Anti-Proliferative Effects of <I>Garcinia</I>

City University of New York (CUNY) CUNY Academic Works

All Dissertations, Theses, and Capstone Projects Dissertations, Theses, and Capstone Projects

2-2018

Anti-Proliferative Effects of Garcinia Fruits in Breast Cancer Cells

Harini Anandhi Senthilkumar The Graduate Center, City University of New York

How does access to this work benefit ou?y Let us know!

More information about this work at: https://academicworks.cuny.edu/gc_etds/2549 Discover additional works at: https://academicworks.cuny.edu

This work is made publicly available by the City University of New York (CUNY). Contact: [email protected]

ANTI-PROLIFERATIVE EFFECTS OF GARCINIA FRUITS IN BREAST CANCER CELLS

Harini Anandhi Senthilkumar

A dissertation submitted to the graduate faculty in Biology towards partial fulfillment of the requirements for the degree of Doctor of philosophy, The City University of New York

2018

Antiproliferative effects of Garcinia fruits in breast cancer cells by Harini Anandhi Senthilkumar

This manuscript has been read and accepted for the Graduate faculty in Biology in satisfaction of the dissertation requirement for the degree of Doctor of Philosophy.

Date Dr. Edward Kennelly Chair of Examining Committee

Date Dr. Cathy Savage-Dunn Executive Officer

Supervisory Committee: Dr. Jimmie Fata Dr. Moira Sauane Dr. Prabodhika Mallikaratchy Dr. Kurt Reynertson

THE CITY UNIVERSITY OF NEW YORK iii

ABSTRACT

Antiproliferative effects of Garcinia fruits in breast cancer cells by Harini Anandhi Senthilkumar

Advisor: Dr. Edward J. Kennelly

Breast cancer continues to be the second leading cause of death in women, even with the recent advances in research that has led to an improved understanding of molecular pathways and targets. Increasing mortality due to recurrence and lack of targeted drugs, especially in aggressive breast cancer subtypes such as triple negative breast cancer, has gained attention from the research community. With nearly 50% of the commonly used cancer drugs being sourced from plants, bioactive small molecules derived from a natural origin have immense potential as therapeutics. This study focuses on the anti-proliferative activities of benzophenones isolated from the edible fruits of Garcinia xanthochymus and Garcinia paucinervis in breast cancer cells, particularly triple negative breast cancer cells. Novel benzophenones paucinones E – I from Garcinia paucinervis were tested on a panel of breast cancer cell lines MDA-MB-231, MCF-7 and SKBR-3 with varying receptor statuses.

Paucinone H was active across the panel of cell lines studied, with IC50 values ranging from

10 to 20 µM. Since the activity was significant on MDA- MB-231, the possible pathways in

MDA-MB-231 that paucinone H could modulate were

iv investigated. Paucinone H induced apoptosis in a dose dependent fashion at IC50 concentration and decreased mitosis and migration at half IC50 concentration in MDA-MB-

231.

In addition to the research described above, a comprehensive review of dietary phytoestrogens and their impact on breast pathophysiology is also presented in Chapter 2. Appendices A and

B discuss the use of UPLC-QTOF-MS based metabolomics to verify bioactivities of various vegetative organs of Garcinia oblongiflia and the identification of novel benzophenones from

Garcinia paucinervis

ACKNOWLEDGEMENTS

My experience in Graduate school has been equally exciting and challenging. It started out because of my love for Science and my inquisitiveness in understanding the biology of

Cancer. After my exposure to natural products in the Phytochemistry course during my first year in school, I developed interest in analyzing the potential use of natural compounds in the prevention and treatment of cancer. I am thankful to Dr. Kennelly and Dr. Fata for their support in helping me carry out my research.

There is no achievement that comes without hard work and support of people who believe in you. In my case, I am grateful to my husband who always stood with me and was my constant source of strength and support through this long journey. I am also grateful to my parents who always believed in me. There were times when my mother’s words have helped me realize my dreams and to overcome challenges. I am thankful to my father for his calm and consoling words. With his professional experience as an Engineer in Research and development of energy efficient automobile engines, he could well relate to the frustrations in my failed experiments. Together we have shared our frustrations and love for Science. I am also thankful to my sister who has cheered me up when I needed a laugh.

I would like to thank my lab mates and good friends Kaushiki Chatterjee, Vanya

Petrova, Adam Negrin and Taylan Morcol for all the technical and non-technical small talks that made my time in lab an enriching experience. I am also thankful to my colleague Ping Li, who was an excellent professional to work with as a co-author. Appendices A and B of this thesis have been published in peer reviewed journals (citations listed below). Chapter 2.1 is a manuscript under review. At the time of completion of this thesis, the second set of reviewer comments have been addressed. vi

Li P, Anandhi Senthilkumar H, Figueroa M, Wu SB, Fata JE, Kennelly EJ, Long C

2016. UPLC-QTOFMS(E)-guided dereplication of the endangered Chinese species Garcinia paucinervis to identify additional benzophenone derivatives. J Nat Prod 79: 1619-1627.

Li P, AnandhiSenthilkumar H, Wu SB, Liu B, Guo ZY, Fata JE, Kennelly EJ, Long CL

2016. Comparative UPLC-QTOF-MS-based metabolomics and bioactivities analyses of

Garcinia oblongifolia. J Chromatogr B Analyt Technol Biomed Life Sci 1011: 179-195

A good research starts with an exciting idea. But thoughtful guidance is the one that leads to achievement of best results. I am thankful to my committee members Dr. Prabodhika

Mallikaratchy, Dr. Kurt Reynertson and Dr. Moira Sauane for their time, guidance and constantly looking out for the best for my career. Their advice has been critical in shaping my research and in educating myself on ways to overcome technical obstacles throughout the course of research and my time in Graduate school.

Finally, I would like to thank my angel son Mukhil whose little feet I never got to touch. Although his life was short, his eternal presence in my heart will be my greatest source of strength for now and forever.

vii

TABLE OF CONTENTS

Abstract ...... iv

Acknowledgements ...... vi

List of Figures ...... x

List of Tables ...... xii

List of Abbreviations ...... xii

Chapter 1: Introduction ...... 1

1.1 Garcinia xanthochymus and Garcinia paucinervis fruits –

Phytochemistry ...... 4

Chapter 2: Breast cancer and bioactive compounds from plants ...... 13

*2.1 Dietary phytoestrogens and their impact on breast biology ...... 14

2.2 Breast cancer pathways modulated by compounds from

Garcinia ...... 39

2.3 Triple negative breast cancer – Introduction ...... 46

*Chapter 3: Molecular mechanisms targeted by benzophenones from Garcinia paucinervis against breast cancer cell lines with varying receptor statuses ...... 51

3.1 Materials and Methods ...... 53

viii

3.2 Results ...... 61

3.3 Discussion ...... 70

Chapter 4: Conclusion ...... 72

Chapter 5: Future studies ...... 75

*Appendix A: Comparative UPLC-QTOF-MS-based metabolomics and bioactivities analyses of Garcinia oblongifolia ...... 79

*Appendix B: UPLC-QTOF-MS guided dereplication of an endangered Chinese Garcinia species to identify cytotoxic benzophenones in triple negative and other breast cancer cell lines ...... 82

Chapter 6: References ...... 86

*Results that have been published in and submitted to peer reviewed journals

ix List of Figures Page

Figure 1: Garcinia paucinervis fruits ...... 3

Figure 2: Garcinia xanthochymus fruits ...... 3

Figure 3: Freeze dried Garcinia xanthochymus fruits, dissected to show seeds ...... 3

Figure 4: 13-carbon skeleton of a benzophenone with A and B rings ...... 4

Figure 5: Bioactive benzophenones from Garcinia xanthochymus –

Xanthochymol, guttiferone E and garcinol ...... 5

Figure 6: Novel benzophenones paucinones E-I isolated from Garcinia paucinervis ...... 11

Figure 7: Structure of 17β-Estradiol (E2) ...... 19

Figure 8: A schematic representation of drug delivery systems that utilize phytoestrogens and their molecular targets ...... 35

Figure 9: Isolation of paucinone H from Garcinia paucinervis seed Fraction D ...... 54

Figure 10: UPLC -QTOFMS profile of Garcinia paucinervis seed fractions obtained from Fraction D ...... 56

Figure 11: UPLC -QTOFMS profile of seed fractions showing paucinone H ...... 57

Figure 12: UPLC -QTOFMS profile of paucinone H after isolation ...... 57

Figure 13: Growth inhibitory effects of paucinone H on MDA-MB-231 ...... 63

x List of Figures Page

Figure 14: Growth inhibitory effects of paucinone H on MDA-MB-231, SKBR-3 and MCF-7 ...... 64

Figure 15: Increase in the percentage of dead cells, 48 hours after treatment with paucinone H on MDA-MB-231 ...... 66

Figure 16: Paucinone H induces a reduction in mitotic marker phospho-histone 3 at half IC50 concentration within 24 hours after treatment on MDA-MB-231 cells ...... 67

Figure 17: Anti migratory effects of Paucinone H on MDA-MB-468 cells ...... 69

Figure 18: Growth inhibiting effects of Paucinone H on MDA-MB-468 cells ...... 77

Figure 19: Morphological changes in MCF-7 cells after treatment with Garcinia oblongifolia extracts ...... 80

Figure 20: A schematic workflow of the study set up to identify bioactive compounds on a panel of breast cancer cell lines with varying receptor status ...... 84

xi List of Tables

Table 1: A list of in vitro studies that discuss pathways targeted by benzophenones from Garcinia in cancer ...... 10

Table 2: Bioassays used in detection and study of phytoestrogens from complex extracts and dietary supplements (2008-2017) ...... 23

Table 3: Clinical studies completed on phytoestrogens and breast cancer intervention

(2008-2017) ...... 34

Table 4: Growth inhibitory effects of novel benzophenones from Garcinia paucinervis..62

List of Abbreviations

HCA Hydroxy citric acid

STAT-3 Signal transducer and activator of transcription -3 DMSO Dimethyl sulfoxide

PI 3K Phosphatidyl inositol 3 kinase

PTEN Phosphatase and tensin homolog deleted on chromosome ten UPLC Ultra-performance liquid chromatography

WST Water soluble tetrazolium salt

PFA Paraformaldehyde

BSA Bovine serum albumin

PH3 Phospho-histone 3

PCNA Proliferating cell nuclear antigen

xii MAPK Mitogen activated phosphor kinase

Mtor Mammalian target of rapamycin

HAT Histone acetylate transferas

xiii

Chapter 1: Introduction

Garcinia belongs to the family Clusiaceae with more than 450 species, widely distributed over tropical Asia, Africa, Madagascar, North East Australia and Polynesia (J. Bennett and Lee

1989). About 30 species are endemic to India while about 20 have been identified to be endemic to China (Kumar et al., 2013). One among the well-studied Garcinia species that is endemic to

India is Garcinia Indica (Kokum), known for the presence of HCA (hydroxy citric acid) which is a popular ingredient in weight loss supplements (Chuah et al., 2013). Polyphenols from Garcinia occur in vegetative organs of these plants, such as their fruits, leaves, stem and bark and their use has been reported in Ayurveda, traditional Chinese medicine and Brazilian and Thai folk medicine (Obolskiy et al., 2009; Hemshekhar et al., 2011). Their medicinal applications vary from the use of fruit infusions to treat skin ailments such as rashes and burns to their use in treatment of peptic ulcer and urinary diseases (Corrêa and Pena 1984). The two primary

Garcinia species, that are the focus of this study are Garcinia xanthochymus and Garcinia paucinervis.

Garcinia xanthochymus is native to India. The fruit is used in preserves, jams, curries and in the preparation of dyes. The dried fruit sap is called gamboge. Fruits of Garcinia xanthochymus are pale orange to dark yellow in color and measure about 9 cm in diameter.

Usually, each fruit has about 2 to 3 seeds (Baggett et al., 2005; Staples and Herbst 2005). The fruits have an acidic taste and strong flavor, while the seeds are bitter. In some cultures, seeds are used in a concoction to treat diarrhea (Barukial and Sarmah 2011). Garcinia paucinervis is a timber species that occurs in limestone mountains in the South East Yunnan and Guangxi provinces in China (Xi-wen Li 1998). Due to its economic importance, overharvesting has led this species to be classified as endangered (Class II). Several vegetative organs such as roots,

2 leaves, bark and wood of Garcinia paucinervis are known to be used in folk medicine as an antidote to treat skin infections, burns and bruises (Fan 2012).

Some of the popular bioactive compounds from Garcinia xanthochymus that have been reported for their cytotoxic/anti-proliferative activity are: xanthochymol, isoxanthochymol, cycloxanthochymol, gambogic acid and gambogenone (Protiva et al., 2008). These compounds have shown the ability to modulate multiple signaling pathways ranging from mTOR,

PI3K/AKT to inhibition of HAT (histone acetylase transferase) (Einbond et al., 2013).

Figure 1: Garcinia paucinervis fruits Figure 2: Garcinia xanthochymus fruits

Figure 3: Freeze-dried Garcinia xanthochymus fruits, dissected to show seeds

1.1 Garcinia xanthochymus & Garcinia paucinervis fruits – Phytochemistry

Phytochemistry of Garcinia species

Three major classes of polyphenols occur in Garcinia species. They are benzophenones, biflavonoids and xanthones. Several studies have reported the bioactivity of these compounds such as ability to increase expression of bax, suppression of bcl-2 and cell proliferation, induction of apoptosis by increased expression of caspase 3 (Guruvayoorappan and Kuttan 2008;

Chantarasriwong et al., 2009; Batova et al., 2010). Among these classes, xanthones and benzophenones have received attention for a wide range of biological activities (Genovese et al.,

2016). With more than 300 candidates reported to date, the unique chemistry of benzophenones is a major reason for the research community’s attention (Wu et al., 2014). Varying levels of benzophenones produced by members of Garcinia indicates their possible use as chemo systematic markers in addition to their significant role as major components of floral resins

(Anholeti et al., 2015).

Structurally, benzophenones have a phenol-carbonyl-phenol skeleton as shown in (Fig 4).

The A ring is a benzene ring (derived from shikimate pathway) while the B ring derived from the malonate pathway undergoes prenylation and cyclization to give rise to a variety of structurally unique compounds with bi, tri and tetra cyclic ring systems (Baggett et al. 2005).

A B

Figure 4: 13-carbon skeleton of a benzophenone with A and B rings

Prenylated benzophenones could further be divided into: Basic benzophenones and

Polyprenylated benzophenones (PPBs) (J. Bennett and Lee 1989). Basic benzophenones are characterized by a thirteen carbon skeleton, with complete A and B rings and varying -OH, prenyl or geranyl groups that are uncyclized or have not undergone more than one cyclization

(Wu et al. 2014). Polyprenylated benzophenones (PPBs) on the other hand, have a complete A ring and their variety is introduced as a result of varying additional groups attached to the B ring.

PPBs are subdivided into types A, B, C or D depending on the location at which the benzoyl group is linked. As of 2013, a total of 80 PPBs have been reported from Garcinia spp with guttiferone being the largest class of PPBs with 22 subtypes (Kumar et al. 2013).

Polyisoprenylated benzophenones from Garcinia xanthochymus that were used in this study are xanthochymol and guttiferone E (Fig 5). Both belong to Type B PPBs.

Garcinol

Figure 5: Bioactive benzophenones from Garcinia xanthochymus – xanthochymol, guttiferone E and garcinol (Baggett et al. 2005) 5

Results of our previous studies, showed that location and arrangement of the prenyl side chains caused a significant difference in the cytotoxicity of these benzophenones (Baggett et al. 2005).

Additionally, guttiferone E has a double bond between C32 and C33 whereas, xanthochymol has a double bond between C33 and C34 (Acuna et al., 2010). This could also explain the reason for guttiferone E being more bioactive than its isomer xanthochymol (Protiva et al. 2008). In another study, the ability of xanthochymol to cause caspase 3 induced apoptosis in human leukemia cell lines was attributed to the presence of 1,3, diketone and a phenolic ring (Matsumoto et al., 2003).

The importance of lipophilic, catechol and enol functional groups was also revealed in a study in which both xanthochymol and guttiferone E as a mixture were tested for their ability to inhibit microtubule disassembly (Roux et al., 2000).

A list of studies that were conducted within the last 12 years, on benzophenones from Garcinia spp. on cancer cell lines and the pathways studied are shown in Table 1. Listed in the order of their publication year, we have included only studies that utilize pure benzophenones and not fruit extracts. Synergistic studies that involve pure compounds and one or more extracts have also been excluded. Structures of the most reported compounds (garcinol, xanthochymol and guttiferone E) are listed in Figure 5.

Concentration Targeted Compound (s) Cell line (s) Reference used Pathway(s)

Garcinol A-549, H-460, H- 2 μM Downregulation of (Wang et 1299, H-1650, H- Aldehyde al., 2017) 358, and HCC-827 Dehydrogenase 1 Family Member A1

Guttiferone K HCC 20 μM Cell migration and (Shen et al., invasion 2016)

Garcinol SCC-4, SCC-9 and 5, 10 and 15 μM Anti-proliferative, (Aggarwal SCC-25 pro-apoptotic, cell- and Das cycle regulatory and 2016) anti-angiogenic

Epigarcinol & HL-60 &PC-3 4 and 76 µg/mL Cell cycle arrest (Pieme et Isogarcinol (G2/S) and al., 2015) antiproliferative Guttiferone K HCT-116 3.4 µmol/L Caspase 3 induced (Li et al., apoptosis 2017)

Garcinol MCF-7 35 μmol/L Down regulation of (Ye et al., Cyclin D,Bcl-2 and 2014) Bcl-xl

Garcinol H-1299, H-460 10 μM Decrease of CDK 2,4 (Yu et al., and 6, p38 MAPK 2014) signaling

Guttiferone K HT-29, CCD-841 5.39 ± 0.22 μM Cell cycle, Cyclin (Kan et al., CoN D1, D3, CDK 4&6 2013) and caspase 3 induced apoptosis

Guttiferone E HCT-116, CCRF- 13.92 μM Activation of (Kuete et CEM, MDA-MB- Caspases 3, 7, 8 and al., 2013) 231BCRP 9, Alteration of Mitochondrial Membrane potential (Protiva et Guttiferone E, HCT-116, HT-29, Gut E- 9-17 μM Cell cycle arrest at al. 2008) Guttiferone H and SW-480 Xanthochymol IC50, Caspase Xanthochymol 10-17 μM activation at IC50*2, mTor

7 Concentration Targeted Compound (s) Cell line (s) Reference used Pathway(s)

Combinatorial effects (Einbond et Guttiferone E and HT-29 Colon 4 μg/ml with sulindac sulfide, al. 2013) Xanthochymol (Pure cancer cells rapamycin, turmeric compounds and and celecoxib synergy with Sulindac) Garcinol CAL27 25 μmol/L Inhibition of STAT-3 (Li et al., activation, Inhibition 2013) of NFĸβ Garcinol MDA-MB-231 25 μmol/L Reversal of EMT, (Ahmad et and BT-549 Inhibition of al., 2012b) NFĸβ,vimentin & nuclear β-catenin Garcinol MDA-MB-231 25 μM STAT-3 (Cheng et phosphorylation, al., 2010; Reduction in Ahmad et invasion and al., 2012a) migration Garcinol LNCaP, C4-2B 18 μM Cell proliferation, (Ahmad et and PC3 and apoptosis and al., 2011) BxPC-3 downregulation of NFĸβ Garcinol BxPC-3 and Panc- 20 μM Anti-angiogenic, (Parasramka 1 anti-metastatic and and Gupta anti-proliferative 2011) Between 10- 25 Cytotoxicity (Yang et al., Guttiferone A and K HCT-116, HT-29, µM (including all 2010) SW-480 the 3 cell lines) Garcinol Hep3B 20 μM ER stress modulator (Cheng et GADD153, Caspase al. 2010) mediated apoptosis, PARP Garcinol HCT-116 15 μM TRAIL mediated (Prasad et apoptosis al., 2010) Garcinol 25 μM Caspase induced (Ahmad et MDA-MB-231 apoptosis, al., 2010) and MCF-7 downregulation of NFĸβ 1 μM Inhibition of STAT-1 (Hong et Garcinol HT-29, HCT-116 al., 2006) and IEC-6 cells

Concentration Targeted Compound (s) Cell line (s) Reference used Pathway(s) 10 μM (Liao et al., Garcinol HT 29 Cell migration, 2005) Invasion, MAPK/ERK & PI3K/Akt (Baggett et Guttiferone H and HCT-116, HT-29, Between 12- Apoptosis and al. 2005) Gambogenone SW-480 188µM (for both antioxidant activity compounds in SW480) Garcinol A-549, H-460, H- 2 μM Downregulation of (Wang et al. 1299, H-1650, H- Aldehyde 2017) 358, and HCC-827 Dehydrogenase 1 Family Member A1

Guttiferone K HCC 20 μM Cell migration and (Shen et al. invasion 2016)

Garcinol SCC-4, SCC-9 and 5, 10 and 15 μM Anti-proliferative, (Aggarwal SCC-25 pro-apoptotic, cell- and Das cycle regulatory and 2016) anti-angiogenic

Epigarcinol & HL-60 &PC-3 4 and 76 µg/mL Cell cycle arrest (Pieme et Isogarcinol (G2/S) and al. 2015) antiproliferative Guttiferone K HCT-116 3.4 µmol/L Caspase 3 induced (Li et al. apoptosis 2017)

Garcinol MCF-7 35 μmol/L Down regulation of (Ye et al. Cyclin D,Bcl-2 and 2014) Bcl-xl

Garcinol H-1299, H-460 10 μM Decrease of CDK 2,4 (Yu et al. and 6, p38 MAPK 2014) signaling

Guttiferone K HT-29, CCD-841 5.39 ± 0.22 μM Cell cycle, Cyclin D1, (Kan et al. CoN D3, CDK 4&6 and 2013) caspase 3 induced apoptosis

Concentration Targeted Compound (s) Cell line (s) Reference used Pathway(s) Guttiferone E HCT-116, CCRF- 13.92 μM Activation of Caspases (Kuete et al. CEM, MDA-MB- 3, 7, 8 and 9, 2013) 231BCRP Alteration of Mitochondrial Membrane potential

Table 1: A list of in vitro studies that discuss the pathways targeted by benzophenones from

Garcinia in relation to cancer

One of the well-studied members of the benzophenone family, garcinol (C38H50O6), shown in Figure 5 is also listed in the table to give a comprehensive idea of the cancer related pathways modulated by this compound. Unlike the compounds used in this study, garcinol is freely available to purchase. Some studies have compared the anti-oxidant and anti-cancer activity of Garcinol against well know compounds such as curcumin (Padhye et al., 2010). The presence of isoprenyl groups is believed to enhance the lipophilicity and hence better cell internalization unlike curcumin, whose bioavailability has long been reported to be one of the challenges to overcome. A list of in-vivo studies on benzophenones in breast cancer cells, including triple negative breast cancer is presented in Chapter 2.

