NOTE TO USERS
The original manuscript received by UMI contains pages with slanted pn'nt. Pages were microfilmed as received.
This reproduction is the best copy available
THEEFFECTS OF PLANT-DERIVED COMPOUNDSON SEXHORMONE RECEPTORS: ~MPLICATIONSFOR HORMONE- DEPENDENT CANCERDEVELOPMENT AND TREATMENT
Rachel Stacey Rosenberg
A thesis submitted in conformity with the requirements for the degree of M.Sc. Graduate Department of Nutritional Sciences University of Toronto
@Copyright by Rachel Rosenberg 1997 ~ibiiographicServices services bibliographiques 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada Your lile Votre raference
Our file Notre réUrence
The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive pennethnt à la National Librq of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique.
The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author' s ou autrement reproduits sans son permission. autorisation. ~MPLICATIONSFOR HORMONE- DEPENDENT CANCERDEVELOPMENT AND f REATMENT Master of Science, 1997 Rachel Stacey Rosenberg Graduate Department of Nutritional Sciences University of Toronto
ABSTRACT
Breast and prostate cancers are leading causes of cancer morbidity and mortality of women and men in North America. This risk is significantly higher than in the
Japanese population, with epidemiologicaf studies suggesting the difference due to high fiavonoid consumption in the latter group. The purpose was to examine effects of plant-derived compounds on androgen (AR) and progesterone receptors (PR). In vitro studies on BT-474 and T47-D breast cancer cells were performed and 7 synthetic antiprogestins and 45 plant-derived compounds were tested. Prostate-specific antigen (PSA) was rneasured as a marker of receptor activity. The strongest blockers of PR were chlorophylline (87 k 7%), p-carotene
(82 f 3%),taxifolin (80 f 5%), and chlorogenic acid (61 -t. 2%). The most potent antagonists of AR were chlorophylline (94 5 3%), hesperetin (79 i 11%), syringic acid (72 + 11 %), and a-tocopherol (65 + 5%). Apigenin had an agonistic effect on these receptors. ------.-.-
Many people have contributed to the inception, planning, and development of this thesis. I would first like to thank Dr David Jenkins for his support, guidance, and patience in al1 aspects of this project and other endeavors. I would also like to thank Dr Eleftherios Diamandis for his teachings and direction, without which 1 would still be struggling with lab technique and data interpretation.
Many thanks to Linda Grass for her help in the lab, as well as for Dr
Gudrun Borchert for aiding me in assay techniques, and especially to Tina
Perera for her administrative, ernotional, and moral support throughout. Thanks to Dr Cyril Kendall and Tom Ransom, for the creation and planning of this project, and to the rest of the group, for their assistance.
1 would like to also thank my family and friends who have provided me with the emotional tools that carried me from day to day. To my parents, thank you for your unconditional love, guidance, and support through the years. Jeff and Jason, thank you for being the brothers I could also count on in times of need. To my grandmother Tillie Binder, I hope that upon reading this, you understand somewhat the work I do, and its importance to me. To Sammy,
Tigger, Ashley, Patch, and Smokey, I hope this thesis does not end up being your biting toy, or worse yet, your kitty litter. And last but not least, to Frank, you have been a source of total support and caring. I plan on making the utmost use of your promise to continue in this rote always ... TABLE OF CONTENTS
Page .. Abstract ...... II Acknowledgements ...... iii List of Abbreviations ...... v i
List of Figures and Tables ...... vii
Chapter 1 lntroduction and Literature Review ...... 1. 1 Introduction ...... 1.2 Literature Review ...... Demographicç and Epidemiology ...... Carcinogenesis ...... Treatment Modalities ...... Sex Hormones ...... Sex Hormone Receptors ...... 1. 2.5. 1 Transactivation Domain: A/B Region .. 1.2.5.2 DNA binding Domain: C Region ...... 1.2.5.3 Hormone binding Domain: E Region ... 1.2.5.4 Mechanism of Action ...... Plant-derived Compounds ...... Components of Various Plant Foods ...... 1.2.7.1 Soya ...... 1.2.7.2 Coffee ...... 1.2.7.3 Tea and Red Wine ...... 1.2.7.4 Fruits and Vegetables ...... Methods of Assessing Sex Hormone Activity in Vitro . 1.2.8.1 Breast Cancer Cells Lines ...... 1.2.8.1 .1 T47-D ...... 1.2.8.1.2 BT-474 ...... 1.2.8.2 Prostate-Specific Antigen ......
