer cell lines

Figure 4.12 showed the flow cytometric quadrant analyses of the mode of cell death induced by GH-EOL in breast cancer cell lines which were determined by using Annexin-

V FITC and propidium iodide staining by flow cytometry. Cells were treated with GH-

EOL at concentration 20 µg/mL and the mode of cell death was assessed within 24 hours of the incubation period. At this time point, cell death was significantly (p < 0.05) induced by GH-EOL at this concentration when compared with untreated cells. The percentage of total apoptotic cells was shown in Figure 4.13. Tamoxifen was used as a positive control at the concentrations of 15 µM and 12.5 µM in MCF-7 and MCF-7/TAMR-1 respectively.

4.4.2 Mode of cell death induced by GH-EOL in MCF-10A cell line

Figure 4.12 showed the flow cytometric quadrant analyses of the mode of cell death induced by GH-EOL in MCF-10A cell line which was determined by using Annexin-V

FITC and propidium iodide staining by flow cytometry. Cells were treated with GH-EOL at concentration 20 µg/mL and the mode of cell death was assessed within 24 hours of the incubation period. The result showed that the tested cells for the apoptosis rate had failed to show significant (p > 0.05) differences after incubation of 24 hours. Tamoxifen was used as a positive control at the concentration of 15 µM. For each quadrant, lower left indicated the viable cells, lower right indicated the early stage of the apoptotic cell, upper right indicated the late stage of apoptotic cells, and upper left indicated the necrotic cells.

45

Figure 4.11: The total percentage of apoptotic cells. Data shown are from a representative experiment repeated three times with similar results. There was a significant difference between treated and untreated (p < 0.05).

46

CHAPTER 5

DISCUSSION

In 2018, approximately 2.1 million females around the globe are diagnosed with breast carcinomas. This disease is the leading cause of cancer among females around the world, both in developed and developing countries, accounting for about 11.6% of cancer- related deaths and the figures continue to increase (Bray et al., 2018). Although the current anticancer drugs continue to play a major role in breast cancer treatment, there are still several subtypes of breast cancer that do not respond effectively to the available therapeutic strategies. Therefore, there is an incentive to identify, test and develop more effective treatments (American Cancer Society, 2017). The present study was aimed to investigate the cytotoxicity and possible modes of action of the essential oil extracted from

G. hombroniana leaves in two different breast tumour cell lines MCF-7 and MCF-

7/TAMR-1.

Several previous studies have recently focused on the potential of plant essential oils as an anticancer treatment in an attempt to overcome the development of resistance drugs. Particularly, plant essential oils from the Clusiaceae family were characterized by high contents of cytotoxic agents (Tan et al., 2019; Blowman et al., 2018). Essential oils from different Garcinia species of this family have been shown to possess a strong anti- proliferative effect against breast cancer cell lines (Choudhury et al., 2018; Mohamed et al., 2017). Hence, in the present study, the essential oil was extracted from leaves of G. hombroniana by hydrodistillation to examine their anti-proliferative activity and

47

characterize their effect in inducing apoptosis against two different breast cancer cell lines

MCF-7 and MCF-7/TAMR-1.

In line with the above studies, the present study showed that the essential oil extracted from the leaves of G. hombroniana (Clusiaceae family) exhibited a strong and selective cytotoxic activity against the ER-positive MCF-7 and MCF-7/TAMR-1 breast cancer cells. Importantly, the MTT assay results showed that GH-EOL had inhibited the cell growth, with IC50 values 35.11 µg/mL in MCF-7, and 17.67 µg/mL MCF-7/TAMR-

1 cells. Based on the IC50 values, it can be concluded that GH-EOL exhibited a higher anti-proliferative effect on MCF-7/TAMR-1 than MCF-7 cell lines. On the other hand,

GH-EOL demonstrated less cytotoxicity effect in non-cancerous MCF-10A cell lines, as indicated by higher IC50 value (76.11 µg/mL) was needed in inhibiting the growth of these cells. The differential effect of GH-EOL between these cell lines may be related to variations in the genetic profile, specific gene expression and distinct cell surface receptors of cancer cells making them more susceptible to the GH-EOL (Dai et al., 2017).

The range of IC50 values obtained from this study were between 17 to 36 µg/mL, which may suggest that GH-EOL could be further developed as a potent anti-cancer agent due to the fact demonstrated in previous literature that an anti-cancer agent derived from plant should have IC50 lower than 100 µg/mL, if not it is only considered to be a chemopreventive agent (Hermawan et al., 2012). As a positive control, the drug tamoxifen was used, and it exerted anti-proliferative effect with IC50 values of 12.5 µM and 15 µM in MCF-7/TAMR-1 and MCF-7 cell lines, respectively.

48

To determine whether the cytotoxic activity of GH-EOL was due to apoptosis, breast cancer cells were treated for 0 – 24 h with the IC50 value of GH-EOL and analysed by flow cytometry. Apoptosis is a programmed cell death that maintains cellular homeostasis between cell division and cell death (Baig et al., 2016). This physiological process induces cellular self-destruction, generating diverse morphological and biochemical features in the nucleus and cytoplasm. Apoptosis is a primary death induced by natural and certain synthetic compounds with antitumoral activities (Koff et al., 2015).

This was confirmed when performed a quantitative analysis of apoptosis using Annexin-

V FITC/propidium iodide staining and flow cytometry analysis. The finding showed that

80-90% of MCF-7 and MCF-7/TAMR-1 cell population is in the early and late stages of apoptosis after exposure to the IC50 value of GH-EOL. Importantly, necrosis was no greater than 10% under all the tested conditions. In addition, GH-EOL induces typical apoptotic morphological changes in MCF-7 and MCF-7/TAMR-1 cells including loss of cellular shape and shrinkage as observed under a light inverted microscope. The changes in cell morphology were increasingly clear as the duration of treatment was increased, suggesting the greater cytotoxic effect was exerted by GH-EOL. Taken together, these findings clearly indicate that leaf essential oil of G. hombroniana had induced cell death in MCF-7 and MCF-7/TAMR-1 breast carcinoma cells by apoptosis rather than necrosis and, therefore, consistent with the goal of a potential anticancer agent.

49

CHAPTER 6

CONCLUSION

6.1 Conclusion

This study concludes that the MCF-7/TAMR-1 cells, which tamoxifen-resistant phenotype, responded even more favourably to the GH-EOL than estrogen receptor- positive, MCF-7 breast cancer cells. The selective cytotoxicity of the essential oils extracted from G. hombroniana leaves was mediated by apoptosis. The leaves oil showed a stronger promising activity on both MCF-7 and MCF-7/TAMR-1 cancer cells. When compared to the breast cancer cells, the finding suggests that much greater concentrations of oil are required to inhibit the growth of the non-cancerous mammary cells (MCF-10A) indicating their efficacy and warrant further investigations for potential pharmaceutical application.

6.2 Recommendation

The current study has investigated the cytotoxic activity and apoptotic mechanism of G. hombroniana essential oils in vitro using human breast cancer cell lines. For future studies, the anti-proliferative and apoptotic effects of G. hombroniana essential oil could be investigated at in vivo level using breast cancer mice model which are resistant towards tamoxifen. This may provide further evidence-based data to confirm the therapeutic efficacy of this essential oil in interacting with various metabolites presence in the living organism. Downstream mechanisms of action by G. hombroniana essential oil such as at

50

genomic, transcriptomic, proteomic and metabolomic levels may as well to be designed and carried out in future.

51

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