As a part of our group’s aim to isolate and study bioactive polyphenols from edible fruits, we recently reported 5 novel benzophenones from Garcinia paucinervis-,paucinones E-I (1-5), and their cytotoxicity across three breast cancer cell lines: MDA-MB-231, SKBR-3 and MCF-7 cell lines (Li et al., 2016a). Paucinone H turned out to be cytotoxic within the concentration ranges of 10 – 20 μM across the panel of cell lines tested. Paucinone G had IC50 values ranging

10 from 15 to 23 μM across the panel of cell lines studied. This was followed by paucinone E, paucinone F and finally paucinone I with IC50 ranges above 50 μM in MDA-MB-231 and MCF-

7 cell lines. The rationale behind choosing the cell lines mentioned above, was to determine if the novel benzophenones displayed cell line specificity.

Paucinone H (C33H40O7) had strong cytotoxicity (at concentrations ranging from 10 - 20

μM), across the panel of breast cancer cell lines. Therefore, paucinone H was selected for a detailed evaluation of its mechanism of action, particularly in triple negative breast cancer cell lines.

Paucinone F Paucinone E

Paucinone H

Paucinone G Paucinone I

Figure 6: Novel benzophenones paucinones E-I isolated from Garcinia paucinervis (Li

et al. 2016a)

The structural difference between paucinones G and H is that paucinone H lacks the hydroxy group attached to C-30 that was present in paucinone G. Other paucinones that have been reported from Garcinia paucinervis include: Paucinones, A, B, C and D (Gao et al., 2010).

In this study, the cytotoxicty of paucinones A, B and D against HeLa cells were attributed to the absence of an ester group at C-2 and C-11.

Chapter 2: Breast cancer and bioactive compounds from plants

2.1. Dietary phytoestrogens and their impact on breast biology

Introduction

Although phytoestrogens have not been reported in Garcinia, the use of dietary phytoestrogens and their impact of breast biology is worth investigating due to the wide spread use of phytoestrogen based dietary supplements. Phytoestrogens are plant-derived small molecules that can act as potential estrogen receptor agonists or antagonists. Some naturally occurring phytoestrogens include coumestans, flavonoids, isoflavonoids, lignans, and stilbenes (Rietjens et al., 2017). Several members of this broad group of plant compounds have been shown to act as natural selective estrogen receptor modulators (SERMs), making them attractive agents for the prevention of postmenopausal osteoporosis and cardiovascular disease (Moutsatsou 2007; Rudy et al., 2012; Poluzzi et al., 2014). Currently prescribed synthetic SERMS such as tamoxifen and raloxifene, are known to cause a number of undesirable side effects such as hot flashes, strokes, cataracts, and most notably, tamoxifen has been reported to increase the risk of uterine and endometrial cancers (Hershman et al., 2002). With the need to develop better alternatives to currently prescribed SERMs, many investigators and clinicians have assessed the effectiveness of several phytoestrogens. However, to date, the clinical benefits of phytoestrogen-based SERMs remains to be established.

The interest in consumption of dietary phytoestrogens, especially soy mainly stems from epidemiological studies that show that the incidence of breast cancer is lower in Asian countries, especially China, Japan, Korea, and Indonesia, thus leading to a hypothesis that women whose diets are rich in phytoestrogens have a reduced risk of developing breast cancer (Adlercruetz

2002). In support of these findings, the rate of breast cancer incidence increases in Asian women who migrate to the west and start consuming a non-traditional diet (Mense et al.,

2008)Therefore, it is clear that genetic makeup alone is not the reason behind lower incidence of breast cancer in Asia. This chapter could serve as a helpful reference for readers who are interested in recent advancements in the field of phytoestrogens specifically in relation to breast pathophysiology.

Classification of Phytoestrogens

The discovery of phytoestrogens was serendipitous through a perplexing case of infertility in sheep, and the cause was ultimately determined to be isoflavones from the red clover in the pastures (Bennets et al., 1946). Phytoestrogens, are comprised of a structurally diverse group of natural products like lignans, flavonoids, isoflavonoids, coumestans, and stilbenes (Ososki and

Kennelly 2003). While these compounds have been reviewed in depth previously (Ososki and

Kennelly 2003), the focus of this review is to inform readers on the different classes of phytoestrogens, their dietary sources and their estrogenic effects, specifically in relation to breast pathophysiology.

Lignans

Lignans contain two phenylpropanoid units linked via a carbon–carbon single bond, and two well-known lignans are secoisolariciresinol and matairesinol (Ososki and Kennelly 2003). Cereal bran, beans, flax seed, nuts, and unrefined grains are some of the important dietary sources of lignans. Certain lignans are known to be metabolized to the estrogenic compounds enterolactone and enterodiol by the action of gut microbial flora (Cornwell et al., 2004). This gut microbial transformation could explain why some women respond to certain phytoestrogens and others do

15 not. Therefore, it can be difficult to establish an effective dose and bioavailability for such phytoestrogens in different individuals.

Isoflavonoids

Isoflavonoids are a class of flavonoids that have been studied extensively for their estrogenic effects. One of the well-studied dietary sources of phytoestrogens is soy (Glycine max) that contains two principle isoflavones, daidzein and genistein. In addition, glycetin, formononetin, and biochanin A are some of the other well studied dietary phytoestrogens (Preedy 2012).

Structurally, the presence of a phenolic ring makes isoflavonoids analogous to estradiol, thus enabling them to bind to the estrogen receptor (Bedel et al., 2014; Sefirin et al., 2014). Although soy is a rich source of isoflavones, other isoflavones have been reported in more than 40 plant species (Ososki and Kennelly 2003). Some examples include legumes, such as alfalfa (Medicago sativa), chickpeas (Cicer arietinum), common bean (Phaseolus vulgaris), and red clover

(Trifolium pratense). and Other edible plants such as licorice (Glycyrrhiza glabra) and certain whole grains, nuts, and berries also have isoflavones (Rowland et al., 1999). Herbs such as

Sophora japonica, Pueraria lobata (kudzu), and Radix pueraria (an ingredient used in traditional

Chinese medicine) are rich in daidzein (El-Halawany et al., 2010; Hong et al., 2011).

The estrogenic benefits of isoflavones appear to be a function of an intricate balance between the bioconversion and bioavailability of isoflavones after they are ingested. The isoflavone daidzein is metabolized into equol by a number of enteric bacteria such as Adlercreutzia,

Asaccharobacter, Eggerthella, Lactococcus, and Slackia (Rafii 2015). Equol is one of the most bioactive isoflavones, capable of binding to both ERα and ERβ receptors (Kelly et al., 2014;

Magee et al., 2014). However, the ability to metabolize daidzein to equol varies from one person

16 to the other, and individuals who are capable of metabolizing daidzein into equol are known as equol producers (Shor et al., 2012). Studies have shown that equol is stronger than daidzein in inducing the mRNA expression of estrogen-responsive protein PS2 in MCF 7 cells

(Sathyamoorthy and Wang 1997). The presence of an enteric bacterial population capable of metabolizing isoflavones plays an important role in their bioavailability

Coumestans

Coumestans are polycyclic aromatic plant secondary metabolites with a carbon-carbon double bond shared by a coumarin moiety and a benzofuran moiety. Their estrogenic and anticancer effects range from modulating pathways related to cell proliferation, synthesis, and survival including ROS production and DNA damage (Nehybova et al., 2014). Coumesterol is an important member of this class, and is found in alfalfa, clover sprouts, and pinto beans (Liu et al., 2012). Trace amounts of coumesterol have also been reported in spinach and Brussel sprouts

(Brassica oleracea) (Cornwell et al. 2004)

Miscellaneous flavonoids

In addition to isoflavonoids and coumestans, another class of estrogenic constituents are prenylflavonoids. One such phytoestrogen is 8-prenylnarigenin, a compound found in hops

(Humulus lupulus ) (Martina et al., 2008), and has sparked considerable interest in studies regarding the potential health benefits of beer consumption.

New phytoestrogens reported for their estrogenic effects in breast cancer

In vivo and in vitro models are both important in the identification of phytoestrogens and their mechanism of action. For example, the erythroidine alkaloids from Erythrina poeppigiana have

17 been reported to be estrogenic, in-vitro using an MCF-7 ER-dependent cell line assay (Lecomte et al., 2017b) .This is the first time that an erythroidine alkaloid demonstrated estrogenic activity.

Transcriptomic analysis of glyceollins (phytoalexins derived enzymatically from daidzein) attributed the antiproliferative effects of these compounds to modulation of the FOXM1 pathway

(Lecomte et al., 2017a). Tanshinone II A, a lipophilic compound isolated from Salvia miltiorrhiza (Danshen), has been analyzed for its potential to bind to ER receptors on vascular endothelial cells (Fang et al., 2011). Some phytoestrogens elicit cytotoxic/antiproliferative effects irrespective of the ER receptor status of a given cell line. For example, arctigenin from burdock (Arctium lappa) showed anti-invasive and antimigratory effects on both ER-positive

(MCF-7) and ER-negative (MDA-MB-231) cell lines (Hsieh et al., 2014). The anti-proliferative effects of arctigenin on MDA-MB-231 have also been reported (Hsieh et al. 2014; Maxwell et al., 2017). The mechanisms targeted by phytoestrogens on triple negative cell lines is discussed in a separate section.

Edible plants continue to be an important group to study since many women may ingest these dietary compounds, either as conventional foods or dietary supplements, without considering or understanding their estrogenic potential. Of recent interest is the coffee constituent trigonelline, reported to be capable of modifying the rate of cell proliferation in vitro, using estrogen receptor positive MCF7 cells (Allred et al., 2009). Diarylheptanoid (DPHD), (3R)-1,7-diphenyl-(4E,6E)-

4,6-heptadien-3-ol, isolated from the rhizomes of Curcuma comosa, showed a protective effect against the deterioration of bone microarchitecture in adult female Sprague-Dawley rats

(Tantikanlayaporn et al., 2013). The phytoestrogen content of foods and dietary supplements continues to be an area of active study due to the important health issues surrounding phytoestrogens.

Phytoestrogens and Estrogen receptors

Estrogens are steroid hormones that that elicit broad physiological responses and most notably play a key role in regulation of the reproductive cycle in women. Estrogens bind to estrogen receptors, which regulate transcription of target genes by binding to specific sequences called estrogen responsive elements (ERE) (Welboren et al., 2007). There are two specific estrogen receptors - ERα and ERβ. The ER ligand binding domains in ERα and ERβ differ by two residues: Met-421(ERα)/Ile-373(ERβ) and Leu-384(ERα)/Met-336(ERβ). ERα is primarily associated with proliferation, inflammation, and malignancy whereas ERβ is counter active to these effects, in the sense that it down regulates the action of ERα regulated genes (Treeck et al.,

2010; Christoforous and Jan-Ake 2011). The binding of certain phytoestrogens to ERβ might explain how phytoestrogens could be beneficial (Turner et al., 2007). For instance, genistein, apigenin, and daidzein have a greater binding affinity to ERβ (Kuiper et al., 1998). Structural similarities of phytoestrogens include the presence of a phenolic hydroxyl group, similar to the hydroxyl group in 17β-estradiol. The position of these hydroxyl groups (4,6, and 7 are more favorable) and their interaction with the ligand binding domains in ERα and ERβ results in activation of the appropriate receptor (Leclercq and Jacquot 2014).

Figure 7: Structure of 17β-Estradiol (E2)(Leclercq and Jacquot 2014)

Although phytoestrogens are capable of binding to ER receptors, their relative binding affinities to a specific type of estrogen receptor varies. This could in turn explain the differences in activity of the same phytoestrogen in different tissue types.

Dietary sources of phytoestrogens known to impact breast pathophysiology and women’s health

The US food supplement industry has grown significantly from $11 million in sales in 1995 to more than $22 billion in 2006 (Bradley 2015). This trend continued further and by 2015, the dietary supplement industry was valued at $36.7 billion (Vatistas and Samuels 2012; Bradley

2015). Flax seed, red clover, soy, and wild yam and are some of the main botanicals containing phytoestrogens that are available in the market to treat menopausal symptoms (Borrelli and Ernst

2010). Single ingredient supplements with chasteberry, cranberry, evening primrose, garlic, hops, and don quai have been examined for their potential role in reducing the risk of breast cancer in postmenopausal women (Boucher et al., 2013). The use of dietary isoflavones such as soy during an individual’s early life has shown to offer protective effects against breast cancer

(Ziaei and Halaby 2017). Black cohosh, is one of the most popular dietary supplements for menopausal women. Although there have been reports that this plant contains the phytoestrogen formononetin, some studies have not been able to verify this

The estrogenic activity of black cohosh has been questioned due to the lack of clinical evidence

(Shulman et al., 2011). However there is no underlying link between consumption of black cohosh and an increased risk of breast cancer (Fritz et al., 2014). Similarly, clinical evidence to support the use of isoflavonoid-based supplements, like red clover, in the treatment of hot flashes and menopausal symptoms have not been significant (Tice et al., 2003). . 20

There is some evidence that shows soy isoflavones like daidzein can enhance cancer characteristics in mouse mammary tumor cell lines (Koo et al., 2015; Yang et al., 2015). Other studies that have analyzed the use of supplements with multiple phytoestrogens suggest a possible interplay between dietary phytoestrogens and increased tumor cell proliferation in vitro, thereby advising consumers to avoid them, especially while undergoing breast cancer treatment

(van Duursen et al., 2013). However, these in vitro models do not completely mimic the conditions in humans since in vivo physiological levels and bioavailability are not accounted for.

To date there is still no strong clinical evidence suggesting phytoestrogens increase breast cancer risk, incidence, or progression as reviewed by Moreira (Moreira et al., 2014)

Assays for phytoestrogens

Detection of phytoestrogens in plant extracts and in dietary supplements is essential for a better evaluation of their activity. Therefore, this section discusses assays for the quantification of phytoestrogens and the measurement of their estrogenic activity, either as purified compounds or as complex mixtures, such as a botanical extract. The limitations of some of these assays are also discussed.

Assays for quantification of phytoestrogens

Radio immunoassays have been in use for the detection of daidzein, genistein and formononetin since 1972 (Cox et al., 1972). However, quantitative estimation of daidzein in human biological fluids such as serum was reported in 1997 (Lapcik et al., 1997). They were found to be an efficient alternative to GC-MS or HPLC methods due to their ease, low cost and their ability to be used in larger epidemiological studies. Transient gene expression assays, E-screen assays,

21 recombinant yeast assays, laser induced fluorescence detection, UV and IR spectroscopy, capillary electrophoresis, MALDI-TOF, ELISA and time-resolved fluoro immunoassays are some of the in-vitro systems used to quantitatively measure phytoestrogens (Lapcik et al. 1997;

Routledge et al., 2000; Garcia-Reyero et al., 2001; Wang et al., 2002). However, each of these assays has drawbacks such as the use of radioactive isotopes, being labor intensive, and the difficulty in arriving at absolute quantification. Some of these drawbacks can be overcome by using quantitative affinity assays based on microchip electrophoresis, that utilize PEG-modified glass microchannel with a short run time (often less than 2 minutes) and a small sample volume

(Chuang et al., 2006)

Bioassays to measure estrogenic activity of phytoestrogens

The classic ligand binding and ER promoter assays are used to test for estrogenic activity.

Ligand binding assays, estimate the binding efficiency of a test compound to receptors ERα or

ERβ, but these assays cannot determine if the compound elicits an estrogenic response (Kuiper et al. 1998). Transactivation assays have been used to detect estrogenic activity. For instance, the estrogenic activity of dietary lignans has been reported using a transgenic estrogen reporter mouse model (Penttinen-Damdimopoulou et al., 2009). Similarly, Hs578T-ERαLuc and

Hs578T-ERβLuc cells have been used to identify and characterize ER subtype selective ligands

(Shanle et al., 2011)

High-throughput screening assays, based on liquid chromatography mass spectrometry (LC-MS) that can provide enhanced sensitivity, selectivity and rapid detailed structural information have been developed (Choi and van Breemen 2008)

Assay Principle Significance Reference Dual cell line GFP Dose response GFP Rapid identification of Xu et al. expression assay intensity in transfected estrogens, antiestrogens (2008) MCF7-GFP and Ishikawa- and selective estrogen GFP. receptor modulators. LC-MS based ERα and β immobilized Binding of receptors to Choi and van magnetic micro magnetic particles to screen magnetic micro particles Breemen particle assay for estrogenic compounds in followed by (2008) Trifolium pretense (red identification of released clover) or Humulus lupus ligands using LC-MS. (hops) extracts. Micro total bioassay Polydimethylsiloxane sheets Enables study of Imura et al. with micro channels that metabolites with shorter (2010) integrate a micro intestine, half-life even at micro liver and target cells. negligible volumes. Intestinal absorption, Shorter run time and hepatic metabolism and lower sample and reagent bioactivity of soy volumes. isoflavones on MCF 7 cells was studied.

Nonspecific An ERα and β binding assay The binding of potent Wang et al. adsorption assay that uses auto fluorescent selective ligands (2014) compounds such as determined using coumesterol using fluorescence polarization. fluorescence polarization. A rapid, simple and homogenous method. HPLC/LC-MS based Quantitative estimation of A rapid method to Andres et al. high throughput isoflavones in dietary estimate the composition (2015) screening assay supplements using tandem of a single phytoestrogen mass spectrometry. in multiple dietary supplement samples.

Table 2: Bioassays used in detection and study of phytoestrogens from complex extracts and dietary supplements (2008-2017)

Examples of such applications include the use of ER immobilized magnetic particles, to eliminate non-specific binding while studying ER-ligand complexes from botanical extracts such

23 as red clover and hops (Choi and van Breemen 2008; Rush et al., 2017). An extension of such

LC-MS based assays have been reported for chemical and biological standardization of botanical extracts with estrogenic compounds (van Elswijk et al., 2004). These methods are particularly useful to evaluate phytoestrogen-based supplements, to help ensure that the levels of phytoestrogenic compounds in a dietary supplement are standardized and consistent. Such standardized extracts would then be a reliable source for clinical studies to assess their safety and efficacy, since there is no possibility of variation in results due to a variation in active ingredients. LC-MS based assays with an ultrafiltration step are extremely useful in screening complex mixtures for ligands and simultaneously estimating their relative binding affinities to specific receptors (Sun et al., 2005). In addition, methods such as online biochemical detection coupled to mass spectrometry for rapid detection and identification of estrogenic compounds from pomegranate peel extract has also been demonstrated (van Elswijk et al. 2004). This method is particularly interesting, as it can be used in screening of complex botanical extracts.

Recently, the use of miniature technology has also been explored to test estrogen-like substances, since most of the in vitro assays do not enable simultaneous analysis of bioavailability of an estrogenic compound. This drawback has been addressed by the use of a micro total bioassay made up of polydimethylsiloxane sheets with micro channels that integrate a–micro intestine, micro liver and target cells to be tested onto a microchip (Imura et al., 2010). The aim of this assay system was to serve as a one-point reference to study intestinal absorption, hepatic metabolism and bioactivity of soy isoflavones on MCF 7 cells. The micro intestine channels had

CaCo2 cells cultured on collagen coated membranes. A fibronectin pre-treated microchannel with HepG2 cells formed the micro liver and a suspension of target cells (MCF7) was cultured on fibronectin coated micro channels. Unlike conventional bioassays, the micro total bioassay,

24 offers benefits such as a reduction in the volume of reagents and samples used. This unique assay system also enables study of sample metabolites with shorter half-life even at negligible volumes. Further, the overall assay time was also reduced to two days. Table 2 shows a list of bioassays reported to detect estrogenic compounds while highlighting their unique capabilities in comparison to other assays discussed, and their mode of action

Enhancing the delivery of phytoestrogens

The low bioavailability of dietary phytoestrogens, suggests that for these compounds to be useful physiologically in humans, drug delivery systems may need to be employed to enhance their efficacy. A number of factors may impact a compound’s availability, such as the mode of administration, presence of certain functional groups, rate of metabolism, activity of binding proteins and hydrophobicity play key roles in determining the appropriate drug dosage (Le

2016). For instance, genistein exhibits poor water solubility, which in turn results in low oral bioavailability (Iwasaki et al., 2008; Lee et al., 2009)In order to overcome this issue, a cationic lipid-based nano carrier system targeting the mitochondria has been designed for use in cancer therapy (Pham et al., 2015). Commercial cationic copolymers such as Eudragit® E have been used in a nanoparticle mediated delivery system to improve oral bioavailability of genistein

(Tang et al., 2011)

Several in vivo and in vitro studies also explain the role of phytoestrogens in the regulation of pro & anti-apoptotic proteins bcl2 and bax. Thus, it can be stated that their ability to modulate multiple regulatory pathways is what makes dietary phytoestrogens an attractive panacea.

Breast physiology and cancer

Although considerable advancements have occurred in the last 20 years, in prevention, detection, and treatment of breast cancer, it is still one among the most common cancers in woman

(Anonymous 2016). The incidence rates of breast cancer in the U.S. has decreased since 2000, yet it is still higher in comparison to the incidence of breast cancer in Asia. Several epidemiological studies hypothesize that the reason for these lower rates of breast cancer among

Asian women can be attributed to their dietary intake of phytoestrogens (Iwasaki et al. 2008).