1.3 Research Hypothesis and Objectives ......
Chapter 2 Synthetic Antiprogestins ...... 2.1 Materials and Methods ...... 2.1 1 Growth and Maintenance of Cells ...... 2.1.2 Harvesting of Supernatant ...... 2.1.3 Experimental Design ...... 2.1 .3.1 Screening for Potential Agonistic Activity 2.1 .3.2 Dose-Response of Agonistic Activity ..... 2.1 .3.3 Assessment of Antagonistic Activity ...... r . I .L) r3n nssay ...... 2.1.5 Statistics ...... Results ...... 2.2.1 Screening for Potential Agonistic Activity ...... 2.2.2 Dose-Response of Agonistic Activity ...... 2.2.3 Assessrnent of Antagonistic Activity ......
Chapter 3 Plant-derived Cornpounds ...... 3.1 Materials and Methods ...... 3.1.1 Experimental Design ...... 3.1 -1-1 Screening for Potential Agonistic Activity 3.1.1.2 Dose-Response of Agonistic Activity ..... 3.1 .1.3 Assessrnent of Antagonistic Activity ...... Results ...... 3.2.7 Screening for Potential Agonistic Activity ...... 3.2.2 Dose-Response for Agonistic Activity ...... 3.2.3 Assessment of Antagonistic Activity ......
Chapter 4 General Discussion and Conclusions ...... 4.1 General Discussion ...... 4.2 Conclusions ...... 4.2.1 Future Studies ...... 4.2.2 Summary ......
Chapter 5 References ...... Page table 2.1 Structures of Synthetic Antiprogestins ...... 41 2.2 Agonistic Activity of Synthetic Antiprogestins in the BT 474 and T 47D Breast Cancer Cell Lines ...... 42 2.3 Antagonistic Activity of Synthetic Antiprogestins ...... 45 3.1 Plant-derived Compounds ...... 47 3.2 Antagonistic Activity of Plant-derived Compounds ...... 54 figure 1.1 Structures of Steroid Hormones ...... 1.2 ldealized Hormone Response Elements ...... f .3 PSA Production ...... 1.4 Structures of Flavonoids ...... 1.5 Cell Cycle ...... 2.1 Creation of Triads for Assessrnent of Antagonistic Activity ..... 2.2 Dose-Response of Synthetic Antiprogestins #2, 5. and 9 ...... 3.1 Structures of Plant-derived Compounds ...... 3.2 Dose-Response of Apigenin ...... 3.3 Antiprogesterogenic Activity of Plant-derived Compounds ... 3.4 Antiandrogenic Activity of Plant-derived Cornpounds ...... 3.5 Antagonistic Activity of Plant-derived Compounds by Groups AR - androgen receptor ER - estrogen receptor PR - progesterone receptor GR - glucocorticoid receptor MR - mineralocorticoid receptor RAR - retinoic acid receptor
E2- estradiol DHT - dihydrotestosterone MPA - medroxyprogesterone acetate MA - megestrol acetate
ER€ - estrogen response element HRE - hormone response element GRE - glucocorticoid response element
PSA - prostate-specific antigen SHBG - steroid hormone-binding globulin hsp - heat shock protein CHD - coronary heart disease CVD - cardiovascular disease lHD - ischernic heart disease
Zn - zinc
NNK - nitrosamine 4-(methylnitrosamino)-1-(3-pyridy1)-1 -butanone NDEA - N-nitrosodiethylamine NMBA - N-nitrosomethylbenzylamine 20-MC- 20-methylcholanthrene
vii CHAPTER ONE
INTRODUCTION
AND
LITERATURE REVIEW 1. INTRODUCTION AND LITERATURE REVIEW
1.1 Introduction
The relationship between diet and cancer has become a topic for much research.
Many issues have corne into the forefront, including the impact of fat, meat, antioxidant vitamins, trace elements, and now flavonoids on cancer development, and prevention
(Gregoire et al. 1991; Tonioli et al. 1994; Hunter et al. 1993; Rohan et al. 1993;
Hirayama 1986; Giovannucci et al. 1993; McKeown-Eyssen et al. 1988; Mills et al.