This is further backed up by ethnobotanical information on the benefits of using phytoestrogen based herbal preparations and nutraceuticals especially in relation to menopause (Depypere and

Comhairea 2015). Some studies point out that the reduced risk of breast cancer due to intake of phytoestrogens such as soy isoflavones is evident only in Asians and not in western population

(Dong and Qin 2011). Also, a higher intake of dietary components such as n-3 PUFA, vitamin D, phytoestrogen, fiber, and folate, in addition to lower intake of saturated fat, n-6 PUFA, grilled meat, and alcohol, has been suggested to be beneficial in reducing the risk of developing breast cancer (Kotepui 2016). Molecular targets of phytoestrogens include cell cycle regulatory proteins and tumor suppressor genes such as cyclin D1, p53, p21 p27, nerve growth factor and ERα

(Sakamoto et al., 2010; Chen and Sun 2012; Lecomte et al. 2017b; Lecomte et al., 2017c). Soy isoflavonoids, especially genistein has shown antiangiogenic effects, including significant reduction in micro vessel density both in vitro and in vivo (Varinska L et al., 2015). Several in vivo and in vitro studies also explain the role of phytoestrogens in the regulation of pro- and anti- apoptotic proteins bcl2 and bax (Rajah et al., 2012). Thus, it can be stated that their ability to modulate multiple regulatory pathways is what makes dietary phytoestrogens an attractive supplement. 26

Phytoestrogens and triple negative breast cancer

Breast cancer is a complex interplay of several biochemical, physiological and molecular factors, giving rise to different subtypes. One of the most aggressive subtypes is triple negative breast cancer (TNBC). This particular subtype is characterized by lack of expression of hormonal receptors, estrogen receptor-α (ER-α), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) (Foulkes et al., 2010). Approximately 15% of all breast cancers turn out to be triple negative (Lara-Medina et al., 2011).TNBC is known to have higher recurrence rates and no approved targeted therapies for these tumor subtypes. There is in-vitro evidence suggesting the possibility of utilizing phytoestrogens as effective therapeutics against TNBC.

These studies range from chemo-protective effects to modulation of key cell survival pathways such as cell cycle, cohesion complex cleavage kinetochore formation, NFκβ, and Ras/MAPK/AP

(Hedelin et al., 2008; Li et al., 2008; Pan et al., 2012; Fang et al., 2016) In addition, gene expression profiling studies using microarrays have been reported in various breast cancer subtypes including triple negative breast cancer. These studies illustrate the ability of phytoestrogens genistein and daidzein have estrogen mimicking activities and alter the expression of genes associated with cell communication, lipid metabolism, signal transduction, fatty acid synthesis and cell growth (Satih et al., 2010; Guo et al., 2016).

Further, a recent review reported that the overall risk factor for triple negative breast cancer in

Asia is the same as in western countries (Wang et al., 2017).

Although several in vitro studies point to the potential of using phytoestrogens in chemoprevention and/or as adjuvants to chemotherapy regimens in aggressive triple negative breast cancers, in vivo studies have not shown such promise. In a TNBC xenograft model tumor

27 growth was not affected by oral gavage administration of phytoestrogen-rich soy extract (Gallo et al., 2006). The use of phytoestrogens for the prevention or treatment of TNBC requires further investigation. Clinical studies are underway to explore the role of phytoestrogens in reducing proliferation of triple negative breast cancer (Lathrop 2017). The results of these studies and the others to come might hold more clues on the exact application of phytoestrogens across a wide demographic.

Phytoestrogens in Hormone replacement therapy

Hormone replacement therapy (HRT) is the substitution of endogenous estrogens with exogenous estrogens, primarily during menopause. Questions about underlying links between hormone replacement therapy in women and an increase in incidence of breast cancer, were raised after the release of the Women’s Health Initiative study in 1998 (Archer 2014). As a consequence, there was an increasing demand for natural alternatives to HRT (Ross et al., 2000;

Mueller and Jungbauer 2008). It is at this juncture that phytoestrogens became the focus of the research community, due to their structural similarity to human estradiol and their ability to modulate the estrogen receptor (Sakamoto et al. 2010). Hormone replacement therapy for postmenopausal woman has shown benefits of decreasing risk of fractures, decreasing incidence of cardiovascular disease, and delaying onset of Alzheimer’s disease. Studies on the benefits of phytoestrogen-rich preparations in perimenopausal women for prevention of atherosclerosis and other climacteric symptoms have been only slightly positive (Kirichenko et al., 2017). The current knowledge on the clinical benefits of phytoestrogens in hormonal replacement therapy is limited (Chen et al., 2015; Franco et al., 2016). The use of phytoestrogens in hormone therapy

28 for menopausal symptoms has also not been successful in breast cancer survivors. (Alipour et al.,

2015). Reviews that have discussed the use of HRT in the treatment of hot flushes in breast cancer survivors have also concluded that each patient should be individually assessed for side effects of HRT (Wisniewska et al., 2016). Therefore, studies that look at long-term exposure to phytoestrogen in a wider demographic might be more invaluable. To this extent, clinical studies that include a varied cohort and longer exposure time, such as the Soy Phytoestrogens as

Replacement Estrogen (SPARE), might help better evaluate the use of phytoestrogens in hormone replacement therapy (Levis et al., 2010). Also, given the wide range of phytoestrogen- based supplements that are promoted for menopausal women, the use of phytoestrogens for these symptoms needs to be better investigated

Phytoestrogens as Selective Estrogen receptor modulators (SERMS)

SERMS are nonsteroidal compounds that serve as ligands for the estrogen receptor while selectively acting as agonists or antagonists. The presence of two different estrogen receptors -

ERα and ERβ, their tissue specific distribution and binding affinities often determines their behavior as an agonist or an antagonist (Jiang et al., 2013). Higher ERαlevels usually correlate with an increase in cell proliferation, because ERα is associated with regulation of gene expression and proliferation in tissues such as the uterus and breast (Philipp et al., 2013). ERβ on the other hand mostly acts as a balance and negates the effects of ERα (Paruthiyil et al., 2009).

However over 70% of breast cancers are known to express both ERα and ERβ (Charn et al.,

2010) SERMS can act as agonists in tissues such as breast and uterus or antagonists in tissues such as cardiovascular cells, bones and brain. This selective specificity of SERMS is the primary reason for their therapeutic potential. Major clinical applications of SERM include prevention and treatment of breast cancer, osteoporosis and maintenance of healthy serum lipid profile 29

(Martinkovich et al., 2014). Recently, prenylated isoflavonoids have received attention due to their ability to act like SERMS. Licorice, hops, and sprouts of leguminous seeds are a rich source of prenylated isoflavonoids (Simons et al., 2012) . Diarylheptanoids from Curcuma comosa have been reported for their anti-estrogenic activity by downregulating ERα signaling and suppressing estrogen-responsive genes (Thongon et al., 2017) Diadzein and genistein are other isoflavones which exhibit SERM like behavior. In other studies, isoflavones such glabidrin, 8- prenylgenistein, kievitone and phaseollin have shown assay dependent estrogenicity i.e. they displayed ER alpha antagonism in a yeast bioassay while they behaved as agonists in human breast cancer cell lines (T47D and MCF-7) (Sotoca et al., 2008). A combination of Glabridin and

Tamoxifen caused inhibition of cell growth in Ishikawa and MCF cell lines. This study points out to the potential use of glabridin and tamoxifen in estrogen replacement therapy while negating the side effect of Tamoxifen to cause endometrial cancer (Jen et al., 2015). Further apigenin, naringenin, quercetin, biochanin A, hesperitin, silymarin and fistein have all reported to influence the expression of breast cancer resistance protein (BCRP) (Kaur and Badhan 2015).

While available data show that phytoestrogens could be used as an alternative to synthetic selective estrogen receptor modulators (SERMs), whether the use of phytoestrogens as SERMs is entirely beneficial remains a question. For example, there is still a need for studies that focus on perinatal exposure to environmental phytoestrogens and their influence on developmental epigenetic patterns (Lecomte et al. 2017b).

Phytoestrogens in breast cancer clinical trials

Several clinical studies have been conducted with phytoestrogens to further evaluate their potential use in humans. A comprehensive list of studies that were completed between 2008 and

2016, and that focused on breast cancer intervention are listed in Table 2. Since the Women’s 30

Health Initiative study that demonstrated a link between hormone replacement therapy (HRT) and breast cancer, there has been an increased interest about whether phytoestrogens would be a good substitution for HRT. In vitro studies suggested that genistein and structurally modified isoflavones induced apoptosis and inhibited cell proliferation pathways such as NFKB and AKT, suggesting their possible role in chemoprevention and sensitization of tumors to chemotherapeutic agents (Sarkar et al., 2006). Data from Mammary carcinoma Risk factor

Investigation (MARIE), showed genistein causes a decrease in tumor proliferation and Ki-67 at high expression levels of Ki-67 (Jaskulski et al., 2017). In contrast no changes were observed in

Ki-67 in a randomized trials investigating the impact of soy on early stage breast cancer patients, however this study also found that transcription of genes associated with proliferation were upregulated in woman treated with soy (Shike et al., 2014). Other studies conducted on the use of soy and flax seed supplements in healthy females, early stage breast cancer patients and postmenopausal women have shown upregulation in the expression of genes associated with cell proliferation as well (Colli et al., 2012). In a randomized trial investigating healthy woman classified as high risk for breast cancer, soy consumption failed to show efficacy in prevention and upregulated proliferation markers in pre-menopausal woman within this study (Khan et al.,

2012). Other randomized studies have shown soy to have no effect on modulating breast density, a known risk factor for breast cancer (Delmanto et al., 2013; Wu et al., 2015). In healthy postmenopausal women, administration of oral doses of soy isoflavones (900 mg/day) proved to be safe in post-menopausal women (Pop et al., 2008). The beneficial outcomes of using seaweed as a prebiotic catalyst in favorably altering estrogen and phytoestrogen metabolism in a smaller ethnic group of 15 women has also been reported (Teas et al., 2009).

In addition to the studies reported here, long term projects such as the Korean Hereditary Breast

Cancer Study (KOHBRA) specifically targeting carriers and non-carriers of BRCA mutations

might hold some invaluable answers to the long-standing debate on the role of dietary

phytoestrogens in lowering the risk of breast cancer and the specific populations that would

benefit from the use of phytoestrogens (Kang et al., 2015). Due to the popularity of

phytoestrogen-based supplements, the need for more detailed clinical trials has led to an increase

in number of ongoing studies. A search for ongoing phytoestrogen clinical trials on

clinicaltrials.gov, showed that results from at least 13 studies are yet to be published.

Phytoe Sample Age Trial design Results Dose Reference strogen size (yrs)

Soy Double blind, Soy intervention 50 mg 102 30 – 75 randomized, was neither Isoflavone (A=44; placebo – beneficial nor tablets per P=41; Wu et al. controlled induced adverse day for 12 W=17) [2015] intervention effects on breast months tissue

Soy Double blind, Soy isoflavone 250 mg of 80 45 Delmanto et randomized, extract did not Soy extract (A = 40; al. placebo – affect breast per day P=40) [2013] controlled density in post- study menopausal women Soy Randomized, Overexpression 25.8 g Soy; 140 50 Shike [2014] placebo of cell Twice/day (A=70; controlled proliferation for 30 days P=70) study genes such as FGFR2

Phytoe Sample Age Trial design Results Dose Reference strogen size (yrs)

Soy Double blind, Unconjugated 150 mg 30 50 Pop et al. isoflavo randomized, isoflavones are genistein (A=18; [2008] nes Phase 1 safe in post- capsules; P=12) menopausal 4/day women, even at higher doses

Soy Randomized Expression of 14 334 mg 98 (A= 25 -55 Khan et al. isoflavo Phase IIB trial genes related to Mixed soy 49;P=49) [2012] nes proliferation, isoflavone apoptosis and capsules 1 estrogenic effect per day was observed Flax Randomized, The flax seed 1 g FSE & 90 53 - 56 Colli et al. seed placebo extract & Flax 90 g (A.FSE= [2012] meal controlled seed meal was FSM/day for 30, and study neither beneficial 6 months FSM=30; flax nor did they P= 30) seed induce adverse extract effects on vaginal epithelium or endometrium Enteroli Multicentric Inverse Lignans – 1060 50 -74 Jaskulski et gnan population association per 100 g al. [2017] and based patient between genistein edible food genistei cohort study and Ki- Enterolignan n using a food 67expression (in – per 100 g frequency post-menopausal ingested food questionnaire breast cancer patients) Isoflav Double blind, Intake of 70 mg 100 50 – 70 Choquett et ones controlled isoflavones along isoflavone (A.ISO= yrs al. [2011] (daidzei study with exercise capsules for 23; n, could be 6 months AEX+IS genistei beneficial to O=16; A. n & overweight EX+P=2 glycitei postmenopausal 5; P=22; n) women W=12)

Phytoe Sample Age Trial design Results Dose Reference strogen size (yrs)

Seawee Double blind, Seaweed Seaweed 15 Teas et al. d randomized favorably alters capsules (5 (SP, GC; 58 – 70 [2009] supple study estrogen & g/d); IS – P) yrs mented phytoestrogen 2g/d for 7 with metabolism weeks soy

Flax 2X2 Factorial Flax seed did not 25 g Ground 24 McCann seed randomized interfere with AI. Flax (AN, 59 – 65 et al. [2014] and intervention But AI reduced seed/day for GFS, yrs anastro design the availability of 13 – 16 days GFS+P, zole circulating GFS+AN (aromat lignans. ; P) ase inhibito r)

Table 3: Clinical studies completed on phytoestrogens and breast cancer intervention (2008- 2017) Abbreviations used: A – Active, P- Placebo, W- Withdrawn, FSE- Flax seed extract, FSM – Flax seed meal, SP – Seaweed powder, GC – Gelatin capsule, GFS- Ground flax seed, AN- Anastrozole

Figure 8: A schematic representation of drug delivery systems that utilize phytoestrogens and

their molecular targets

One of the key challenges when working with complex botanical extracts is to achieve consistent quality control per batch to ensure bioactive compounds do not vary greatly, given that environmental factors such as seasonal changes, biotic and abiotic stress, and extraction techniques, can have a significant impact on the expression of phytoestrogenic compounds.

Quality assurance issues with botanical dietary supplements include standardizing the concentration and consistency of active constituents and screening for the presence of potential impurities and toxic compounds. Recent advances in the biochemical analysis of phytoestrogens, such as on-line biochemical detection coupled to mass spectrometry, use of high-throughput screening methods such as microchips, micro bioassay systems that mimic the human intestinal lumen and biosensors that help detect phytoestrogens in complex plant extracts can help overcome these challenges (van Elswijk et al. 2004; Chuang et al. 2006; Imura et al. 2010).

However, in the United States, many dietary supplement manufacturers are small companies

35 without the resources to conduct such expensive analyses. Unless the US Food and Drug

Administration requires standardization to a bioactive compound, significant levels of variation will likely continue to be seen in phytoestrogenic supplements like soy and red clover. Some groups like the United States Pharmacopeia have developed standards for certain herbal dietary supplements, but U.S. manufacturers are not required to follow these.

While there is some evidence in animal studies pointing to the benefits of phytoestrogens, data available from studies with human subjects are inconclusive as to whether consumption of phytoestrogens is entirely beneficial. This debate holds true even for well-studied phytoestrogens such as genistein. For instance, in vitro studies that show incidence of uterine carcinoma, multiple oocyte follicle formation, and several studies with conflicting results in human subjects on the inhibitory and stimulatory effects of soy on the growth of breast cancer cells emphasizes the fact that phytoestrogens should be ingested cautiously with appropriate clinical guidance

(Duffy et al., 2007; Cederroth and Nef 2009). In addition, the use of dietary phytoestrogens such as isoflavones to prevent breast cancer has not shown encouraging results (Sak 2017). Studies have even pointed out that genistein interferes with the inhibitory effects of tamoxifen on breast cancer cell growth (Liu et al., 2005; Spagnuolo et al., 2015). A meta-analysis of prospective studies suggested that high consumption of soy isoflavone decreases risk of breast cancer incidence in Asian populations but not in Western populations (Dong and Qin 2011)

However, a subsequent meta-analysis indicated that soy consumption associates with protective effects against breast cancer irrespective of nationality, and may reduce recurrence and mortality

(Fritz et al., 2013) . The Komen Foundation suggests that breast cancer patients and survivors are not required to completely avoid soy. They can include soy based products in their diet, while the intake of soy supplements is not recommended. For a comprehensive article highlighting human 36 studies and the relationship between dietary phytoestrogens such as isoflavones and breast cancer we also suggest reading the following review (Sak 2017).

An important question that then remains is whether phytoestrogens would have a protective effect at prolonged exposures in lower doses. Studies like this would help determine the long- term effects of widely marketed phytoestrogen-based dietary supplements and the impact of exposure to dietary phytoestrogens. In addition, there is lack of clinical studies on the exposure of a fetus to phytoestrogens. So, the risks associated with phytoestrogen consumption in pregnant women remains unclear (Todaka et al., 2005). While the general picture is that phytoestrogens have beneficial effects and do not induce endometrial or breast cancer, these studies suffer from lack of sufficient sample size (Eden 2012). Therefore, additional randomized clinical trials with a larger sample cohort would help better understand the effects of these phytoestrogens in a pharmacologically relevant dose.

Since an individual’s ability to metabolize certain phytoestrogens is dependent on the nature of the individual’s gut microbial flora, the interplay between an administered dosage and its bioavailability can induce significant variability in results. Future studies could therefore utilize genetic screening techniques in a personalized medicine approach to understand the genetic predisposition of an individual to metabolize phytoestrogens. The completion of the Cancer

Genome Atlas – a collaborative venture of The National Cancer Institute (NCI) and National

Human Genome Research Institute (NHGRI) is sure to help better under the molecular signatures of various cancer subtypes. This in turn could be helpful in understanding how the intake of phytoestrogen supplements would affect breast cancer survivors with specific subtypes such as triple negative breast cancer (Tomczak et al., 2015).

The use of phytoestrogen-based dietary supplements in the United States has gained great popularity in the past two decades since the passage of the Dietary Supplement and Education

Act (DSHEA) of 1994, although many of their molecular targets and specificity remains under investigation. Under DSHEA, botanical supplements, unlike drugs, are not required to demonstrate efficacy before being brought onto the marketplace. Some products include health claims to describe a relationship between a dietary supplement and a disease, but such claims are not required. Manufacturers are required to clearly state in a disclaimer, the weakness in scientific evidence (if any), such as mixed research study results or lack of clinical information.

Disclaimers that state whether or not the FDA approves the claim made by a manufacturer with regards to the health benefits of consuming a product/supplement must also to be mentioned in the product label (Schneeman 2011). Due to the 1 DSHEA, the FDA’s ability to regulate botanical supplements is restricted.

Therefore, consumers should be aware of these issues when selecting a phytoestrogen-based supplement. Whether consumption of phytoestrogen-based supplements is purely beneficial is still debatable. So consumers should discuss their individual risk factors such as family history of breast cancers with their physicians prior to consumption. Overall, the existing evidence on phytoestrogens is encouraging as they appear to be safe when consumed as dietary components even in breast cancer survivors. However, there is not enough clinical evidence at this point of time to support the use of concentrated or purified phytoestrogens in clinical doses as chemo preventive agents. Due to their widespread presence in the human diet, in both conventional foods and dietary supplement products, phytoestrogens remain an important area of study, especially for women’s health.

2.2. Breast cancer pathways modulated by compounds from Garcinia

Breast cancer continues to be the second leading cause of death in women (Pan et al.,

2015). About 80% of the drugs approved by the FDA for treatment of cancer, within the last three decades are directly sourced from plants (Bishayee and Sethi 2016). Natural compounds commonly have the ability to modulate multiple oncogenic drivers and pathways which makes them ideal for use by themselves or in synergy with existing chemotherapeutic drugs

(Shanmugam et al., 2011). Some of the well-known drugs obtained from natural sources are:

Anthracyclines: Doxorubicin, epirubicin and donorubicin. Liposome encapsulated forms of doxorubicin help overcome the issue of cardiotoxicity (Karikas 2010). Similarly Paclitaxel isolated from the bark of Taxus brevifolia is continued to be used in the treatment of metastatic breast cancer, although it was originally approved for treatment of ovarian cancer (Altmann and

Gertsch 2007). Vinorelbine, a semi synthetic tubulin binding alkaloid that is used in the treatment of advanced breast cancer (Jordan and Wilson 2004). Vinca alkaloids are known for their ability to stabilize microtubules thus destroying cancer cells. While the recent developments in cancer treatment are focused on more patient centric approaches such as immunotherapy, the use of natural compounds in cancer management is still an important area of study. Some pathways investigated as targets to treat breast cancer are:

1. Apoptosis

2. Invasion and Metastasis

3. Angiogenesis

5. Cell proliferation

6. Self-sufficiency in growth signals

Compounds from Garcinia that have been studied and reported under each of the above- mentioned pathways in breast cancer cell lines are discussed here:

Apoptosis

Apoptosis or “programmed cell death” involves execution of damaged or unwanted cells by the human body, primarily with the use of enzymes known as caspases (Kritsanawong et al., 2016).

The two major pathways of apoptosis are: Intrinsic pathway and Extrinsic pathway. The extrinsic pathway involves activation of initiator caspases-8 mediated through the activation of ligands such as tumor necrosis factor-α (TNF-α), TNF-related apoptosis inducing ligand (TRAIL) and cluster of differentiation 95 ligand (CD95L/Fas ligand (FasL)(Mellier et al., 2010). The intrinsic pathway on the other hand begins with DNA damage and depolarization of mitochondrial membrane, involving Bcl2 proteins, cytochrome C and finally the activation of caspases 3 or 9

(Kim 2005).

Camptothecin, etoposide, paclitaxel and vinblastine are some of the plant derived chemotherapeutic drugs whose mechanism of action is apoptosis (Feng et al., 2014). Plant derived polyphenols have been known to induce endoplasmic reticulum related stress contributing to apoptosis. Being a rich source of polyphenols, several members of Garcinia species have been shown to induce activation of caspases thus initiating the mechanism of apoptosis (Acuña UM et al., 2009). α mangostin, a xanthone that occurs in a few Garcinia species like Garcinia mangostana has been reported to induce apoptosis by the activation of caspases 3 and 9 by modulating HER2/PI3K,Akt and ERK1/2 (Kritsanawong et al. 2016).

Gambogic acid and guttiferone K are other compounds in Garcinia species that are known to induce apoptosis by activation of caspases 3,8 and 9 (Shi et al., 2013);(Kan et al. 2013). In

40 addition, oblongifolins F, G and xanthones from Garcinia oblongifolia have been reported to initiate apoptosis by activation of caspase 3 (Feng et al. 2014). Triterpenoids from Garcinia celebica have also reported to induce apoptosis by increasing the expression of PARP and inhibiting Akt (Subarnas et al., 2016). Cambogin, gambogenic acid, neogambogic acid and gambogic acid are other polyphenols from Garcinia have also shown to induce apoptosis by inhibition of STAT-3 phosphorylation, increased expression of caspases 3, 8 and 9 and increased expression of Bax/Bcl2 (Prasad et al., 2011; Wang et al., 2011; Zhou et al., 2013). Literature points out to the apoptosis inducing potential of Garcinia polyphenols including benzophenones

(Protiva et al. 2008).Therefore, the possibility of caspase mediated apoptosis in TNBC cells by the novel benzophenones was considered to be important. The approach and results are discussed in detail in Chapter 3.

Invasion and metastasis

About 50% of women diagnosed with breast cancer are likely to develop metastasis (Chun and

Kim 2013). The leading cause of deaths due to cancer are in fact due to metastasis and not from the primary tumor itself (Melzer et al., 2017).The term metastasis refers to the process by which tumor cells detach from the primary site, migrate to one or more distant sites an form secondary tumors (Tsai and Yang 2013). Metastasis involves the following series of events: i) Detachment of cells from the primary tumor involving epithelial mesenchymal transition (EMT) ii) Migration of tumor cells into the blood or lymphatic system mediated by matrix metalloproteinases and iii)

Interaction of tumor cells with target organs such as lungs, liver and bones (Chun and Kim

2013). Often patients who present metastatic disease are incurable. Therefore, there is a need for

41 dietary agents which by themselves or in synergy with chemotherapeutics are capable of suppressing metastasis (Doi et al., 2009). Garcinol in synergy with Taxol has shown to inhibit metastasis by modulating epithelial mesenchymal transition (EMT) in mouse mammary carcinoma 4T1 cells (Tu et al., 2017). In another study α mangostin inhibited metastasis of 12-O- tetradecanoylphorbol-13-acetate (TPA) induced matrix metalloproteinase-2 and 9 in MCF7 cells

(Lee et al., 2010). In addition, administration of Panaxanthone (which is a combination of 78% α mangostin and 16%γ mangostin) inhibited metastasis in a mouse mammary cancer model via inhibition of proliferating cell nuclear antigen (PCNA) (Doi et al. 2009). Similarly, garcinol reversed epithelial-to-mesenchymal transition in MDAM-MB-231 and BT-549 cells. The mechanism of action was found to be through upregulation of E cadherin and down regulation of markers vimentin, ZEB-1 and ZEB-2 (Ahmad et al. 2012b). As evident from the above- mentioned studies, garcinol is the only Garcinia derived polyphenol that has been explored for its antimigratory effects in breast cancer cells. The scarce literature available about this aspect of

Garcinia benzophenones, makes it worth investigating

Angiogenesis

Angiogenesis is the formation of new blood vessels from preexisting micro vessels (Pandya et al., 2006). Studies on the anti-angiogenic activity of compounds from Garcinia on breast cancer cells are limited. Although the total number of studies reported on the anti-angiogenic activity of

Garcinia compounds is 25. In addition to these studies, use of Garcinia compounds in anti- psoriatic treatments and other carcinomas have been reported (Li et al. 2013). But only two studies reported the use of purified Garcinia compounds on breast cancer cell lines. In one of these studies, garcinol in combination with Taxol induced cell cycle arrest (G2/M) and caused inhibition of caspase-3/cytosolic Ca2+-independent phospholipase A2 (iPLA2) and nuclear 42 factor-κB (NF-κB)/Twist-related protein 1 (Twist1) which in turn inhibited angiogenesis in a mouse 4T1 breast model (Tu et al. 2017). In another study, α mangostin caused a significant decrease in Akt phosphorylation in mammary tumors carrying a p53 mutation. Consequently, α mangostin inhibited angiogenesis among other key cell survival pathways (Shibata et al., 2011)

Anti-proliferative action

Gambogic acid is a known inhibitor of signal transducer and activator of transcription 3 (STAT-

3) signaling, in addition to its targets such as vimentin, keratin 18 and calumenin in MDA-MB-

231 cells, thereby causing a decrease in cell proliferation in a dose dependent manner (Sulaiman et al., 2016);(Li et al., 2015). Phosphorylated STAT-3 (p - STAT3) – the active form of STAT-3 is often elevated in cancers, thus promoting cell proliferation and tumorigenesis by promoting resistance to apoptosis (Page et al., 2011). In addition, the expression of STAT-3 is transient in normal tissues (Furtek et al., 2016). Therefore, STAT-3 inhibitors could be used to selectively target tumor cells. Garcinol is the only benzophenone from Garcinia that has been reported to inhibit STAT-3 (Ahmad et al. 2012a). Therefore, study of small molecule STAT-3 inhibitors from Garcinia could be helpful.

Mitogen activated protein kinase (MAPK) signaling pathway is involved in cell proliferation, differentiation and motility and is often mutated in human cancers (Rauch et al., 2016). This makes them one of the well-studied targets for the development of anti-cancer drugs (Caunt et al., 2015). Inhibition of cell proliferation via modulation of HER2/MAPK signaling by α mangostin has also been reported in T47D (Human Breast Carcinoma) cells (Kritsanawong et al.

2016). Panaxanthone, a xanthone from the pericarp of Garcinia mangostana reported for the first time, showed significant anti-proliferative activity on a mouse metastatic mammary cancer

43 model via inhibition of proliferating cell nuclear antigen PCNA (Doi et al. 2009). Fruit extracts of Garcinia mangostana also induced antiproliferative effects on SKBR-3 cells (Moongkarndi et al., 2004). Friedolanostane triterpenoid from the leaves from Garcinia subeliptica has been reported for its dose dependent antiproliferative effects on MCF-7 cells (Subarnas et al. 2016).

Two prenylated xanthones from the branch extracts of Garcinia achachairu have also been reported for their anti-proliferative and cytotoxic effects on MCF-7 cells (Mariano et al., 2016).

While these studies focus on anti-proliferative effects of compounds derived from Garcinia on several breast cancer cells, none of them specifically studied anti-proliferative pathways and associated markers only in triple negative cell lines. Other studies in which anti-proliferative effects were attributed to cell cycle arrest and apoptosis are discussed in the appropriate sections.

Modulation of protein transport and cell cycle arrest

Cyclin dependent kinase (CDK) 4/6 inhibitors have most recently gained attention as selective drugs to induce G1 phase cell cycle arrest and cause inhibition of tumor progression (de Groot et al., 2017). Interestingly, several compounds isolated from Garcinia have shown to be successful inhibitors of cell cycle proteins such as the cyclins. These studies are discussed in this section.

Gambogic acid which is currently in clinical trials in China has been identified as an inhibitor of nucleocytoplasmic transport of proteins by regulating nucelophosphin and Nup88. This results in apoptosis and cell cycle arrest in HL60, Jurkat cells an U937 cells (Shu et al., 2008). Increased

G1 phase and a reduction in S phase population was observed in mouse mammary carcinoma

BJMC3879luc2 cells treated with α mangostin (Shibata et al. 2011). The xanthone isobractin from Garcinia bractea has been shown to induce G0/G1 phase cell cycle arrest via reduced expression of cyclins D1 and E (Shen et al., 2014). One of the well-known xanthones α mangostin was shown to cause G1 phase arrest and a reduction in the S phase in MDA-MB-231 44 cells. Increased expression of p21cip1 and CHEK2 lead to reduced expression of CDKs and cyclins (Kurose et al., 2012). Inhibition of G0/G1 phase of the cell cycle in MCF7 cells has also been reported on treatment with neogambogic acid (Wang et al. 2011). A G1 phase arrest and S phase suppression of the cell cycle were observed in a mouse mammary metastatic cancer model upon treatment with α mangostin (Shibata et al. 2011). Panaxanthone isolated form the pericarp of Garcinia mangostana caused arrest of G1 phase accompanied by significant decreases in cell population in the S and G2 phase (Doi et al. 2009). Stabilization of microtubules is an established mechanism of action of Taxol, which is currently used in the treatment of cancer.

Gambogic acid, induced cell cycle arrest in the G2/M phase in MCF-7 cells by stabilization of microtubules (Chen et al., 2008). As evident from the studies listed above, most of the Garcinia benzophenones and even xanthones have been able to cause a G1 phase arrest and a decrease in

M phase of the cell cycle. Often the G1 phase accumulation is influenced by the inhibition of cyclins. The catechol and enol functional groups and the presence of prenyl groups have been associated with the activity on tubulin and microtubules which could explain the decrease in M phase of the cell cycle (Wu et al. 2014). Given the lack of the three common receptors ER, PR and HER2 in TNBC, the use of a compound that selectively targets cell cycle progression in tumor cells could be of interest.

As mentioned above, there are multiple pathways related to cancer, that compounds from

Garcinia can modulate. Since the scope of this study was limited to TNBC, pathways that are relevant to the etiology and progression of TNBC were chosen for detailed analysis.

2.3 Triple negative breast cancer – Introduction

TNBC (Triple negative breast cancer) is characterized by the lack of 3 cell surface receptors: Estrogen receptor (ER), Progesterone receptor (PgR) and Human epidermal growth factor receptor (HER2). About 15 to 20% of all diagnosed breast cancers are triple negative and are prevalent in younger women, especially of African American or Hispanic origi n (Carey et al., 2010). The lack of expression of the 3 common receptors (ER, PgR and HER2) which are also the known molecular targets, leaves the only treatment option for TNBC to be conventional chemotherapy (Bianchini et al., 2016). Although patients with a mutation in BRAC1 often are diagnosed as triple negative, all triple negative breast cancers are not BRCA1 positive (Bulfoni et al., 2016; Massihnia et al., 2016). Recent studies such as the Cancer Genome Atlas, have revealed more information that has led to subcategorization within the broad umbrella of triple negative breast cancer (Herold and Anders 2013). Basal like subtypes BL1 and BL2,

Immunomodulatory subtype, mesenchymal subtype, luminal androgen receptor subtype are the categories reported, based on gene expression profiling (Prat and Perou 2011). The heterogenicity of these cancers makes chemotherapy the only treatment option. Therefore development of more targeted approaches to treat TNBC is a topic that the research community is focused on at the moment.

Molecular profiling studies have revealed that the common genetic alterations found in

TNBC are loss of tumor protein TP53, activation of PI3K pathways, alterations in DNA damage repair genes and loss of RB1 and BRCA1 (Oualla et al., 2017). Now that these pathways are known and studied in TNBC, targeting these pathways could help in stopping the progression of

TNBC. To this extent, platinum salts and poly ADP-ribose polymerase (PARP) inhibitors, use of monoclonal antibodies to inhibit growth factors such as EGFR, VEGFR and FGFR are currently

46 being explored for use in treatment against TNBC (Corkery et al., 2009). Other molecular targets that are being explored for the development of targeted therapeutics are JAK2/STAT3 pathway, inhibition of notch signaling and blockage of Androgen Receptor (AR) (Cancer Genome Atlas

2012).

Several natural compounds have been explored for their role in treatment of TNBC by themselves or in an adjuvant setting. Experimental results reported within the last seven years, that discuss the specific pathways modulated by natural compounds against triple negative breast cancer are discussed below.

A sesquiterpene lactone analog DETD-35 obtained from Elephantopus scaber L, a member of traditional Chinese medicine induced significant oxidative stress followed by ER stress mediated apoptosis or paraptosis like cell death in an MDAMB-231 xenograft mouse model (Shiau et al., 2017). This study is particularly interesting as DETD-35 proved to be a promising agent for the treatment of apoptosis resistant cancer and TNBC. In another study, manuka honey showed specific antitumor effects against MDA-MB231 and MCF-7 while sparing normal breast epithelial cells MCF-10A (Aryappalli et al., 2017). The mechanism of action was via activation of caspases 8,9,6,3 and 7. A significant reduction in tyrosine phosphorylated STAT-3 was also observed in both the cell lines studied. This further coincided with a decrease in production of interleukin-6 (IL-6). STAT-3 pathway has been known for its role in oncogenesis of breast cancer. Also, MDA-MB-231 cells express constitutively activated

STAT-3 (Banerjee and Resat 2016). The cancer specific activities of manuka honey in this study were attributed to its flavonoids and phenolic constituents. Several molecules have been screened for cytotoxic effects in vitro on TNBC cells (Bao et al., 2017; Dziwornu et al., 2017). An indolin-2-core novel molecule was reported to be a potent inhibitor of TNBC cells (Zhou et al., 47

2017). Curcumin the polyphenol that has been well studied for its ability to modulate multiple signaling pathways has also been explored for its therapeutic potential against TNBC. To this extent two analogs of curcumin have been reported for the first time in selectively inducing ROS mediated apoptosis in a TNBC xenograft model on intraperitoneal administration (Pignanelli et al., 2017).

Some studies also report the use of plant extract and one or more pure compounds for their activity against TNBC. Leaf extracts of Cynara cardunculus and its active ingredient cynaropicrin caused upregulation of p21Waf1/Cip1 , G2 phase cell cycle arrest via modulation of cyclin B and CDK1 as well as downregulation of phospho(Ser 473)-Akt in MDA-MB-231

(Ramos et al., 2016). Ethanol extract of propolis and caffeic acid phenethyl ester exhibited significant cytotoxicity on MDA-MB-231 and Hs578T cells (Rzepecka-Stojko et al., 2015) .

Piperine an alkaloid isolated from Piper nigrum and Piper longum has shown TRAIL induced cytotoxicity in TRAIL sensitive and resistant TNBC (Abdelhamed et al., 2014). Microtubule targeting agents like the current chemotherapeutic drugs such as paclitaxel, vinblastine and vincristine are also being explored for their use as TNBC therapeutics. In a recent study, acridones were studied on MDAM-MB-231 cells and identified as being able to cause cell cycle arrest in G2/M phase in addition to inducing apoptosis and disruption of microtubules by binding to the colchicine binding site of tubulin (Magalhaes et al., 2016). Basal like TNBCs have been known for their sensitivity to DNA damaging agents relative to other TNBC subtypes (Lehmann et al., 2011). Maximiscin displayed selective cytotoxicity against MDA-MB-468 in comparison with other TNBC cell types. In addition to being cytotoxic, maximiscin caused an accumulation of G1 phase in cell cycle followed by phosphorylation of checkpoint kinases 1 and 2 (CHK1 and

2) which are involved in DNA damage check points. In addition to its in-vitro effects,

48 maximiscin also inhibited growth of MDA-MB-468 xenografts. Defective DNA repair systems in addition to higher basal levels of DNA and high proliferation rates were discussed as causes of increased BL1 sensitivity to DNA damaging agents (Robles et al., 2016). Similarly, a compound reported for the first time - Zj6413 in synergy with chemotherapeutics showed synergistic antitumor effects in vitro and in vivo on MDA-MB-453 cells via inhibition of Poly(ADP-ribose) polymerases (Zhou et al., 2016). In addition to mechanisms that target DNA damage, compounds derived from plants have also been studied for their anti-migratory effects against

TNBC. A piper amide like compound (NED-135) caused significant inhibition of matrix metalloproteinase (MMP)-9 in MDA-MB-231cells (Kim et al., 2015). In addition, NED-135 also inhibited activation of signaling molecules Akt, ERKs and p38 MAPK in sphingosine-1- phosphate (SP1) induced MCF10A cells (Kim et al., 2011). Similarly, diallyl trisulfide (DATS) isolated from garlic inhibited the protein and enzyme activities of MMP2/9 via downregulation of NFĸB and MAPK pathways in MDA-MB-231 cells (Liu et al., 2015). In another study, a compound 3,4 methyenedioxy-β-nitrostyrene, exhibited strong anti-metastatic effect on TNBC cells without affecting cell viability. The underlying mechanism was reported to be inhibition of

Focal adhesion kinase (FAK) phosphorylation as a result of reducing β1 integrin activation

(Chen et al., 2015a).

The number of studies that report compounds isolated form Garcinia for their activity on

TNBC cells are limited. These studies have already been discussed in Chapter 2.1. The closest structural relative of the benzophenones that are discussed in this research is garcinol. Although the studies on garcinol’s anti-cancer effects on TNBC cells are listed in Chapter 1, important studies that served as a guide for my research will be discussed in this section. Garcinol is known to induce apoptosis visa down regulation of NF-ĸB. In addition, it has also been reported to

49 reverse epithelial-mesenchymal transition in MDA-MB-231 and BT-549 breast cancer cells

(Ahmad et al. 2012b; Tu et al. 2017).

Triple negative breast cancer cell lines used in my research were: MDA-MB-231 and

MDA-MB-468. MDA-MB-231 is classified as basal type 2 with mesenchymal features and vimentin expression. Whereas MDA-MB-468 is epithelial basal type 1 with keratin 5 expression.

Further, 231 has a wild type PI3K pathway and 468 has a PTEN homo deletion and amplified

EGFR (Chavez et al., 2010). Other characteristics of the basal subtype are : loss of TP53,RB1,

BRCA 1, MYC amplification and activation of phosphatidyl inositol 3 kinase (PI3K) (Herold and Anders 2013). Although the availability of limited quantities of the novel compound paucinone H only enabled studying its effects on MDA-MB-231 cells, results of a cell viability study showed that the IC50 ranges of paucinone H on both MDA-MB-468 and MDA-MB-231 varied from 19 to 25 µM. The study and its results are discussed in detail in Chapter 3.

Chapter 3: Molecular mechanisms targeted by benzophenones from Garcinia paucinervis against breast cancer cell lines with varying receptor status

Five novel compounds: paucinone E (1), paucinone F (2), paucinone G (3), paucinone H (4), and paucinone I (5) were tested on a panel of breast cancer cell lines: MDA-MB-231, MCF-7 and

SKBR-3. Among the compounds that were screened, paucinone H exhibited significant antiproliferative, apoptotic and anti-migratory effects against MDA-MB-231. IC50 values of paucinone H ranged from 10 to 19 µM across the panel of cell lines. At half the IC50 concentration (10 µM), paucinone H induced significant anti-migratory effects and a reduction in mitosis. Further analysis of paucinone H could help understand the potential application of paucinone H by itself or in synergy with other therapeutic agents against TNBC.

Garcinia species have been used in traditional Chinese and Ayurvedic preparations for their wide range of biological activities (Hemshekhar et al. 2011; Kumar et al. 2013). Compounds from Garcinia have been studied for their ability to modulate multiple signaling pathways ranging from caspase dependent/independent apoptosis to MEK pathway (Protiva et al. 2008;

Einbond et al. 2013). Especially, the cytotoxicity of selected xanthones from Garcinia spp. have been reported in breast cancer cell lines (Kritsanawong et al. 2016; Mariano et al. 2016; Sun et al., 2016; Mohamed et al., 2017; Youn et al., 2017). However, another class of polyphenols known as benzophenones that have a unique structure and diversity have been least explored, particularly in triple negative breast cancer cells (Wu et al. 2014). Only a total of two studies have explored the use of benzophenone garcinol against triple negative breast cancer. One of them analyzed the synergistic effects of existing chemotherapeutic agent Taxol (Tu et al. 2017).

This study revealed that low doses of Taxol in combination, with garcinol in a metastatic mouse mammary carcinoma model could downregulate key cell survival events such as cell cycle, angiogenesis and metastasis. In another study, garcinol was found to reverse epithelial- mesenchymal transition in MDA-MB-231 cells (Ahmad et al. 2012b). The possible mechanism

52 of action was suggested to be upregulation of E-cadherin and downregulation of vimentin, ZEB-

1 and ZEB-2. Recently we reported the identification of five novel benzophenones from

Garcinia paucinervis and their cytotoxicity against a panel of 3 breast cancer cell lines with varying receptor statuses: MDA-MB-231 ER-,PR-,HER2-; SKBR-3 HER2+ and MCF-7 ER+

(Li et al. 2016a). During our initial studies, we identified that one of these benzophenones was active against MDAM-MB-231. This was the rationale behind our interest to delve deeper into the mechanism of action of these compounds

3.1 Materials and Methods

Plant material and extraction

Garcinia paucinervis seeds were collected from Xishuangbanna, Yunnan Province, in People’s

Republic of China, the specimens were identified by one of the authors Dr. Chun lin Long. The voucher specimens (YN20144538) were deposited in the Herbarium of Minzu University of

China in Beijing, People’s Republic of China. Air dried Garcinia paucinervis seeds were the starting material for the extraction process. Powdered seeds were extracted twice with 2L of 95% ethanol and allowed to soak for two days. This solution was then filtered and concentrated under a reduced pressure of 40◦C from which 320 g of crude extract was obtained. The extract was then dissolved in 800 ml of water. After a solvent-solvent portioning with petroleum ether, 140g of the upper phase was removed. The residue (120 g) was then purified by silica gel column chromatography (120x10 cm) and eluted with a solvent system of petroleum ether and acetone

(100:0 to 0:100 and 800 ml of each fraction was collected and concentrated, to yield 9 combined fractions as follows: Fr. A-Fr. I,0.5,2.2,2.1,1.5,2.0,7.8,13.2,5.1, and 6.0 g respectively). Each fraction was later analyzed by UPLC-QTOFMS before commencing preparative-scale isolation.

Isolation of Paucinone H from Garcinia paucinervis seed fraction

Additional amounts of paucinone H was isolated based on need for use in bioassays. Since collection of Garcinia paucinervis fruits was not feasible, a previously isolated fraction of

Garcinia paucinervis seed was used to isolate additional amounts of paucinone H needed for bioassay studies. The scheme of isolation is shown in Figure 9.

Yield: 332.5 mg Fraction = 0.3 mg paucinone H

Figure 9: Isolation of paucinone H from Garcinia paucinervis seed Fraction D

Briefly, 332.5 mg of fraction D was fractionated over Sephadex LH-20. A total of 40 fractions were obtained and scanned for paucinone H as reported (Li et al. 2016a). Fractions 15,

20, 24, 29 and 35 were scanned for paucinone H using a UPLC-QTOF Acquity UPLC system

(Waters Corporation, Milford, MA, USA) coupled with QTOF-MS (Xevo G2 QTOF, Waters MS

Technologies, Manchester, UK). The fractions are shown in Figure 10. Out of the scanned fractions 24, 29 and 35 showed presence of paucinone H as marked in Figure 10. These selected fractions were then recombined and dried down to be weighed and used in preparative scale isolation of paucinone H. The purity of paucinone H isolated by this method was estimated to be

≤ 98%. The isolated compound was then sealed in a vial and stored at -4◦C until use in bio assays. The UPLC -QTOFMS profile of the isolated compound is shown in Figure 11.

Figure 10: UPLC-QTOFMS profile of Garcinia paucinervis seed fractions obtained from

Fraction D.

The forty fractions obtained by Sephadex separation were scanned for paucinone H. Shown in the figure above are fractions 24, 29 and 35 with their total ion content and the chromatograms extracted for paucinone H. Fractions 15 and 20 did not have paucinone H

Figure 11: UPLC-QTOFMS profile of seed fractions showing paucinone H. a, b and c denote

fractions 35, 24 and 29 respectively. Paucinone H is marked with an arrow.

Figure 12: UPLC-QTOFMS profile of paucinone H after isolation.

A stock solution of 2 mM concentration was prepared with DMSO, for use in bioassays.

This stock solution would then be diluted to the appropriate concentration required for each experiment. Care was taken to ensure that the concentration of DMSO never exceed 0.01% in the treatment wells. This stock solution was sealed in a vial and stored at -4◦C. The time frame between isolation of the compound and use in bioassays was approximately 4 months.