1989; Le Marchand et al. 1994; Greoire et al. 1989). Epidemiological studies evaluating nutrient intakes demonstrate positive associations between meat and fat intake and cancer development, as well as negative associations between vitamin, rnineral, and flavonoid consumption and carcinogenesis (Franceschi et al. 1996; Ishimoto et al. 1994;
Garland et al. 1993; Tominaga et al. 1995, Sirnopoulos 1987; Paganini-Hill et al. W87;
Hsing et al. 1990a). Flavonoids may also be important. Research has expanded in terms of carcinogenesis, and is beginning to give us new insights into the possible benefits of plant foods in cancer prevention.
Research into the ability of flavonoids to alter hormonal balance is relatively new.
Its earlier days appeared somewhat irrelevant to the human situation, Le. reproductive failures in sheep eating clover from certain fields, and cheetahs fed soy products at the
Cincinnati Zoo (Bennetts et al. 1946; Setchell et al. 1987), however such
"phytoestrogenic" effects soon proved to be relevant to humans. The follicular phase of
Japanese women were measured to be longer than their American counterparts, related to lower breast cancer risk (Henderson et al. 1985). This was shown to be manipulatable, as feeding American premenopausal women diets high in soy increased follicular phases by 1-2 days (Cassidy et al. 1994).
Phytoestrogens as they relate to breast and prostate cancer prevention have since been studied in vitro and in vivo (Messina et al. 1994; Adlercreutz et al. 1992; Lee et al. 1991; Setchell et al. 1988). This work includes their inhibitory activities on enzyme systems, the estrogen receptor pet se, and cytostasis (Pelissero et al. 1996; Adlercreutz et al. 1993; Makela et al. 1995; Miksicek et al. 1993; Kohlmeier et al. 1995; Martin et al.
1996). However, the effects of these compounds on progesterone and androgen receptors have not yet been studied. As hormone-dependent cancer development is related to an imbalance of steroid hormone production and metabolism, the possible effect on these hormones by flavonoids must be understood in order to determine the full potential of plant-derived compounds in prevention andfor treatrnent of these cancers. 1.2 Literature Review
1.2.1 Demoaraphics and Epiderniology
In 1997, hormone-dependent (e.g. breast and prostate) cancers made up the most frequently diagnosed carcinomas in women and men, respectively. The incidences of both are on the rise for the aging population. At the present time, 1 in 9 women and men will develop these diseases, mainly after the age of 50 years, with 1 in
25 Canadian women, and 1 in 27 Canadian men dying from these tumors or from their metastases (NCIC 1997). These risks are 5-10 fold higher than those seen in Japan and China. Heredity accounts for 5-1 0% of these cancers (Skolnick et al. 1997), and studies show that within two generations after migration to a new country (e.g.
Japanese in the United States), incidences of developing these cancers reach the same levels as the native population (Haenszel 1961; BueII et al. 1965; Haenszel et al. 1968;
Tominaga 1985; Thomas et al. 1987). For these reasons, environmental explanations
(including diet) are being pursued.
Dietary differences believed to be responsible include lower fat and higher fiber consumption by lower risk groups, high monounsaturated fat intake relative to saturated fat: less ingestion of animal protein, and diets high in vegetables and fruits. Wine and tea have also been associated with the lower incidence of hormone-dependent cancers in populations that make these beverages a regular staple (Block et al. 1992; Negri et a!. 1994; La Vecchia et al. 1988; Hertog et al. 1993). 1.2.2 Carcinoaenesis
Carcinogenesis is a multi-step process that takes place over 30 years or more from initial insult to turnor formation (Miller et al. 1981 ). Although progressive over tirne, carcinogenesis occurs through necessary steps occurring on a specific time course.
These steps are known as initiation, promotion, and progression.
Initiation is the initial insult to DNA through endogenous or exogenous (chernical, ultraviolet, radiation) means. Usually this darnage is repaired via excision of the error, or through apoptosis (programmed cell death) of the cell. However, if DNA replication and cell division occur before this repair or apoptosis takes place, the damage may be converted into a stable genomic error (Miller et al. 1981). This process of increased celi proliferation is known as promotion. Agents that are classified as good promoters tend to be non-genotoxic on their own, but act as irritants. Estrogens are known to be good promoters.