Cell culture

MDA-MB-231 and MCF-7 cells were purchased from American Type Culture Collection

(Rockville, MD) and cultured in DMEM glucose medium (Gilbco, USA) supplemented with

10% fetal calf serum and 100 µL of antibiotics (penicillin and streptomycin). SKBR-3 cells were cultured in DMEM F12 glutamine medium supplemented with 10% fetal calf serum and 100 µL of antibiotics (penicillin and streptomycin). All cell lines were maintained in an incubator at 37

°C with 5% CO2.

Growth inhibitory effects on a panel of breast cancer cells

All compounds tested in vitro were dissolved in DMSO and diluted with required volumes of respective cell culture media before each treatment. To determine the cytotoxic effects of test compounds on the three cell lines used in this study, exponentially growing cells (12 × 103 cells/well) were seeded in 96 well multitier plates. After 24 h, each test compound was added to each cell line used in his study. Concentrations of the compounds studied ranged from 5 to 200

µM. Doxorubicin was used as a positive control. After 48 hours of treatment, a cell viability assay (WST) was performed. Each well was washed twice with serum-free medium followed by the addition of a 150 µL of 10% WST solution. The plates were wrapped in aluminum foil and 58 incubated for 1 h, their absorbance was then read at 440 nm using a Spectra Max plate reader.

Each experiment was performed in triplicate. Average absorbance and the percentage inhibition relative to control was calculated. Results were analyzed using Graph Pad Prism (v 6.01) (Graph

Pad Software, San Diego, CA, USA), and IC50 values were estimated.

Apoptosis assay to identify increase in apoptotic cells after treatment with paucinone H

Propium Iodide was used to identify apoptotic cells. MDA-MB-231 cells were plated at a density of 12 × 103 cells/well in a 96 well plate. Each well was subjected to treatment with Control, paucinone H 5 and 20 µM respectively 24 hours after plating. After a treatment time of 48 hours, 5 µL of PI buffer was added to the wells. Each well was then imaged three times to capture fluorescence. Average fluorescence relative to control was then used to calculate percentage of apoptotic cells.

Immunostaining for mitotic marker phospho-histone 3

MDA-MB-231 cells were seeded at a density of 12 × 103 cells/well in chamber slides and allowed to form a monolayer. After seeding, paucinone H was added to the wells at a concentration of 10 µM. 24 hours after treatment, cell culture medium was removed, and cells were fixed with 4% Paraformaldehyde (PFA) followed by incubation with a blocking buffer comprising of 2% BSA in PBS. The primary antibody Phosphohistone (SER 28) (Cell signaling;

1:200) was then added to the set up and allowed to incubate overnight in a wet chamber at 4 °C.

DAPI was used to counterstain DNA. The secondary antibody used in this procedure was Alexa

Fluor® 488 anti-rabbit IgG (Cell signaling; 1:1000). Images were captured using a Zeiss live cell fluorescent imaging system. Each treatment well was imaged 3 times and percentage mitotic

59 cells were calculated relative to control. Statistical analysis was done using Graph Pad Prism (v

6.01) (Graph Pad Software, San Diego, CA, USA)

Wound healing assay

MDA-MB-231 cells were allowed to form a confluent monolayer 24 hour prior to start of the experiment. A 20-200 µL pipette tip was then used to create a scratch ˜ 1mm in width. The wells were gently washed twice with PBS and replenished with specific concentrations of paucinone H in FCS free media. Wound closure was monitored over 48 hours and images were captured using a live cell imager (Magnification=10X). Wound closure relative to control was then calculated using the measure and analyze tools in Graph Pad Prism (v 6.01). Results were verified in two individual experiments with duplicates.

Statistical analysis

All data presented here are from at least 2 independent experiments done in triplicate. Statistical analysis was performed using Graph Pad Prism (v 6.01). Statistically significant differences between the control and treatment groups were calculated by Student’s t test except for the increase in apoptotic cells in which case ANOVA was used. P values < 0.05 were statistically significant.

3.2 Results

Growth inhibitory effects of paucinones on a panel of breast cancer cell lines with varying receptor statuses

The five isolated compounds (1-5) were evaluated for cytotoxicity against a panel of human breast cancer cells including MDA-MB-231, SKBR-3 and MCF-7 using a WST assay (Table 1).

Cell lines for this study were chosen based on their varying receptor status i.e. MDA-MB-231 are ER-, PR-, HER2- and SKBR-3 is HER2+ while MCF-7 is ER+. Cells were treated with test compounds for 48 hours. Compounds (2), (3) and (4), had IC50 values <50 µM on the panel of cell lines used. Interestingly, paucinone H (4), was the most active among the panel of compounds tested (IC50 values ranging from 10 to 19 µM). Doxorubicin was used as a positive control (IC50 < 3µM) in our experiments. Compounds (1) and (5) required concentrations of more than 50 µM to elicit cytotoxicity on MCF-7 and MDA-MB-231

Paucinone H (4) was found to be actively inducing cell death in MDA-MB-231 at 20 µM concentration, 48 hours after treatment when compared with untreated control cells (Fig 2). We also noticed that paucinone H (4) had cytotoxic effects on MCF-7.

Table 4: Growth inhibitory effects of novel benzophenones from Garcinia paucinervis. Each of the cell lines under study were treated with novel benzophenones at concentrations: 5,10, 20, 30, 40, 50, 100 and 200 µM respectively for 48 hours. A WST assay was performed to measure cell viability. Results were analyzed using Graph Pad Prism (v 6.01) and found to be statistically significant (P ≤ 0.05)

Figure 13: Growth inhibitory effects of paucinone H on MDA-MB-231. Images a, b, c, d, e and f denote control, 5, 10, 20, 40 and 100 µM concentrations of paucinone H, 48 hours after treatment.

Magnification: 10X. Scale bar = 10µm

Paucinone G (3) also showed some capacity to induce cell death. But none of the other paucinones tested were as effective as paucinone H on the panel of cell lines. Interestingly we noticed that paucinone H at IC50 concentrations caused apoptotic bodies characterized by nuclear chromatin condensation and cellular shrinkage as shown in Figure 14. Condensation of nuclear chromatin, membrane blebbing and cellular shrinkage have been reported to be hallmarks of apoptosis (Vermes et al., 2000)

Figure 14: Growth inhibitory effects of paucinone H on MDA-MB-231, SKBR-3 and MCF-7. Paucinone H was the most potent among the novel benzophenones isolated from Garcinia paucinervis. Images a, b and c show cytotoxicity of paucinone H on MDA-MB-231, SKBR-3 and

MCF -7 48 hours after treatment with 20 µM paucinone H. Red arrows indicate dead cells. Magnification: 10X. Scale bar = 10µm

Paucinone H causes an increase in apoptosis in MDA-MB-231 cells

Since our cytotoxicity studies revealed that paucinone H was effective in inducing cell death in

MDA-MB-231 cells at IC50 concentrations and MDA-MB-231 is a basal subtype of triple negative breast cancer, we wanted to understand if the mechanism of action involves apoptosis.

The use of Propium iodide to access cell death has been reported earlier (Vermes et al. 2000). A simple apoptosis assay using Propium iodide was used to identify increase in apoptotic cells after treatment with paucinone H (4) at IC50 concentration. To show a dose responsive increase in apoptosis, cells were also treated with concentrations 5 and 20 µM and compared against the control. The fluorescence emitted by Propium iodide was captured using a live cell imager with fluorescent filter. Each treatment well was imaged thrice and the percentage increase in apoptotic cells was calculated. A dose dependent increase in apoptosis was observed and the percentage of cells undergoing apoptosis increased by 50% after treatment with 20 µM paucinone H (4) in comparison with the control (Figure 14). All the hallmarks of apoptosis such as membrane blebbing, cellular shrinkage and nuclear condensation were prominent at this concentration. This is particularly interesting since breast cancer cells such as MDA-MB-231 with basal characteristics have been shown in the past to be responsive to drugs that cause DNA damage

(Kalimutho et al., 2015)

Figure 15: Increase in the percentage of dead cells, 48 hours after treatment with paucinone H on MDA-MB-231. Images a, b and c represent Control, paucinone H 5 and 20 µM respectively are shown here. Results shown here are from three images per well per concentration. Average fluorescence relative to control was used to calculate percentage of apoptotic cells.

Magnification: 10X. Scale bar = 10 µm. Statistical significance: P ≤ 0.01

Decrease in the expression of mitotic marker phospho-histone 3 in MDA-MB-231 cells after treatment with paucinone H

Mitotic activity is a key factor that is used in histological grading of breast carcinomas (Elston and Ellis 1991; Veras et al., 2009; Cui et al., 2015). One of the major constituents of chromatin is

Histone H3. Phosphorylation of Histone H3 marks the initiation of mitosis (Hendzel et al., 1997;

Perez-Cadahia et al., 2009). The use of phospho-histone 3(PH3) as a prognostic marker for

66 proliferation in triple negative breast cancer has been discussed earlier (Skaland et al., 2009;

Klintman et al., 2013).

Figure 16: Paucinone H induces a reduction in mitotic marker phospho-histone 3 at half IC50 concentration within 24 hours after treatment on MDA-MB-231 cells. Images a and b show control and paucinone H 10 µM respectively after immunostaining for mitotic marker phospho- histone 3. Blue nuclei denote DAPI. Magnification =10X. Results shown here are representative of two independent experiments with triplicate. Statistical significance: P ≤ 0.05

The prognostic value of phospho-histone 3(PH3) and the underlying link to prognosis in patients with a low PH3 index has also been reported (Skaland et al., 2007). In this context, we analyzed the expression of phospho-histone 3 in MDA-MB-231 cells after treatment with paucinone H at

10 µM concentration. A significant reduction (50%) in the percentage of mitotic cells was observed within 24 hours after treatment (Figure 16). The experiment was repeated twice with triplicate and the percentage decrease in mitotic cells were estimated relative to control. Changes in PH3 has been proven to be useful in estimating mitotic activity in tumors, paucinone H could be a useful agent in inducing a decrease in mitotic activity even in solid tumors (Cui et al. 2015;

Ginter et al., 2016). Also, the expression levels of PH3 helps in the evaluation of mitotic inhibitors in early stage drug development (Sun et al., 2012)

Inhibition of migration in MDAM-MB231 cells post treatment with paucinone H

TNBC is known to be one of the most metastatic types. Recurrence could often be attributed to metastasis (Kalimutho et al. 2015). To test if paucinone H can arrest migration, a scratch assay

(wound healing assay) was performed. Wound closure was monitored for 48 hours and bright field images of the wound were captured. Each treatment had two replicates and the experiment was repeated twice. Migration of cells was calculated as the rate of wound closure relative to control. Wound width was normalized across all the wells to account for a wider scratch in the 10

µM treatment well. A significant reduction in migration was observed in cells treated with paucinone H at a concentration of 10 µM (Figure 16). Continued imaging up to 3 days showed that a completely closed wound in the control well as opposed to the treated well which showed significant anti-migratory effects.

Figure 17: Anti-migratory effects of paucinone H on MDA-MB-231 cells. Images were recorded

48 hours after treatment. Control and paucinone H 10 µM are shown here. White lines indicate the boundaries of the wound. Red arrows indicate cells (wound closure) in the control well.

Results presented here are from two individual experiments with duplicates for each treatment.

Statistical significance: P ≤ 0.01.

Bright field imaging was continued past 48 hours to find the time taken for the control well to close completely. At 96 hours, the control well closed completely whereas the well with 10 µM paucinone H did not.

3.3 Discussion

In this study, the activity of novel benzophenones isolated form Garcinia pacucinervis were presented for the first time across three different cell lines with varying receptor statuses. Since one of the compounds - paucinone H exhibited significant cytotoxicity on MDAM-MB-231, identifying the mechanism of action of Paucinone H was the next step. While were able to observe significant anti-proliferative and anti-migratory effects of paucinone H on MDA-MB-

231, further analysis into the downstream targets that elicit these responses would be the future direction for a more comprehensive study. Although benzophenones have been proven to be effective at modulating multiple anti-cancer pathways, their unique chemistry also comes with a challenging isolation process, since most of them are isomers. Previously, our lab has focused on the identification, isolation and purification of benzophenones from Garcinia xanthochymus

(Baggett et al. 2005). However, since the compounds reported in this study were isolated from an endangered medicinal species, sample collection for a massive isolation process was impossible.

Thus, compound availability was a limiting factor in our study. However, future studies involving this compound could utilize chemical synthesis methods which would help scaling up synthesis, that in turn would address the issue of compound unavailability.

Once this is accomplished, future studies could involve study of specific pathways that could be modulated by paucinone H in order to elicit anti-proliferative effects. STAT-3 could be a good pathway of choice given that the role of STAT-3 in cell migration has been reported previously in MDA-MB-231 (Xing et al., 2015). A recent study has reported the use of xanthones from

Garcinia xanthochymus in the inhibition of intracellular STAT-3 phosphorylation in MDA-MB-

231 cells. Given that benzophenones are chemically different from xanthones, this could be a particularly exciting avenue to focus, since a member of the benzophenone class was reported to 70 be potent against colon cancer cells that developed resistance to xanthones and other conventional antitumor agents (Kan et al. 2013). The second pathway worth exploring would be

PI3K/AKT/Mtor, due to its underlying link in regulating cell proliferation, motility and the fact that it is often over expressed in TNBC patients (Janku et al., 2012). While the inhibitory effects of structurally similar benzophenones on Mtor pathway has been reported in colon cancer cells, no such study exists on triple negative breast cancer and benzophenones (Einbond et al. 2013).

Given that the need for a targeted therapy to decrease mortality rates due to triple negative breast cancer is the need of the hour, this study would serve as a reference for researchers in understanding the use of benzophenones and their possible pathways of action in triple negative breast cancer. This in turn could lead to the use of paucinone H by itself or in synergy with existing chemotherapeutic drugs in the treatment of triple negative breast cancer.

Chapter 4: Conclusion

The increasing number of TNBC cases and the lack of targeted therapies, makes TNBC one of the major focus areas of the research community. Currently there are about 439 clinical studies at various stages, that explore the use of existing and new strategies to treat

TNBC(ClinicalTrials.gov 2017). Novel benzophenones from Garcinia paucinervis, especially paucinones G and H were effective against triple negative breast cancer (TNBC). Given the nature of TNBC and the need for a targeted agent against TNBC, future studies could utilize novel benzophenones presented in this study in the development of a targeted therapeutic agent.

The limited availability of the bioactive compound (paucinone H) is an issue that needs to be addressed before commencing future studies. Garcinia paucinervis has also been declared as an endangered species and been placed in the IUCN list of threatened species (Sun 1998). This poses a challenge for sample collection. In addition, paucinones were isolated from the seed extract of Garcinia paucinervis and paucinones H and G appeared as isomers. In an earlier study, the Kennelly lab reported that guttiferone E is more bioactive in than its isomer xanthochymol

(Protiva et al. 2008). Guttiferone E has a double bond between C32 and C33 whereas, xanthochymol has a double bond between C33 and C34 (Acuna et al. 2010). This could explain the reason for greater cytotoxicity and the corresponding anti-apoptotic activity of guttiferone E.

Similarly, in the present study paucinone H which lacks a hydroxy group was comparatively more bioactive than its isomer. One of the challenges of working with isomers is their separation from their structurally similar counterparts. Although separation was accomplished as mentioned by (Li et al. 2016a), the use of UPLC QTOF was inevitable in this process. Given these limitations, care was taken to design experiments which would help give a basic insight into the possible pathways that paucinone H is capable of modulating in TNBC. The cell line that was used in all these experiments was MDA-MB-231. In addition to inducing significant cell death at

20µM concentration, paucinone H was also able to cause significant decrease in expression of phospho-histone 3 (a mitotic marker) at half IC50 concentration. Paucinone H also caused significant inhibition of migration of cells in a scratch assay.

Chapter 5: Future studies

To understand the ability of paucinone H to induce cytotoxicity in other TNBC cell lines, MDA-

MB-468 was also used as part of the study. In contrast with MDA-MB-231 which has wild type

PI3K, MDA-MB-468 is PTEN negative and has high epidermal growth factor receptor (EGFR)

(She et al., 2008). This difference between the selected cell lines could later help figure out if the mode of action of paucinone H is through PI3K pathway. During the initial studies to screen for activity of paucinone H on MDA-MB-468, it was noticed that at 20 µM cell death was not as pronounced as in MDA-MB-231.

Studies from the Kennelly lab have shown that three Garcinia derived benzophenones – xanthochymol, guttiferone E and guttiferone H caused growth inhibition on a panel of colon cancer cell lines (Einbond et al. 2013). The mode of action of these benzophenones, especially for xanthochymol was partly stated to be via activation of endoplasmic stress response and inhibition of mammalian target of rapamycin (mtoR) cell survival pathway. Especially mTOR

C2, since the involvement of ER IP3R was reported. Due to the limitations mentioned earlier, the possibility of inhibition of mTOR by paucinone H was not studied. This could well be a future study

Figure 18: Growth inhibitory effects of paucinone H on MDA-MB-468. Images a, b, c, d, e and f denote control, 5, 10, 20, 40 and 100µM concentrations of paucinone H, 48 hours after treatment. IC50 was estimated to be 20 µM. Magnification: 10X. Scale bar = 10µm

PI3K/AKT/mTOR is the essential signaling pathway involved in survival, cell proliferation, metabolism and regulation of motility (Gonzalez-Angulo and Blumenschein 2013). PI3K is one of the targets explored in the development of therapeutics against TNBC (Ciruelos Gil 2014).

Almost 60% of TNBC patients show over activation of the PI3K pathway, mostly with a

77 mutation or deletion of PTEN and loss of heterozygosity at the INPP4B locus (Shah et al., 2012).

AKT is an important downstream target of the PI3K pathway and it regulates proliferation, growth, cell survival, proliferation apoptosis and glycogen metabolism (Ma et al., 2016). mTOR

(The mammalian target of rapamycin) – another downstream component of the PI3K/AKT pathway is a serine/threonine kinase, occurring as two distinct complexes – mTOR C1 and mTOR C2 (Massihnia et al. 2016). mTOR C1 is a growth regulator that is composed of mTOR,

Raptor, GβL and DEPTOR while mTOR C2 is composed of mTOR, Rictor, GβL, Sin1,

PRR5/Protor-1, and DEPTOR (Dowling et al., 2010). Since previous studies with xanthochymol a Garcinia derived benzophenone have pointed out to the possibility of xanthochymol altering mTOR C2, future studies with paucinone H could be focused in this direction.

Appendix A: Comparative UPLC-QTOF-MS-based metabolomics and bioactivities analyses of Garcinia oblongifolia

Additional projects on the in-vitro activity of Garcinia spp. that resulted in two peer reviewed publications are discussed in Appendices A and B respectively. One of these studies involved analyzing the activity of the vegetative organs of Garcinia oblongifolia against breast cancer.

Although the medicinal value of Garcinia species is well studied, there were no reports on their effects against breast cancer. This study involved, comparative metabolic profiling and bioactivity analysis of vegetative organs of Garcinia oblongifolia. All vegetative organs used in the study were collected, stored and extracted as described previously (Li et al., 2016b). Briefly, leaves and branches collected were air dried, while fruits were freeze dried before beginning the extraction process. The dried samples were then extracted with 80 % methanol, concentrated in vacuo and stored at -20◦C until use. UPLC-QTOF-MS and MSE followed by heatmap analysis helped identify the metabolite profile of the extracts under study. This was followed by use of bioassays to understand cytotoxicity of the studied extracts. The cytotoxic effects of plant extracts was evaluated in estrogen receptor positive breast cancer, using MCF 7 cell line. Human

MCF 7 cells were cultured in DMEM Glucose medium (Gibco, BRL Life Technologies, Inc.,

◦ Rockville, MD) and maintained in an incubator at 37 C and 5% CO2. Cell growth media was supplemented with 10% FCS and 100 µl penicillin/ streptomycin.

Cytotoxicity of the extracts under study was evaluated using a WST-12-(4-iodophenyl)-3-(4- nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt assay. A concentration of

12, 000 cells per well were seeded in 96 well plates after which they were treated with branch,

79 fruit, leaf or seed extract respectively. Cells were treated at concentrations: 31.25, 62.50, 125,

250, 500 and 1000 µgml-1 , 24 hours after plating. An untreated well and a well with 0.01%

DMSO were used as controls.

2 3

Figure 19. 1) Morphological changes in MCF-7 cells after treatment with Garcinia oblongifolia extracts. Images a, b, c and d represent control, branch, fruit and leaf extract at 500 µgml-1 respectively. Mild vacuolization observed after treatment with leaf extract and extensive vacuolization observed with the branch extract are marked with a triangle and an asterisk respectively. A dead cell is marked in figure b with a black arrow. Graphs 1, 2 and 3 show the

80 growth inhibitory effects of the branch, leaf and fruit extracts on MCF-7 cells 24 hours after treatment. Magnification:10X; Scale bar = 100 µm

24 hours after treatment, the contents in each well were removed and washed thoroughly with serum free media. This step was followed by the addition of 150 µl of 10% WST solution. The

96 well plates were then wrapped in aluminum foil and incubated for an hour. Absorbance was read at 440 nm using a Spectra Max (340 PC) plate reader. The percentage inhibition relative to control was calculated from the average absorbance values. All experiments were performed in triplicate. Results were then analyzed using Graph Pad Prism.

Changes in cell morphology as shown in Figure 19.1 were captured using a high objective fully enclosed inverted microscope (Zeiss Axio Observer, Thornwood, NY). Images were captured up to 48 hours after treatment. Controls used were the same as that of the bioassays. The results revealed that the branch extract was the most bioactive in comparison with the leaf and fruit extracts (Figure 19 Graphs 1, 2 and 3).

The cytotoxicity results observed, coincided with principal component analysis (PCA) of the metabolite profiles of different vegetative organs studied. A total of 12 compounds which included 10 xanthones and 2 biflavonoids were unique to the branch extract. In contrast, leaves showed the least amount of biflavonoids and fruits were low in xanthones. Therefore, the higher activity of branch extract in cytotoxicity assays could be attributed to the high concentration of xanthones. The approach handled in this study proved to be a time consuming and effective screening strategy for bioactive constituents from medicinal plant extracts.

Appendix B: UPLC-QTOF-MS guided dereplication of an endangered Chinese Garcinia species to identify cytotoxic benzophenones in triple negative and other breast cancer cell lines

This study describes a new strategy in which UPLC-QTOF-MSE technique was used to identify benzophenone derivatives using characteristic fragment ions. Garcinia species has previously been studied by our group to contain several bioactive compounds mostly belonging to the class of benzophenones(Baggett et al. 2005; Einbond et al. 2013). Several other Garcinia members such Garcinia xanthochymus, Garcinia kola, Garcinia atroviridis, Garcinia mangostana,

Garcinia bractea and Garcinia celebica have been studied in several in-vitro assays and in-vivo models (Suksamrarn et al., 2006; Subarnas et al. 2016; Mohamed et al. 2017; Tan et al., 2017).

But this is the first study that reported Garcinia paucinervis – an endangered species for its cytotoxicity and possible mechanisms of action against multiple breast cancer cell lines irrespective of their receptor status

UPLC-QTOF-MS guided targeted isolation of unidentified compounds, resulted in the identification of five novel compounds that belong to the class of benzophenones, paucinones E -

I. Their structures were elucidated using MS, NMR and ECD spectroscopy as described in our recent publication (Li et al. 2016a)

All five compounds were treated on 3 different breast cancer cell lines with varying receptor status to screen for their activity in bioassays. MDA-MB-231, MCF-7 and SKBR-3 were the cell lines used in this study. Cells were cultured in DMEM glucose medium (Gilbco, USA) 82 supplemented with 10% fetal calf serum and 100 µL of antibiotics (penicillin and streptomycin).

SKBR3 cells were cultured in DMEM F12 glutamine medium supplemented with 10% fetal calf serum and 100 µL of antibiotics (penicillin and streptomycin). All cell lines were maintained at

37 °C in an incubator with 5% CO2.

An in-vitro assay was done to determine the cytotoxicity of the compounds on the cell lines under study. All compounds were dissolved in DMSO and diluted with required volumes of respective cell culture media before each treatment. To determine the cytotoxicity effects of test compounds on the three cell lines used in this study, exponentially growing cells (12 × 103 cells/well) were seeded in 96 well multitier plates. After 24 h, each test compound was added to each cell line used in his study. Concentrations of the compounds studied ranged from 5 to 200

µM. Doxorubicin was used as a positive control. After 48 hours of treatment, a cell viability assay (WST) was performed. Each well was washed twice with serum-free medium followed by the addition of a 10% WST solution. This solution (150 µL) was then added to each well. The plates were wrapped in aluminum foil and incubated for 1 h, their absorbance was then read at

440 nm using a Spectra Max plate reader. Each experiment was performed in triplicate. Average absorbance and the percentage inhibition relative to control were calculated. The results were analyzed using Graph Pad Prism Software (v 6.01)(Graph Pad Software, San Diego, CA, USA), and IC50 values were estimated. Doxorubicin was used as the positive control in these studies. A schematic representation of the study set up is shown below in Figure 20.

Figure 20: A schematic representation of the study set up.

Paucinone H was identified to be the most bioactive compound. Although the overall activity level was similar around the IC50 range (19.05 µM), the compound was particularly active in triple negative cell line MDA-MB-231 and estrogen receptor positive cell line MCF-7. We later studied the possible targets of this compound and its mode of action in triple negative breast cancer cell lines

Chapter 6: References

Abdelhamed S, Yokoyama S, Refaat A, Ogura K, Yagita H, Awale S, Saiki I 2014. Piperine enhances the efficacy of TRAIL-based therapy for triple-negative breast cancer cells. Anticancer

Res 34: 1893-1899.

Acuña UM, Jancovski N, EJ K 2009. Polyisoprenylated benzophenones from Clusiaceae: potential drugs and lead compounds. Curr Top Med Chem. 9: 1560-1580.

Acuna UM, Figueroa M, Kavalier A, Jancovski N, Basile MJ, Kennelly EJ 2010.

Benzophenones and biflavonoids from Rheedia edulis. J Nat Prod 73: 1775-1779.

Adlercruetz H 2002. Phytoestrogens and breast cancer. J Steroid.Biochem.Mol.Biol 83: 113-118.

Aggarwal S, Das SN 2016. Garcinol inhibits tumour cell proliferation, angiogenesis, cell cycle progression and induces apoptosis via NF-kappaB inhibition in oral cancer. Tumour Biol 37:

7175-7184.

Ahmad A, Sarkar SH, Aboukameel A, Ali S, Biersack B, Seibt S, Li Y, Bao B, Kong D,

Banerjee S, Schobert R, Padhye SB, Sarkar FH 2012a. Anticancer action of garcinol in vitro and in vivo is in part mediated through inhibition of STAT-3 signaling. Carcinogenesis 33: 2450-

2456.

Ahmad A, Sarkar SH, Bitar B, Ali S, Aboukameel A, Sethi S, Li Y, Bao B, Kong D, Banerjee S,

Padhye SB, Sarkar FH 2012b. Garcinol regulates EMT and Wnt signaling pathways in vitro and in vivo, leading to anticancer activity against breast cancer cells. Mol Cancer Ther 11: 2193-

2201.

Ahmad A, Wang Z, Ali R, Maitah MY, Kong D, Banerjee S, Padhye S, Sarkar FH 2010.

Apoptosis-inducing effect of garcinol is mediated by NF-kappaB signaling in breast cancer cells.

J Cell Biochem 109: 1134-1141.

Ahmad A, Wang Z, Wojewoda C, Ali R, Kong D, Maitah MY, Banerjee S, Bao B, Padhye S,

Sarkar FH 2011. Garcinol-induced apoptosis in prostate and pancreatic cancer cells is mediated by NF- kappaB signaling. Front Biosci (Elite Ed) 3: 1483-1492.

Alipour S, Jafari-Adli S, Eskandari A 2015. Benefits and harms of phytoestrogen consumption in breast cancer survivors. Asian Pac J Cancer Prev 16: 3091-3396.

Allred KF, Yackley KM, Vanamala J, Allred CD 2009. Trigonelline is a novel phytoestrogen in coffee beans. J Nutr 139: 1833-1838.

Altmann KH, Gertsch J 2007. Anticancer drugs from nature--natural products as a unique source of new microtubule-stabilizing agents. Nat Prod Rep 24: 327-357.

Anholeti MC, Paiva SR, Figueiredo MR, Kaplan MA 2015. Chemosystematic aspects of polyisoprenylated benzophenones from the genus Clusia. An Acad Bras Cienc 87: 289-301.

Anonymous. 2016. U.S. Breast Cancer Statistics, from http://bit.ly/1l92g3W.

Archer DF 2014. Commentary on "Menopausal hormone treatment in postmenopausal women: risks and benefits". Southern Medical Journal 107: 696-697.

Aryappalli P, Al-Qubaisi SS, Attoub S, George JA, Arafat K, Ramadi KB, Mohamed YA, Al-

Dhaheri MM, Al-Sbiei A, Fernandez-Cabezudo MJ, Al-Ramadi BK 2017. The IL-6/STAT3 signaling pathway is an early target of Manuka honey-induced suppression of human breast cancer cells. Front Oncol 7: 167.

Baggett S, Protiva P, Mazzola EP, Yang H, Ressler ET, Basile MJ, Weinstein IB, Kennelly EJ

2005. Bioactive benzophenones from Garcinia xanthochymus fruits. J Nat Prod 68: 354-360.

Banerjee K, Resat H 2016. Constitutive activation of STAT3 in breast cancer cells: A review. Int

J Cancer 138: 2570-2578.

Bao N, Ou J, Xu M, Guan F, Shi W, Sun J, Chen L 2017. Novel NO-releasing plumbagin derivatives: Design, synthesis and evaluation of antiproliferative activity. Eur J Med Chem 137:

88-95.

Barukial J, Sarmah JN 2011. Ethnomedicinal plants used by the people of Golaghat District,

Assam, India. Int. J. Med. Arom. Plants 1: 203-2111.

Batova A, Altomare D, Chantarasriwong O, Ohlsen KL, Creek KE, Lin YC, Messersmith A, Yu

AL, Yu J, Theodorakis EA 2010. The synthetic caged garcinia xanthone cluvenone induces cell stress and apoptosis and has immune modulatory activity. Mol Cancer Ther 9: 2869-2878.

Bedel S, Nachtigall M, Naftolin F 2014. The pros and cons of plant estrogens for menopause. J

Steroid Biochem Mol Biol. 139: 225-236.

Bennets HW, Underwood EJ, Shier A 1946. A specific breeding problem of sheep on subterranean clover pastures in Western Australia. Aust. Vet. J. 22: 2-12.

Bianchini G, Balko JM, Mayer IA, Sanders ME, Gianni L 2016. Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease. Nat Rev Clin Oncol 13: 674-690.

Bishayee A, Sethi G 2016. Bioactive natural products in cancer prevention and therapy: Progress and promise. Semin Cancer Biol 40-41: 1-3.

Borrelli F, Ernst E 2010. Alternative and complementary therapies for the menopause. Maturitas

66: 333-343.

Boucher BA, Cotterchio M, Anderson LN, Kreiger N, Kirsh VA, Thompson LU 2013. Use of isoflavone supplements is associated with reduced postmenopausal breast cancer risk. Int J

Cancer 132: 1439-1450.

Bradley J. 2015. NBJ: The US supplement industry is $37 billion and not $12 billion. from http://bit.ly/1KLytJP.

Bulfoni M, Gerratana L, Del Ben F, Marzinotto S, Sorrentino M, Turetta M, Scoles G, Toffoletto

B, Isola M, Beltrami CA, Di Loreto C, Beltrami AP, Puglisi F, Cesselli D 2016. In patients with metastatic breast cancer the identification of circulating tumor cells in epithelial-to-mesenchymal transition is associated with a poor prognosis. Breast Cancer Res 18: 30.

Cancer Genome Atlas N 2012. Comprehensive molecular portraits of human breast tumours.

Nature 490: 61-70.

Carey L, Winer E, Viale G, Cameron D, Gianni L 2010. Triple-negative breast cancer: disease entity or title of convenience? Nat Rev Clin Oncol 7: 683-692.

Caunt CJ, Sale MJ, Smith PD, Cook SJ 2015. MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road. Nat Rev Cancer 15: 577-592.

Cederroth CR, Nef S 2009. Soy, phytoestrogens and metabolism: A review. Mol Cell Endocrinol

304: 30-42.

Chantarasriwong O, Cho WC, Batova A, Chavasiri W, Moore C, Rheingold AL, Theodorakis

EA 2009. Evaluation of the pharmacophoric motif of the caged Garcinia xanthones. Org Biomol

Chem 7: 4886-4894.

Charn TH, Liu ET, Chang EC, Lee YK, Katzenellenbogen JA, Katzenellenbogen BS 2010.

Genome-wide dynamics of chromatin binding of estrogen receptors alpha and beta: mutual restriction and competitive site selection. Mol Endocrinol 24: 47-59.

Chavez KJ, Garimella SV, Lipkowitz S 2010. Triple negative breast cancer cell lines: one tool in the search for better treatment of triple negative breast cancer. Breast Dis 32: 35-48.

Chen IH, Chang FR, Wu YC, Kung PH, Wu CC 2015a. 3,4-Methylenedioxy-beta-nitrostyrene inhibits adhesion and migration of human triple-negative breast cancer cells by suppressing beta1 integrin function and surface protein disulfide isomerase. Biochimie 110: 81-92.

Chen J, Gu HY, Lu N, Yang Y, Liu W, Qi Q, Rong JJ, Wang XT, You QD, Guo QL 2008.

Microtubule depolymerization and phosphorylation of c-Jun N-terminal kinase-1 and p38 were involved in gambogic acid induced cell cycle arrest and apoptosis in human breast carcinoma

MCF-7 cells. Life Sci 83: 103-109.

Chen J, Sun L 2012. Formononetin-induced apoptosis by activation of Ras/p38 mitogen- activated protein kinase in estrogen receptor-positive human breast cancer cells. Horm Metab

Res 44: 943-948.

Chen MN, Lin CC, Liu CF 2015. Efficacy of phytoestrogens for menopausal symptoms: a meta- analysis and systematic review. Climacteric 18: 260-269.

Cheng AC, Tsai ML, Liu CM, Lee MF, Nagabhushanam K, Ho CT, Pan MH 2010. Garcinol inhibits cell growth in hepatocellular carcinoma Hep3B cells through induction of ROS- dependent apoptosis. Food Funct 1: 301-307.

Choi Y, van Breemen RB 2008. Development of a screening assay for ligands to the estrogen receptor based on magnetic microparticles and LC-MS. Comb Chem High Throughput Screen.

11: 1-6.

Christoforous T, Jan-Ake G 2011. The different roles of ER subtypes in cancer biology and therapy. Nature Reviews 11: 597-608.

Chuah LO, Ho WY, Beh BK, Yeap SK 2013. Updates on antiobesity effect of Garcinia Origin (-

)-HCA. Evid Based Complement Alternat Med 2013: 751658.

Chuang YJ, Huang JW, Makamba H, Tsai ML, Li CW, Sh C 2006. Electrophoretic mobility shift assay on poly(ethylene glycol)-modified glass microchips for the study of estrogen responsive element binding. Electrophoresis 27: 4158-4165.

Chun J, Kim YS 2013. Platycodin D inhibits migration, invasion, and growth of MDA-MB-231 human breast cancer cells via suppression of EGFR-mediated Akt and MAPK pathways. Chem

Biol Interact 205: 212-221.

Ciruelos Gil EM 2014. Targeting the PI3K/AKT/mTOR pathway in estrogen receptor-positive breast cancer. Cancer Treat Rev 40: 862-871.

ClinicalTrials.gov. 2017. Triple negative breast cancer drugs. from https://clinicaltrials.gov/ct2/results?cond=Triple+Negative+Breast+Cancer&term=drug&cntry1=

&state1=&Search=Search.

Colli MC, Bracht A, Soares AA, de Oliveira AL, Boer CG, de Souza CG, Peralta RM 2012.

Evaluation of the efficacy of flaxseed meal and flaxseed extract in reducing menopausal symptoms. J Med Food 15: 840-845.

Corkery B, Crown J, Clynes M, O'Donovan N 2009. Epidermal growth factor receptor as a potential therapeutic target in triple-negative breast cancer. Ann Oncol 20: 862-867.

Cornwell T, Cohick W, Raskin I 2004. Dietary phytoestrogens and health. Phytochemistry 65:

995-1016.

Corrêa MP, Pena LdA. 1984. Dicionário das plantas úteis do Brasil e das exóticas cultivadas.

Ministério da Agricultura, Instituto Brasileiro de Desenvolvimento Florestal, Rio de Janeiro

Cox RI, Wong MS, Braden AW, Trikojus VM, Lindner HR 1972. The formation and specificity of antibodies to phyto-estrogens in the sheep. J Reprod Fertil 28: 157-158.

Cui X, Harada S, Shen D, Siegal GP, Wei S 2015. The utility of Phosphohistone H3 in breast cancer grading. Appl Immunohistochem Mol Morphol 23: 689-695. de Groot AF, Kuijpers CJ, Kroep JR 2017. CDK4/6 inhibition in early and metastatic breast cancer: A review. Cancer Treat Rev 60: 130-138.

Delmanto A, Nahas-Neto J, Traiman P, Uemura G, Pessoa EC, Nahas EA 2013. Effects of soy isoflavones on mammographic density and breast parenchyma in postmenopausal women: a randomized, double-blind, placebo-controlled clinical trial. Menopause 20: 1049-1054.

Depypere FH, Comhairea HT 2015. Hormones, herbal preparations and nutriceuticals for a better life after the menopause: part II. Climacteric 18: 358-363.

Doi H, Shibata MA, Shibata E, Morimoto J, Akao Y, Iinuma M, Tanigawa N, Otsuki Y 2009.

Panaxanthone isolated from pericarp of Garcinia mangostana L. suppresses tumor growth and metastasis of a mouse model of mammary cancer. Anticancer Res 29: 2485-2495.

Dong JY, Qin LQ 2011. Soy isoflavones consumption and risk of breast cancer incidence or recurrence: a meta-analysis of prospective studies. Breast Cancer Res Treat 125: 315-323.

Dowling RJ, Topisirovic I, Fonseca BD, Sonenberg N 2010. Dissecting the role of mTOR: lessons from mTOR inhibitors. Biochim Biophys Acta 1804: 433-439.

Duffy C, Perez K, Partridge A 2007. Implications of phytoestrogen intake for breast cancer. CA

Cancer J. Clin. 57: 260-277.

Dziwornu GA, Caira MR, Mare JA, Edkins AL, Bolton JJ, Beukes DR, Sunassee SN 2017.

Isolation, characterization and antiproliferative activity of new metabolites from the South

African endemic red algal Species Laurencia alfredensis. Molecules 22.

Eden JA 2012. Phytoestrogens for menopausal symptoms: a review. Maturitas 72: 157-159.

Einbond LS, Mighty J, Kashiwazaki R, Figueroa M, Jalees F, Acuna UM, Le Gendre O, Foster

DA, Kennelly EJ 2013. Garcinia benzophenones inhibit the growth of human colon cancer cells and synergize with sulindac sulfide and turmeric. Anticancer Agents Med Chem 13: 1540-1550.

El-Halawany AM, Chung MH, Abdallah HM, Nishihara T, Hattori M 2010. Estrogenic activity of a naringinase-treated extract of Sophora japonica cultivated in Egypt. Pharm Biol 48: 177-

181.

Elston CW, Ellis IO 1991. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up.

Histopathology 19: 403-410.

Fan QN, Z.; Hu, H.; Xu, Y.; Tang, T 2012. Chinese Tradiitonal Herbal Drugs 43: 436−439

Fang G, Zhu Y, Guo H, Wang X, Wang Ha, Gao X 2011. Direct vasorelaxation by a novel phytoestrogen tanshinone IIA is mediated by nongenomic action of estrogen receptor through endothelial nitric oxide synthase activation and calcium mobilization. Journal of Cardiovascular

Pharmacology 57: 340-347.

Fang Y, Zhang Q, Wang X, Yang X, Wang X, Huang Z, Jiao Y, Wang J 2016. Quantitative phosphoproteomics reveals genistein as a modulator of cell cycle and DNA damage response pathways in triple-negative breast cancer cells. Int J Oncol 48: 1016-1028.

Feng C, Huang SX, Gao XM, Xu HX, Luo KQ 2014. Characterization of proapoptotic compounds from the bark of Garcinia oblongifolia. J Nat Prod 77: 1111-1116.

Foulkes WD, Smith IE, Reis-Filho JS 2010. Triple-negative breast cancer. N Engl J Med 363:

1938-1948.

Franco OH, Chowdhury R, Troup J, Voortman T, Kunutsor S, Kavousi M, Oliver-Williams C,

Muka T 2016. Use of plant-based therapies and menopausal symptoms: A systematic review and meta-analysis. JAMA 315: 2554-2563.

Fritz H, Seely D, Flower G, Skidmore B, Fernandes R, Vadeboncoeur S, Kennedy D, Cooley K,

Wong R, Sagar S, Sabri E, Fergusson D 2013. Soy, red clover, and isoflavones and breast cancer: a systematic review. PLoS One 8: e81968.

Furtek SL, Backos DS, Matheson CJ, Reigan P 2016. Strategies and approaches of targeting

STAT3 for cancer treatment. ACS Chem Biol 11: 308-318.

Gallo D, Ferlini C, Fabrizi M, Prislei S, Scambia G 2006. Lack of stimulatory activity of a phytoestrogen-containing soy extract on the growth of breast cancer tumors in mice.

Carcinogenesis 27: 1404-1409.

Gao X-M, Yu T, Lai FSF, Pu J-X, Qiao C-F, Zhou Y, Liu X, Song J-Z, Luo KQ, Xu H-X 2010.

Novel polyisoprenylated benzophenone derivatives from Garcinia paucinervis. Tetrahedron

Letters 51: 2442-2446.

Garcia-Reyero N, Grau E, Castillo M, Lopez de Alda MJ, Barcelo D, Pina B 2001. Monitoring of endocrine disruptors in surface waters by the yeast recombinant assay. Environ Toxicol Chem

20: 1152-1158.

Genovese S, Fiorito S, Taddeo VA, Epifano F 2016. Recent developments in the pharmacology of prenylated xanthones. Drug Discov Today 21: 1814-1819.

Ginter PS, Shin SJ, Liu Y, Chen Z, D'Alfonso TM 2016. Phosphohistone H3 expression correlates with manual mitotic counts and aids in identification of "hot spots" in fibroepithelial tumors of the breast. Hum Pathol 49: 90-98.

Gonzalez-Angulo AM, Blumenschein GR, Jr. 2013. Defining biomarkers to predict sensitivity to

PI3K/Akt/mTOR pathway inhibitors in breast cancer. Cancer Treat Rev 39: 313-320.

Guo X, Cai Q, Bao P, Wu J, Wen W, Ye F, Zheng W, Zheng Y, Shu XO 2016. Long-term soy consumption and tumor tissue MicroRNA and gene expression in triple-negative breast cancer.

Cancer 122: 2544-2551.

Guruvayoorappan C, Kuttan G 2008. Amentoflavone stimulates apoptosis in B16F-10 melanoma cells by regulating bcl-2, p53 as well as caspase-3 genes and regulates the nitric oxide as well as proinflammatory cytokine production in B16F-10 melanoma cells, tumor associated macrophages and peritoneal macrophages. J Exp Ther Oncol 7: 207-218.

Hedelin M, Lof M, Olsson M, Adlercreutz H, Sandin S, Weiderpass E 2008. Dietary phytoestrogens are not associated with risk of overall breast cancer but diets rich in coumestrol are inversely associated with risk of estrogen receptor and progesterone receptor negative breast tumors in Swedish women. J Nutr 138: 938-945.

Hemshekhar M, Sunitha K, Santhosh MS, Devaraja S, Kemparaju K, Vishwanath BS, Niranjana

SR, Girish KS 2011. An overview on genus garcinia: phytochemical and therapeutical aspects.

Phytochemistry Reviews 10: 325-351.

Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, Brinkley BR, Bazett-Jones DP,

Allis CD 1997. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106: 348-360.

Herold CI, Anders CK 2013. New targets for triple-negative breast cancer. Oncology (Williston

Park) 27: 846-854.

Hershman D, Sundararajan V, Jacobson JS, Heitjan DF, Neugut AI, Grann VR 2002. Outcomes of Tamoxifen chemoprevention for breast cancer in very high-risk women: A cost-effectiveness analysis. Journal of clinical oncology 20: 9-16.

Hong J, Sang S, Park HJ, Kwon SJ, Suh N, Huang MT, Ho CT, Yang CS 2006. Modulation of arachidonic acid metabolism and nitric oxide synthesis by garcinol and its derivatives.