Over time, tumors becorne more aggressive and acquire increased malignant potential. The process by which this occurs is defined as progression. lncreasing malignancy, characterized by rapid growth, invasiveness, and the ability to metastasize progresses slowly over many years. It is related to the sequential appearance of subpopulations of cells that differ with respect to several phenotypic attributes including invasiveness, rate of growth, metastatic ability, karyotype, hormonal responsiveness, and susceptibility to antineoplastic drugs. These cancerous cells become hetergeneous, which most likely results from multiple mutations that accumulate independently in different cells, thus generating subclones with different characteristics frorn original cells, as well as from each other. Progression ultimately leads to invasion, metastasis, and death (Miller et al. 7 981).
1.Z.3 Treatment Modalities
Three treatment modalities are usually ernployed when dealing with breast and prostate cancer. They are surgery, radiotherapy, and chemotherapy, including hormone blockade.
Surgery has been used since 1890, where Halsted showed that breast cancer may not necessarily be fatal (Halsted 1890). Surgery is used today as one of several options for the management of breast and prostate cancers, with cure possible if al1 tumor cells are removed. In sorne patients, surgery may just postpone the inevitable mortality frorn metastases or frorn extremely large tumors. As with al1 treatments, the decision for surgery, is made based on patient factors, tumor characteristics and stage of disease.
Radiation is another treatment option for these cancers. Some consider it to be the most effective treatment modality currently available to control local disease
(Rutqvist et al. 1990;Rutqvist et al. 1994). Large studies including the National Surgery
Adjuvant Breast Program Trial (Fisher et al. 1989) and the Swedish Uppsala-Orebro
Trial (Uppsala-Orebro IWO) have demonstrated radiotherapy to decrease the number of breast cancer recurrences.
Hormone blockade is a third option. For breast cancer, estradiol is the hormone usually blocked, and for prostate cancer, it is dihydrotestosterone. This is accomplished through use of tamoxifen and finasteride, respectively. Up to 60% of ER-positive tumors respond to hormone therapy, but only 10% of ER-negative tumors respond
(Allegra et al. 1980; Samaan et al. 1981; Williams et al. 1987). Diethylstilbestrol and flutamide have response rates of 50-60% as primary treatment in prostate cancer patients (Chang et al. 1996). The theory behind hormone blockade is discussed in the next section.
1.2.4 Sex Hormones
Al1 steroid hormones have in common the 17-carbon cyclopentanoperhydrophenanthrene structure with four rings (A-D) (fig. 1.1). Additional carbons at positions 10, 13, or side chains attached to C17are found, as number and type of substituted group, number and location of double bonds, and stereochemical configuration al1 allow for differences in function of the groups of steroid hormones. All mammalian steroid hormones are formed from cholesterol through reactions in the mitochondria or endoplasmic reticulum of adrenal cells (Granner 1996).
Estrogens are a family of hormones synthesized in a variety of tissues. 17P- estradiol is the primary estrogen, formed mainly by aromatization of testosterone, in the ovaries of women and in the adipose tissue of men. Estrone is formed from arornatization of androstenedione in adipocytes. Estrogens have several mechanisms of action by which they can induce growth of human breast and other tissues. They can directly stimulate growth of cells, or induce nuclear transcription factors to increase the expression of genes regulated by the estrogen response element (ERE). Alternately, in 17p-Estradiol Testosterone Progesterone
Figure 1.1 Structure of Steroid Hormones
The three sex steroids (estrogen, testosterone, and estrogen) are al1 17-carbon cyclopentanoperhydrophenanthrene structures with four rings. Additional carbons at positions
10, 13, or side chains attached to CjTare found, as number and type of substituted group, number and location of double bonds, and sterochemical configuration altow for differences in function of hormone. ER-positive breast cancer cells, estrogens can stimulate their proliferation independently of interactions with other cells or tissues (Rochefort 1994).
Tamoxifen is a triphenylethylene derivative that functions mainly as an estrogen antagonist, but may have other mechanisrns of action as well. It competitively binds to the ER, forrning a complex recognized by the ERE similar to the estradiol- (E2) ER complex (Kumar et al. 1988; Miller et al. 1986), but induces alternate conformations.
Thus, transcription-activating factors are not activated, and RNA is not produced.