Carcinogenesis 27: 278-286.

Hong YH, Wang SC, Hsu C, Lin BF, Kuo YH, Huang CJ 2011. Phytoestrogenic compounds in alfalfa sprout (Medicago sativa) beyond coumestrol. J Agric Food Chem 59: 131-137.

Hsieh CJ, Kuo PL, Hsu YC, Huang YF, Tsai EM, Hsu YL 2014. Arctigenin, a dietary phytoestrogen, induces apoptosis of estrogen receptor-negative breast cancer cells through the

ROS/p38 MAPK pathway and epigenetic regulation. Free Radic Biol Med 67: 159-170.

Imura Y, Sato K, Yoshimura E 2010. Micro total bioassay system for ingested substances: assessment of intestinal absorption, hepatic metabolism, and bioactivity. Anal Chem 82: 9983-

9988.

Iwasaki M, Inoue M, Otani T, Sasazuki S, Kurahashi N, Miura T, Yamamoto S, Tsugane S 2008.

Plasma isoflavone level and subsequent risk of breast cancer among Japanese women: a nested case-control study from the Japan public health center-based prospective study group. J Clin

Oncol 26: 1677-1683.

J. Bennett G, Lee H-H. 1989. Xanthones from Guttiferae.28: 967-998

Janku F, Wheler JJ, Westin SN, Moulder SL, Naing A, Tsimberidou AM, Fu S, Falchook GS,

Hong DS, Garrido-Laguna I, Luthra R, Lee JJ, Lu KH, Kurzrock R 2012. PI3K/AKT/mTOR inhibitors in patients with breast and gynecologic malignancies harboring PIK3CA mutations. J

Clin Oncol 30: 777-782.

Jaskulski S, Jung AY, Rudolph A, Johnson T, Thone K, Herpel E, Sinn P, Chang-Claude J 2017.

Genistein and enterolactone in relation to Ki-67 expression and HER2 status in postmenopausal breast cancer patients. Mol Nutr Food Res.

Jen SH, Wei MP, Yin AC 2015. The Combinatory Effects of Glabridin and Tamoxifen on

Ishikawa and MCF-7 Cell Lines. Natural Product Communications 10: 1573-1576.

Jiang Y, Gong P, Madak-Erdogan Z, Martin T, Jeyakumar M, Carlson K, Khan I, Smillie TJ,

Chittiboyina AG, Rotte SC, Helferich WG, Katzenellenbogen JA, Katzenellenbogen BS 2013.

Mechanisms enforcing the estrogen receptor beta selectivity of botanical estrogens. Faseb j 27:

4406-4418.

Jordan MA, Wilson L 2004. Microtubules as a target for anticancer drugs. Nat Rev Cancer 4:

253-265.

Kalimutho M, Parsons K, Mittal D, Lopez JA, Srihari S, Khanna KK 2015. Targeted therapies for triple-negative breast cancer: combating a stubborn disease. Trends Pharmacol Sci 36: 822-

846.

Kan WL, Yin C, Xu HX, Xu G, To KK, Cho CH, Rudd JA, Lin G 2013. Antitumor effects of novel compound, guttiferone K, on colon cancer by p21Waf1/Cip1-mediated G(0) /G(1) cell cycle arrest and apoptosis. Int J Cancer 132: 707-716.

Kang E, Seong MW, Park SK, Lee JW, Lee J, Kim LS, Lee JE, Kim SY, Jeong J, Han SA, Kim

SW, Korean Hereditary Breast Cancer Study G 2015. The prevalence and spectrum of BRCA1 and BRCA2 mutations in Korean population: recent update of the Korean Hereditary Breast

Cancer (KOHBRA) study. Breast Cancer Res Treat 151: 157-168.

Karikas GA 2010. Anticancer and chemopreventing natural products: some biochemical and therapeutic aspects. J buon 15: 627-638.

Kaur M, Badhan RK 2015. Phytoestrogens modulate breast cancer resistance protein expression and function at the blood-cerebrospinal fluid barrier. J Pharm Pharm Sci 18: 132-154.

Kelly LA, Seidlova-Wuttke D, Wuttke W, O’Leary JJ, Norris LA 2014. Estrogen receptor alpha augments changes in hemostatic gene expression in HepG2 cells treated with estradiol and phytoestrogens. Phytomedicine 21: 155-158.

Khan SA, Chatterton RT, Michel N, Bryk M, Lee O, Ivancic D, Heinz R, Zalles CM,

Helenowski IB, Jovanovic BD, Franke AA, Bosland MC, Wang J, Hansen NM, Bethke KP, Dew

A, Coomes M, Bergan RC 2012. Soy isoflavone supplementation for breast cancer risk reduction: a randomized phase II trial. Cancer Prev Res (Phila) 5: 309-319.

Kim ES, Cho H, Lim C, Lee JY, Lee DI, Kim S, Moon A 2015. A natural piper-amide-like compound NED-135 exhibits a potent inhibitory effect on the invasive breast cancer cells. Chem

Biol Interact 237: 58-65.

Kim ES, Kim JS, Kim SG, Hwang S, Lee CH, Moon A 2011. Sphingosine 1-phosphate regulates matrix metalloproteinase-9 expression and breast cell invasion through S1P3-Galphaq coupling.

J Cell Sci 124: 2220-2230.

Kim R 2005. Recent advances in understanding the cell death pathways activated by anticancer therapy. Cancer 103: 1551-1560.

Kirichenko TV, Myasoedova VA, Orekhova VA, Ravani AL, Nikitina NA, Grechko AV,

Sobenin IA, Orekhov AN 2017. Phytoestrogen-rich natural preparation for treatment of climacteric syndrome and Atherosclerosis prevention in perimenopausal women. Phytother Res

31: 1209-1214.

Klintman M, Strand C, Ahlin C, Beglerbegovic S, Fjallskog ML, Grabau D, Gudlaugsson E,

Janssen EA, Lovgren K, Skaland I, Bendahl PO, Malmstrom P, Baak JP, Ferno M 2013. The prognostic value of mitotic activity index (MAI), phosphohistone H3 (PPH3), cyclin B1, cyclin

A, and Ki67, alone and in combinations, in node-negative premenopausal breast cancer. PLoS

One 8: e81902.

Koo J, Cabarcas-Petroski S, Petrie JL, Diette N, White RJ, Schramm L 2015. Induction of proto- oncogene BRF2 in breast cancer cells by the dietary soybean isoflavone daidzein. BMC Cancer

15: 905.

Kotepui M 2016. Diet and risk of breast cancer. Contemp Oncol (Pozn) 20: 13-19.

Kritsanawong S, Innajak S, Imoto M, Watanapokasin R 2016. Antiproliferative and apoptosis induction of alpha-mangostin in T47D breast cancer cells. Int J Oncol 48: 2155-2165.

Kuete V, Tchakam PD, Wiench B, Ngameni B, Wabo HK, Tala MF, Moungang ML, Ngadjui

BT, Murayama T, Efferth T 2013. Cytotoxicity and modes of action of four naturally occuring benzophenones: 2,2',5,6'-tetrahydroxybenzophenone, guttiferone E, isogarcinol and isoxanthochymol. Phytomedicine 20: 528-536.

Kuiper GG, Lemmen J, Carlsson B, Corton J, Safe S, van der Saag P, van der Burg B,

Gustafsson J 1998. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139: 4252-4263.

Kumar S, Sharma S, Chattopadhyay SK 2013. The potential health benefit of polyisoprenylated benzophenones from Garcinia and related genera: ethnobotanical and therapeutic importance.

Fitoterapia 89: 86-125.

Kurose H, Shibata MA, Iinuma M, Otsuki Y 2012. Alterations in cell cycle and induction of apoptotic cell death in breast cancer cells treated with alpha-mangostin extracted from mangosteen pericarp. J Biomed Biotechnol 2012: 672428.

Lapcik O, Hampl R, Al-Maharik N, Salakka A, Wahala K, Adlercreutz H 1997. A novel radioimmunoassay for daidzein. Steroids 62: 315-320.

100

Lara-Medina F, Perez-Sanchez V, Saavedra-Perez D, Blake-Cerda M, Arce C, Motola-Kuba D,

Villarreal-Garza C, Gonzalez-Angulo AM, Bargallo E, Aguilar JL, Mohar A, Arrieta O 2011.

Triple-negative breast cancer in Hispanic patients: high prevalence, poor prognosis, and association with menopausal status, body mass index, and parity. Cancer 117: 3658-3669.

Lathrop K (2017). S-equol in women with triple negative breast cancer, Clinical trials.gov.

Le J. 2016. Drug bioavailability. Merck Manual from https://mrkmnls.co/2hVVzsc.

Leclercq G, Jacquot Y 2014. Interactions of isoflavones and other plant derived estrogens with estrogen receptors for prevention and treatment of breast cancer-considerations concerning related efficacy and safety. J Steroid Biochem Mol Biol 139: 237-244.

Lecomte S, Chalmel F, Ferriere F, Percevault F, Plu N, Saligaut C, Surel C, Lelong M,

Efstathiou T, Pakdel F 2017a. Glyceollins trigger anti-proliferative effects through estradiol- dependent and independent pathways in breast cancer cells. Cell Commun Signal 15: 26.

Lecomte S, Demay F, Ferrière F, Pakdel F 2017b. Phytochemicals targeting estrogen receptors: beneficial rather than adverse effects? International Journal of Molecular Sciences 18: 1381.

Lecomte S, Lelong M, Bourgine G, Efstathiou T, Saligaut C, Pakdel F 2017c. Assessment of the potential activity of major dietary compounds as selective estrogen receptor modulators in two distinct cell models for proliferation and differentiation. Toxicol Appl Pharmacol 325: 61-70.

Lee SA, Shu XO, Li H, Yang G, Cai H, Wen W, Ji BT, Gao J, Gao YT, Zheng W 2009.

Adolescent and adult soy food intake and breast cancer risk: results from the Shanghai Women's

Health Study. Am J Clin Nutr 89: 1920-1926.

Lee YB, Ko KC, Shi MD, Liao YC, Chiang TA, Wu PF, Shih YX, Shih YW 2010. alpha-

Mangostin, a novel dietary xanthone, suppresses TPA-mediated MMP-2 and MMP-9 expressions

101 through the ERK signaling pathway in MCF-7 human breast adenocarcinoma cells. J Food Sci

75: H13-23.

Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, Pietenpol JA 2011.

Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest 121: 2750-2767.

Levis S, Strickman-Stein N, Doerge DR, Krischer J 2010. Design and baseline characteristics of the soy phytoestrogens as replacement estrogen (SPARE) study--a clinical trial of the effects of soy isoflavones in menopausal women. Contemp Clin Trials 31: 293-302.

Li D, Song XY, Yue QX, Cui YJ, Liu M, Feng LX, Wu WY, Jiang BH, Yang M, Qu XB, Liu X,

Guo DA 2015. Proteomic and bioinformatic analyses of possible target-related proteins of gambogic acid in human breast carcinoma MDA-MB-231 cells. Chin J Nat Med 13: 41-51.

Li F, Shanmugam MK, Chen L, Chatterjee S, Basha J, Kumar AP, Kundu TK, Sethi G 2013.

Garcinol, a polyisoprenylated benzophenone modulates multiple proinflammatory signaling cascades leading to the suppression of growth and survival of head and neck carcinoma. Cancer

Prev Res (Phila) 6: 843-854.

Li H, Meng XX, Zhang L, Zhang BJ, Liu XY, Fu WW, Tan HS, Lao YZ, Xu HX 2017.

Oblongifolin C and guttiferone K extracted from Garcinia yunnanensis fruit synergistically induce apoptosis in human colorectal cancer cells in vitro. Acta Pharmacol Sin 38: 252-263.

Li P, Anandhi Senthilkumar H, Figueroa M, Wu SB, Fata JE, Kennelly EJ, Long C 2016a.

UPLC-QTOFMS(E)-guided dereplication of the endangered Chinese species Garcinia paucinervis to identify additional benzophenone derivatives. J Nat Prod 79: 1619-1627.

102

Li P, AnandhiSenthilkumar H, Wu SB, Liu B, Guo ZY, Fata JE, Kennelly EJ, Long CL 2016b.

Comparative UPLC-QTOF-MS-based metabolomics and bioactivities analyses of Garcinia oblongifolia. J Chromatogr B Analyt Technol Biomed Life Sci 1011: 179-195.

Li Z, Li J, Mo B, Hu C, Liu H, Qi H, Wang X, Xu J 2008. Genistein induces G2/M cell cycle arrest via stable activation of ERK1/2 pathway in MDA-MB-231 breast cancer cells. Cell Biol

Toxicol 24: 401-409.

Liao CH, Sang S, Ho CT, Lin JK 2005. Garcinol modulates tyrosine phosphorylation of FAK and subsequently induces apoptosis through down-regulation of Src, ERK, and Akt survival signaling in human colon cancer cells. J Cell Biochem 96: 155-169.

Liu B, Edgerton S, Yang X, Kim A, Ordonez-Ercan D, Mason T, Alvarez K, McKimmey C, Liu

N, Thor A 2005. Low-dose dietary phytoestrogen abrogates tamoxifen-associated mammary tumor prevention. Cancer Research 65: 879-886.

Liu MM, Huang Y, Wang J 2012. Developing phytoestrogens for breast cancer prevention.

Anticancer Agents Med Chem 12: 1306-1313.

Liu Y, Zhu P, Wang Y, Wei Z, Tao L, Zhu Z, Sheng X, Wang S, Ruan J, Liu Z, Cao Y, Shan Y,

Sun L, Wang A, Chen W, Lu Y 2015. Antimetastatic therapies of the polysulfide diallyl trisulfide against triple-negative breast cancer (TNBC) via suppressing MMP2/9 by blocking

NF-kappaB and ERK/MAPK signaling pathways. PLoS One 10: e0123781.

Ma CX, Sanchez C, Gao F, Crowder R, Naughton M, Pluard T, Creekmore A, Guo Z, Hoog J,

Lockhart AC, Doyle A, Erlichman C, Ellis MJ 2016. A phase I study of the AKT inhibitor MK-

2206 in combination with hormonal therapy in postmenopausal women with estrogen receptor- positive metastatic breast cancer. Clin Cancer Res 22: 2650-2658.

103

Magalhaes LG, Marques FB, da Fonseca MB, Rogerio KR, Graebin CS, Andricopulo AD 2016.

Discovery of a series of acridinones as mechanism-based tubulin assembly inhibitors with anticancer activity. PLoS One 11: e0160842.

Magee PJ, Allsopp P, Samaletdin A, Rowland IR 2014. Daidzein, R-(+)equol and S-(-)equol inhibit the invasion of MDA-MB-231 breast cancer cells potentially via the down-regulation of matrix metalloproteinase-2. Eur J Nutr 53: 345-350.

Mariano LN, Vendramini-Costa DB, Ruiz AL, de Carvalho JE, Correa R, Cechinel Filho V,

Delle Monache F, Niero R 2016. In vitro antiproliferative activity of uncommon xanthones from branches of Garcinia achachairu. Pharm Biol 54: 1697-1704.

Martina B, Julie C, Wolfgang W 2008. Effects of long-term treatment with 8-prenylnaringenin and oral estradiol on the GH–IGF-1 axis and lipid metabolism in rats. J Endocrinol 198: 395-

401.

Martinkovich S, Shah D, Planey SL, Arnott JA 2014. Selective estrogen receptor modulators: tissue specificity and clinical utility. Clinical Interventions in Aging 9: 1437-1452.

Massihnia D, Galvano A, Fanale D, Perez A, Castiglia M, Incorvaia L, Listi A, Rizzo S, Cicero

G, Bazan V, Castorina S, Russo A 2016. Triple negative breast cancer: shedding light onto the role of pi3k/akt/mtor pathway. Oncotarget 7: 60712-60722.

Matsumoto K, Akao Y, Kobayashi E, Ito T, Ohguchi K, Tanaka T, Iinuma M, Nozawa Y 2003.

Cytotoxic benzophenone derivatives from Garcinia species display a strong apoptosis-inducing effect against human leukemia cell lines. Biol Pharm Bull 26: 569-571.

Maxwell T, Chun SY, Lee KS, Kim S, Nam KS 2017. The anti-metastatic effects of the phytoestrogen arctigenin on human breast cancer cell lines regardless of the status of ER expression. Int J Oncol 50: 727-735.

104

Mellier G, Huang S, Shenoy K, Pervaiz S 2010. TRAILing death in cancer. Mol Aspects Med 31:

93-112.

Melzer C, von der Ohe J, Hass R 2017. Breast carcinoma: From initial tumor cell detachment to settlement at secondary sites. Biomed Res Int 2017: 8534371.

Mense SM, Hei TK, Ganju RK, Bhat HK 2008. Phytoestrogens and breast cancer prevention: possible mechanisms of action. Environ Health Perspect 116: 426-433.

Mohamed GA, Al-Abd AM, El-Halawany AM, Abdallah HM, Ibrahim SR 2017. New xanthones and cytotoxic constituents from Garcinia mangostana fruit hulls against human hepatocellular, breast, and colorectal cancer cell lines. J Ethnopharmacol 198: 302-312.

Moongkarndi P, Kosem N, Luanratana O, Jongsomboonkusol S, Pongpan N 2004.

Antiproliferative activity of Thai medicinal plant extracts on human breast adenocarcinoma cell line. Fitoterapia 75: 375-377.

Moreira AC, Silva AM, Santos MS, Sardao VA 2014. Phytoestrogens as alternative hormone replacement therapy in menopause: What is real, what is unknown. J Steroid Biochem Mol Biol

143: 61-71.

Moutsatsou P 2007. The spectrum of phytoestrogens in nature: our knowledge is expanding.

Hormones 6: 173-193.

Mueller M, Jungbauer A 2008. Red clover extract: a putative source for simultaneous treatment of menopausal disorders and the metabolic syndrome. Menopause 15: 1120-1131.

Nehybova T, Smarda J, Benes P 2014. Plant coumestans: recent advances and future perspectives in cancer therapy. Anticancer Agents Med Chem 14: 1351-1362.

Obolskiy D, Pischel I, Siriwatanametanon N, Heinrich M 2009. Garcinia mangostana L.: a phytochemical and pharmacological review. Phytother Res 23: 1047-1065.

105

Ososki AL, Kennelly EJ 2003. Phytoestrogens: a review of the present state of research.

Phytother Res 17: 845-869.

Oualla K, El-Zawahry HM, Arun B, Reuben JM, Woodward WA, Gamal El-Din H, Lim B,

Mellas N, Ueno NT, Fouad TM 2017. Novel therapeutic strategies in the treatment of triple- negative breast cancer. Ther Adv Med Oncol 9: 493-511.

Padhye S, Ahmad A, Oswal N, Dandawate P, Rub RA, Deshpande J, Swamy KV, Sarkar FH

2010. Fluorinated 2′-hydroxychalcones as garcinol analogs with enhanced antioxidant and anticancer activities. Bioorganic & Medicinal Chemistry Letters 20: 5818-5821.

Page BD, Fletcher S, Yue P, Li Z, Zhang X, Sharmeen S, Datti A, Wrana JL, Trudel S,

Schimmer AD, Turkson J, Gunning PT 2011. Identification of a non-phosphorylated, cell permeable, small molecule ligand for the STAT-3 SH2 domain. Bioorg Med Chem Lett 21:

5605-5609.

Pan H, Zhou W, He W, Liu X, Ding Q, Ling L, Zha X, Wang S 2012. Genistein inhibits MDA-

MB-231 triple-negative breast cancer cell growth by inhibiting NF-kappaB activity via the

Notch-1 pathway. Int J Mol Med 30: 337-343.

Pan MH, Chiou YS, Chen LH, Ho CT 2015. Breast cancer chemoprevention by dietary natural phenolic compounds: specific epigenetic related molecular targets. Mol Nutr Food Res 59: 21-

35.

Pandya NM, Dhalla NS, Santani DD 2006. Angiogenesis--a new target for future therapy. Vascul

Pharmacol 44: 265-274.

Parasramka MA, Gupta SV 2011. Garcinol inhibits cell proliferation and promotes apoptosis in pancreatic adenocarcinoma cells. Nutr Cancer 63: 456-465.

106

Paruthiyil S, Cvoro A, Zhao X, Wu Z, Sui Y, Staub RE, Baggett S, Herber CB, Griffin C,

Tagliaferri M, Harris HA, Cohen I, Bjeldanes LF, Speed TP, Schaufele F, Leitman DC 2009.

Drug and cell type-specific regulation of genes with different classes of estrogen receptor beta- selective agonists. PLoS One 4: e6271.

Penttinen-Damdimopoulou PE, Power KA, Hurmerinta TT, Nurmi T, van der Saag PT, Makela

SI 2009. Dietary sources of lignans and isoflavones modulate responses to estradiol in estrogen reporter mice. Mol Nutr Food Res 53: 996-1006.

Perez-Cadahia B, Drobic B, Davie JR 2009. H3 phosphorylation: dual role in mitosis and interphase. Biochem Cell Biol 87: 695-709.

Pham J, Grundmann O, Elbayoumi T 2015. Mitochondriotropic nanoemulsified genistein-loaded vehicles for cancer therapy. Methods Mol Biol 1265: 85-101.

Philipp Y, Maximov TM, Lee V, Craig J 2013. The discovery and development of selective estrogen receptor modulators(SERMS) for clinical practice. Current Clinical Pharmacology 8:

135 - 155.

Pieme CA, Ambassa P, Yankep E, Saxena AK 2015. Epigarcinol and isogarcinol isolated from the root of Garcinia ovalifolia induce apoptosis of human promyelocytic leukemia (HL-60 cells).

BMC Res Notes 8: 700.

Pignanelli C, Ma D, Noel M, Ropat J, Mansour F, Curran C, Pupulin S, Larocque K, Wu J,

Liang G, Wang Y, Pandey S 2017. Selective targeting of cancer cells by oxidative vulnerabilities with novel curcumin analogs. Sci Rep 7: 1105.

Poluzzi E, Piccinni C, Raschi E, Rampa A, Recanatini M, De Ponti F 2014. Phytoestrogens in postmenopause: the state of the art from a chemical, pharmacological and regulatory perspective.

Curr Med Chem 21: 417-436.

107

Pop EA, Fischer LM, Coan AD, Gitzinger M, Nakamura J, Sh Z 2008. Effects of a high daily dose of soy isoflavones on DNA damage, apoptosis, and estrogenic outcomes in healthy postmenopausal women: a phase I clinical trial. Menopause 4: 684-692.

Prasad S, Pandey MK, Yadav VR, Aggarwal BB 2011. Gambogic acid inhibits STAT3 phosphorylation through activation of protein tyrosine phosphatase SHP-1: potential role in proliferation and apoptosis. Cancer Prev Res (Phila) 4: 1084-1094.

Prasad S, Ravindran J, Sung B, Pandey MK, Aggarwal BB 2010. Garcinol potentiates TRAIL- induced apoptosis through modulation of death receptors and antiapoptotic proteins. Mol Cancer

Ther 9: 856-868.

Prat A, Perou CM 2011. Deconstructing the molecular portraits of breast cancer. Mol Oncol 5: 5-

23.

Preedy V. 2012. Isoflavones: chemistry, analysis, function and effects. RSC Publishing, London

United Kingdom.2012

Protiva P, Hopkins ME, Baggett S, Yang H, Lipkin M, Holt PR, Kennelly EJ, Bernard WI 2008.

Growth inhibition of colon cancer cells by polyisoprenylated benzophenones is associated with induction of the endoplasmic reticulum response. Int J Cancer 123: 687-694.

Rafii F 2015. The role of colonic bacteria in the metabolism of the natural isoflavone daidzin to equol. Metabolites 5: 56-73.

Rajah TT, Peine KJ, Du N, Serret CA, Drews NR 2012. Physiological concentrations of genistein and 17β-estradiol inhibit MDA-MB-231 breast cancer cell growth by increasing

BAX/BCL-2 and reducing pERK1/2. Anticancer Res 32: 1181-1191.

108

Ramos PA, Guerra AR, Guerreiro O, Santos SA, Oliveira H, Freire CS, Silvestre AJ, Duarte MF

2016. Antiproliferative effects of Cynara cardunculus L. var. altilis (DC) lipophilic extracts. Int

J Mol Sci 18.

Rauch N, Rukhlenko OS, Kolch W, Kholodenko BN 2016. MAPK kinase signalling dynamics regulate cell fate decisions and drug resistance. Curr Opin Struct Biol 41: 151-158.

Rietjens I, Louisse J, Beekmann K 2017. The potential health effects of dietary phytoestrogens.

Br J Pharmacol 174: 1263-1280.

Robles AJ, Du L, Cichewicz RH, Mooberry SL 2016. Maximiscin induces DNA damage, activates DNA damage response pathways, and has selective cytotoxic activity against a subtype of triple-negative breast cancer. J Nat Prod 79: 1822-1827.

Ross RK, Paganini-Hill A, Wan PC, Pike MC 2000. Effect of hormone replacement therapy on breast cancer risk: estrogen versus estrogen plus progestin. J Natl Cancer Inst 92: 328-332.

Routledge EJ, White R, Parker MG, Sumpter JP 2000. Differential effects of xenoestrogens on coactivator recruitment by estrogen receptor (ER) alpha and ERbeta. J Biol Chem 275: 35986-

35993.

Roux D, Hadi HA, Thoret S, Guenard D, Thoison O, Pais M, Sevenet T 2000. Structure-activity relationship of polyisoprenyl benzophenones from Garcinia pyrifera on the tubulin/microtubule system. J Nat Prod 63: 1070-1076.

Rowland I, Wiseman H, Sanders T, Adlercreutz H, Bowey E 1999. Metabolism of oestrogens and phytoestrogens: role of the gut microflora. Biochem Soc Trans 27: 304-308.

Rudy S, Harry G, Toine FHB, Marian AV, Vincken J-P 2012. Prenylated isoflavonoids from plants as selective estrogen receptor modulators (PhytoSERMS). Food & Function 3: 810-827.

109

Rush MD, Walker EM, Prehna G, Burton T, van Breemen RB 2017. Development of a magnetic microbead affinity selection screen (MagMASS) using mass spectrometry for ligands to the retinoid X receptor-alpha. J Am Soc Mass Spectrom 28: 479-485.

Rzepecka-Stojko A, Kabala-Dzik A, Mozdzierz A, Kubina R, Wojtyczka RD, Stojko R,

Dziedzic A, Jastrzebska-Stojko Z, Jurzak M, Buszman E, Stojko J 2015. Caffeic Acid phenethyl ester and ethanol extract of propolis induce the complementary cytotoxic effect on triple- negative breast cancer cell lines. Molecules 20: 9242-9262.

Sak K 2017. Epidemiological evidences on dietary flavonoids and breast cancer risk: A narrative review. Asian Pac J Cancer Prev 18: 2309-2328.

Sakamoto T, Horiguchi H, Oguma E, F K 2010. Effects of diverse dietary phytoestrogens on cell growth, cell cycle and apoptosis in estrogen-receptor-positive breast cancer cells. J Nutr

Biochem. 21: 856-864.

Sarkar FH, Adsule S, Padhye S, Kulkarni S, Li Y 2006. The role of genistein and synthetic derivatives of isoflavone in cancer prevention and therapy. Mini Rev Med Chem 6: 401-407.

Sathyamoorthy N, Wang TT 1997. Differential effects of dietary phyto-oestrogens daidzein and equol on human breast cancer MCF-7 cells. Eur. J. Cancer: 2384–2389.

Satih S, Chalabi N, Rabiau N, Bosviel R, Fontana L, Bignon YJ, Bernard-Gallon DJ 2010. Gene expression profiling of breast cancer cell lines in response to soy isoflavones using a pangenomic microarray approach. Omics 14: 231-238.

Schneeman OB. 2011. Letter responding to health claim petition dated January 27, 2004: Green tea and reduced risk of cancer health claim from http://bit.ly/2w3RUOm.

110

Sefirin D, Maria H, Dieudonné N, George K, George L, Josephine H, Francesca MR, Emmanuel

M, Alexios-Leandros S, Günter V 2014. Erythroidine alkaloids: a novel class of phytoestrogens.

Planta Med 2014: 861-869.

Shah SP, Roth A, Goya R, Oloumi A, Ha G, Zhao Y, Turashvili G, Ding J, Tse K, Haffari G,

Bashashati A, Prentice LM, Khattra J, Burleigh A, Yap D, Bernard V, McPherson A, Shumansky

K, Crisan A, Giuliany R, Heravi-Moussavi A, Rosner J, Lai D, Birol I, Varhol R, Tam A, Dhalla

N, Zeng T, Ma K, Chan SK, Griffith M, Moradian A, Cheng SW, Morin GB, Watson P, Gelmon

K, Chia S, Chin SF, Curtis C, Rueda OM, Pharoah PD, Damaraju S, Mackey J, Hoon K, Harkins

T, Tadigotla V, Sigaroudinia M, Gascard P, Tlsty T, Costello JF, Meyer IM, Eaves CJ,

Wasserman WW, Jones S, Huntsman D, Hirst M, Caldas C, Marra MA, Aparicio S 2012. The clonal and mutational evolution spectrum of primary triple-negative breast cancers. Nature 486:

395-399.

Shanle EK, Hawse JR, Xu W 2011. Generation of stable reporter breast cancer cell lines for the identification of ER subtype selective ligands. Biochem Pharmacol 82: 1940-1949.

Shanmugam MK, Kannaiyan R, Sethi G 2011. Targeting cell signaling and apoptotic pathways by dietary agents: role in the prevention and treatment of cancer. Nutr Cancer 63: 161-173.

She QB, Chandarlapaty S, Ye Q, Lobo J, Haskell KM, Leander KR, DeFeo-Jones D, Huber HE,

Rosen N 2008. Breast tumor cells with PI3K mutation or HER2 amplification are selectively addicted to Akt signaling. PLoS One 3: e3065.

Shen K, Xi Z, Xie J, Wang H, Xie C, Lee CS, Fahey P, Dong Q, Xu H 2016. Guttiferone K suppresses cell motility and metastasis of hepatocellular carcinoma by restoring aberrantly reduced profilin 1. Oncotarget 7: 56650-56663.

111

Shen T, Li W, Wang YY, Zhong QQ, Wang SQ, Wang XN, Ren DM, Lou HX 2014.

Antiproliferative activities of Garcinia bracteata extract and its active ingredient, isobractatin, against human tumor cell lines. Arch Pharm Res 37: 412-420.

Shi X, Chen X, Li X, Lan X, Zhao C, Liu S, huang H, Liu N, Liao S, Song W, Zhou P, Wang S,

Xu L, Wang X, Liu PDaJ 2013. Gambogic acid induces apoptosis in Imatinib resistandt chronic myleoid leukemia cells via proteaosome inhibition and caspas dependent Bcr-Abl downregulation. Cancer Therapy: Preclincial.

Shiau JY, Nakagawa-Goto K, Lee KH, Shyur LF 2017. Phytoagent deoxyelephantopin derivative inhibits triple negative breast cancer cell activity by inducing oxidative stress- mediated paraptosis-like cell death. Oncotarget 8: 56942-56958.

Shibata MA, Iinuma M, Morimoto J, Kurose H, Akamatsu K, Okuno Y, Akao Y, Otsuki Y 2011.

Alpha-mangostin extracted from the pericarp of the mangosteen (Garcinia mangostana Linn) reduces tumor growth and lymph node metastasis in an immunocompetent xenograft model of metastatic mammary cancer carrying a p53 mutation. BMC Med 9: 69.

Shike M, Doane AS, Russo L, Cabal R, Reis-Filho JS, Gerald W, Cody H, Khanin R, Bromberg

J, Norton L 2014. The effects of soy supplementation on gene expression in breast cancer: a randomized placebo-controlled study. J Natl Cancer Inst 106.

Shor D, Sathyapalan T, Atkin SL, Thatcher NJ 2012. Does equol production determine soy endocrine effects? Eur J Nutr 51: 389-398.

Shu W, Chen Y, Li R, Wu Q, Cui G, Ke W, Chen Z 2008. Involvement of regulations of nucleophosmin and nucleoporins in gambogic acid-induced apoptosis in Jurkat cells. Basic Clin

Pharmacol Toxicol 103: 530-537.

112

Shulman LP, Banuvar S, Fong HHS, Farnsworth NR 2011. Discussion of a well-designed clinical trial which did not demonstrate effectiveness: UIC center for botanical dietary supplements research study of black cohosh and red clover. Fitoterapia 82: 88-91.

Simons R, Gruppen H, Bovee TF, Verbruggen MA, Vincken JP 2012. Prenylated isoflavonoids from plants as selective estrogen receptor modulators (phytoSERMs). Food Funct 3: 810-827.

Skaland I, Janssen EA, Gudlaugsson E, Hui Ru Guo L, Baak JP 2009. The prognostic value of the proliferation marker phosphohistone H3 (PPH3) in luminal, basal-like and triple negative phenotype invasive lymph node-negative breast cancer. Cell Oncol 31: 261-271.

Skaland I, Janssen EA, Gudlaugsson E, Klos J, Kjellevold KH, Soiland H, Baak JP 2007.

Phosphohistone H3 expression has much stronger prognostic value than classical prognosticators in invasive lymph node-negative breast cancer patients less than 55 years of age. Mod Pathol 20:

1307-1315.

Sotoca AM, Ratman D, van der Saag P, Strom A, Gustafsson JA, Vervoort J, Rietjens IM, Murk

AJ 2008. Phytoestrogen-mediated inhibition of proliferation of the human T47D breast cancer cells depends on the ERalpha/ERbeta ratio. J Steroid Biochem Mol Biol 112: 171-178.

Spagnuolo C, Russo GL, Orhan IE, Habtemariam S, Daglia M, Sureda A, Nabavi SF, Devi KP,

Loizzo MR, Tundis R, Nabavi SM 2015. Genistein and cancer: current status, challenges, and future directions. Adv Nutr 6: 408-419.

Staples G, Herbst DR. 2005. A Tropical Garden Flora: Plants Cultivated in the Hawaiian Islands and Other Tropical Places. Bishop Museum Press

Subarnas A, Diantini A, Abdulah R, Zuhrotun A, Nugraha PA, Hadisaputri YE, Puspitasari IM,

Yamazaki C, Kuwano H, Koyama H 2016. Apoptosis-mediated antiproliferative activity of

113 friedolanostane triterpenoid isolated from the leaves of Garcinia celebica against MCF-7 human breast cancer cell lines. Biomed Rep 4: 79-82.

Suksamrarn S, Komutiban O, Ratananukul P, Chimnoi N, Lartpornmatulee N, Suksamrarn A

2006. Cytotoxic prenylated xanthones from the young fruit of Garcinia mangostana. Chem

Pharm Bull (Tokyo) 54: 301-305.

Sulaiman NB, Mohan CD, Basappa S, Pandey V, Rangappa S, Bharathkumar H, Kumar AP,

Lobie PE, Rangappa KS 2016. An azaspirane derivative suppresses growth and induces apoptosis of ER-positive and ER-negative breast cancer cells through the modulation of

JAK2/STAT3 signaling pathway. Int J Oncol 49: 1221-1229.

Sun A, Zhou W, Lunceford J, Strack P, Dauffenbach LM, Kerfoot CA 2012. Level of phosphohistone H3 among various types of human cancers. BMJ Open 2.

Sun W (1998). Garcinia paucinervis. The IUCN Red List of Threatened Species 1998: e.T32352A9700403.

Sun Y, Gu C, Liu X, Liang W, Yao P, Bolton JL, van Breemen RB 2005. Ultrafiltration tandem mass spectrometry of estrogens for characterization of structure and affinity for human estrogen receptors. J Am Soc Mass Spectrom 16: 271-279.

Sun Y, Li D, Jia C, Xue C, Bai J, Li Z, Hua H 2016. Three new xanthones from the leaves of

Garcinia lancilimba. J Nat Med 70: 173-178.

Tan WN, Lim JQ, Afiqah F, Nik Mohamed Kamal NNS, Abdul Aziz FA, Tong WY, Leong CR,

Lim JW 2017. Chemical composition and cytotoxic activity of Garcinia atroviridis Griff. ex T.

Anders. essential oils in combination with tamoxifen. 1-5.

Tang J, Xu N, Ji H, Liu H, Wang Z, Wu L 2011. Eudragit nanoparticles containing genistein: formulation, development, and bioavailability assessment. Int J Nanomedicine 6: 2429-2435.

114

Tantikanlayaporn D, Wichit P, Weerachayaphorn J, Chairoungdua A, Chuncharunee A,

Suksamrarn A, Piyachaturawat P 2013. Bone sparing effect of a novel phytoestrogen diarylheptanoid from Curcuma comosa Roxb. in ovariectomized rats. PLoS One 8: e78739.

Thongon N, Boonmuen N, Suksen K, Wichit P, Chairoungdua A, Tuchinda P, Suksamrarn A,

Winuthayanon W, Piyachaturawat P 2017. Selective Estrogen Receptor Modulator (SERM)-like activities of diarylheptanoid, a phytoestrogen from Curcuma comosa, in breast cancer cells, pre- osteoblast cells, and rat uterine issues. J Agric Food Chem 65: 3490-3496.

Tice JA, Ettinger B, Ensrud K, Wallace R, Blackwell T, Cummings SR 2003. Phytoestrogen supplements for the treatment of hot flashes: the Isoflavone Clover Extract (ICE) Study: a randomized controlled trial. JAMA 290: 207-214.

Todaka E, Sakurai K, Fukata H, Miyagawa H, Uzuki M, Omori M, Osada H, Ikezuki Y,

Tsutsumi O, Iguchi T, Mori C 2005. Fetal exposure to phytoestrogens--the difference in phytoestrogen status between mother and fetus. Eviron Res. 99: 195-203.

Tomczak K, Czerwinska P, Wiznerowicz M 2015. The Cancer Genome Atlas (TCGA): an immeasurable source of knowledge. Contemp Oncol (Pozn) 19: A68-77.

Treeck O, Lattrich C, Springwald A, Ortmann O 2010. Estrogen receptor beta exerts growth- inhibitory effects on human mammary epithelial cells. Breast Cancer Res Treat 120: 557-565.

Tsai JH, Yang J 2013. Epithelial-mesenchymal plasticity in carcinoma metastasis. Genes Dev 27:

2192-2206.

Tu SH, Chiou YS, Kalyanam N, Ho CT, Chen LC, Pan MH 2017. Garcinol sensitizes breast cancer cells to Taxol through the suppression of caspase-3/iPLA2 and NF-kappaB/Twist1 signaling pathways in a mouse 4T1 breast tumor model. 8: 1067-1079.

115

Turner JV, Agatonovic-Kustrin S, Glass BD 2007. Molecular aspects of phytoestrogen selective binding at estrogen receptors. J Pharm Sci 96: 1879-1885. van Duursen MB, Smeets EE, Rijk JC, Nijmeijer SM, van den Berg M 2013. Phytoestrogens in menopausal supplements induce ER-dependent cell proliferation and overcome breast cancer treatment in an in vitro breast cancer model. Toxicol Appl Pharmacol 269: 132-140. van Elswijk DA, Schobel UP, Lansky EP, Irth H, van der Greef J 2004. Rapid dereplication of estrogenic compounds in pomegranate (Punica granatum) using on-line biochemical detection coupled to mass spectrometry. Phytochemistry 65: 233-241.

Varinska L, P G, G M, Mirossay L, J M 2015. Soy and breast cancer: focus on angiogenesis.

International Journal of Molecular Science 16: 11728-11749.

Vatistas TJ, Samuels JG 2012. The regulation of dietary supplements in the United States: advocating for a reasonable approach, protecting patient safety, and the role of nursing. Policy

Polit Nurs Pract 13: 113-116.

Veras E, Malpica A, Deavers MT, Silva EG 2009. Mitosis-specific marker phospho-histone H3 in the assessment of mitotic index in uterine smooth muscle tumors: a pilot study. Int J Gynecol

Pathol 28: 316-321.

Vermes I, Haanen C, Reutelingsperger C 2000. Flow cytometry of apoptotic cell death. J

Immunol Methods 243: 167-190.

Wang C, Kar S, Lai X, Cai W, Arfuso F, Sethi G, Lobie PE, Goh BC, Lim LHK, Hartman M,

Chan CW, Lee SC, Tan SH, Kumar AP 2017. Triple negative breast cancer in Asia: An insider's view. Cancer Treat Rev 62: 29-38.

Wang CC, Prasain JK, Barnes S 2002. Review of the methods used in the determination of phytoestrogens. J Chromatogr B Analyt Technol Biomed Life Sci 777: 3-28.

116

Wang J, Wang L, Ho CT, Zhang K, Liu Q, Zhao H 2017. Garcinol from Garcinia indica

Downregulates Cancer Stem-like Cell Biomarker ALDH1A1 in Nonsmall Cell Lung Cancer

A549 Cells through DDIT3 Activation. J Agric Food Chem 65: 3675-3683.

Wang K, Tang Y, Sun M, Lu B, Zhu H, Ji O, Shen Q 2011. The mechanism of neogambogic acid-induced apoptosis in human MCF-7 cells. Acta Biochim Biophys Sin (Shanghai) 43: 698-

702.

Welboren WJ, Stunnenberg HG, Sweep FC, Span PN 2007. Identifying estrogen receptor target genes. Mol Oncol 1: 138-143.

Wisniewska I, Jochymek B, Lenart-Lipinska M, Chabowski M 2016. The pharmacological and hormonal therapy of hot flushes in breast cancer survivors. Breast Cancer 23: 178-182.

Wu AH, Spicer D, Garcia A, Tseng C-C, Hovanessian-Larsen L, Sheth P, Martin SE, Hawes D,

Russell C, donald HM, Tripathy D, Su M-Y, Ursin G, Pike MC 2015. Double-Blind Randomized

12-Month Soy Intervention Had No Effects on Breast MRI Fibroglandular Tissue Density or

Mammographic Density. Cancer Prevention Research 8: 942-951.

Wu SB, Long C, Kennelly EJ 2014. Structural diversity and bioactivities of natural benzophenones. Nat Prod Rep 31: 1158-1174.

Xi-wen Li JL, Norman K. B. Robson & Peter Stevens. 1998. Garcinia paucinervis. eFlora of

China 2016, from http://www.efloras.org/florataxon.aspx?flora_id=2&taxon_id=200014182.

Xing WJ, Liao XH, Wang N, Zhao DW, Zheng L, Zheng DL, Dong J, Zhang TC 2015. MRTF-A and STAT3 promote MDA-MB-231 cell migration via hypermethylating BRSM1. IUBMB Life

67: 202-217.

117

Yang H, Figueroa M, To S, Baggett S, Jiang B, Basile MJ, Weinstein IB, Kennelly EJ 2010.

Benzophenones and biflavonoids from Garcinia livingstonei fruits. J Agric Food Chem 58:

4749-4755.

Yang X, Belosay A, Hartman JA, Song H, Zhang Y, Wang W, Doerge DR, Helferich WG 2015.

Dietary soy isoflavones increase metastasis to lungs in an experimental model of breast cancer with bone micro-tumors. Clin Exp Metastasis 32: 323-333.

Ye X, Yuan L, Zhang L, Zhao J, Zhang CM, Deng HY 2014. Garcinol, an acetyltransferase inhibitor, suppresses proliferation of breast cancer cell line MCF-7 promoted by 17beta-estradiol.

Asian Pac J Cancer Prev 15: 5001-5007.

Youn UJ, Sripisut T, Miklossy G, Turkson J, Laphookhieo S, Chang LC 2017. Bioactive polyprenylated benzophenone derivatives from the fruits extracts of Garcinia xanthochymus.

Bioorg Med Chem Lett 27: 3760-3765.

Yu SY, Liao CH, Chien MH, Tsai TY, Lin JK, Weng MS 2014. Induction of p21(Waf1/Cip1) by garcinol via downregulation of p38-MAPK signaling in p53-independent H1299 lung cancer. J

Agric Food Chem 62: 2085-2095.

Zhou A, Yan L, Lai F, Chen X, Goto M, Lee KH, Xiao Z 2017. Design, synthesis and biological evaluation of novel indolin-2-ones as potent anticancer compounds. Bioorg Med Chem Lett 27:

3326-3331.

Zhou J, Luo YH, Wang JR, Lu BB, Wang KM, Tian Y 2013. Gambogenic acid induction of apoptosis in a breast cancer cell line. Asian Pac J Cancer Prev 14: 7601-7605.

Zhou Q, Ji M, Zhou J, Jin J, Xue N, Chen J, Xu B, Chen X 2016. Poly (ADP-ribose) polymerases inhibitor, Zj6413, as a potential therapeutic agent against breast cancer. Biochem

Pharmacol 107: 29-40.

118

Ziaei S, Halaby R 2017. Dietary Isoflavones and Breast Cancer Risk. Medicines (Basel) 4: 18 -

29.

119

Anti-Proliferative Effects of &lt;I&gt;Garcinia&lt;/I&gt;

Anti-Proliferative Effects of <I>Garcinia</I>