Tarnoxifen hence inhibits estrogen-stimulated cell division. Stilbestrol is an estrogen agonist used in the treatment of prostate cancer. It raises estrogen levels in men, hence diminishing the testosterone:estrogen ratio believed to be of major factor in the development and progression of this disease (Walsh 975; Resnick et al. 1975).
Progesterone is produced and secreted by the corpus luteum of the ovaries, from pregnenolone (Granner 1996) and generally it decreases the proliferative activities of estrogens. Progestins usually require previous or concurrent presence of estrogens to function, and the two classes of steroid hormones rnay act synergistically or antagonistically, depending on tissue and particular function (Spicer et al. 1992). Very high doses of progestins are used in breast cancer as first- or second-line therapy, and have been demonstrated to be as effective (30% response rate) as tarnoxifen in the treatment of advanced breast cancer (Sedlack et al. 1994; Canobbio et al. 1987; Howell et al. 1987; Lundgren et al. 1989; Gundersen et al. 1990; Parnes et al. 1991; Pronzato et al. 1990). They are well-tolerated, with relatively low toxicity (Henderson et al. 1989). and are currently experiencing a resurgence in use (McGuire et al. 1985; McGuire et al. 1989).
Antiprogestins are now also being used in the treatment of breast cancer. RU
486 (mifepristone) an 11 P-substituted steroid derivative utilized clinically and experimentally (Weiss 1993), and has become a starting point for the synthesis of a second generation of drugs with similar antiprogesterogenic, but weaker antiglucocorticoid activity than RU 486 (Horowitz j992).
Pregnenolone is also a precursor to testosterone, and is synthesized to such through either the progesterone or dehydroepiandrosterone (DHEA) pathway in adrenal glands, ovaries and testis. Dihydrotestosterone (DHT), a more potent androgen is then produced in men by the enzyme 5a-reductase in target tissues such as the prostate
(Granner 1996). Finasteride (Proscar), a 5a-reductase inhibitor, is used clinically in the management and treatment of prostate cancer. As such, it inhibits the reduction of testosterone to the more active form DHT (CPS 1992). Androgen agonists have been successfully used in the treatment of breast cancer (Kennedy 1958; Co-operative
Breast Cancer Group 1964; Gordon et al. 1973; Tormey et al. 1983), however, severe side effects were seen with this treatment, and hence it was withdrawn in the 1970s.
1.Z.5 Sex Hormone Receptors
The androgen and progestin receptors are two members of a group known as the nuclear receptor superfamily. Other receptors within this ciass include the rnineralocorticoid and glucocorticoid receptors, as well as the estrogen receptor, thyroid receptor, vitamin D3 receptor, and various orphan receptars with unknown functions (Berger et al. 1989; Evans 1988; Beato 1989; O'Malley 1990; Fuller 1991 ; Wahli 1991).
Nuclear receptors consist of at least three functional regions (Wahli 1991). Starting from the N-terminus of the protein, these regions are:
1.2.5.1 Transactivation Domain: NBReaion
This region is responsible for optimizing the transactivation capability of the receptor, i.e. regulating and activating transcription factors such as DNA-dependent
RNA polymerase (Evans 1988; Beato 1989; O'Malley 1990; Fuller 1991; Wahli 1991). It also recognizes specific sequences in transcriptional areas of the genes to which it binds. Because this domain does not contain a conserved region, steroid receptors are able to interact and bind with other cellular proteins (Wahli et al. 1991). This region is promoter and cell-type specific.
1.2.5.2 DNA bindina Domain: C Reaion
This 66-68 amino acid-long region recognizes specific DNA sequences known as hormone-response elements (HRE) (Evans 1988; Beato 1989; O'Malley 1990; Fuller
1991; Wahli 1991). These sequences are specific for the type or subfamily of receptor and are made up of a 15 nucleotide-long partially palindromic sequence, usuaily found at the 5'4anking region of the gene. These 15 base pairs are arranged as two hexameric half-sites separated by three bases (fig. 1.2) (Ham et al. 1988; Cato et al.
1988).
The DNA-binding dornain is cysteine rich and is well conserved among categories within the nuclear superfamily (Amero et al. 1992; Laudet et al. 1992). This Consensus Hormone Response Element (PRE, ARE, GRE)
Estrogen Response Element (ERE)
AGGTCAnnnTGACCT TCCAGTnnnACTGGA
Figure 1.2 ldealized Hormone Response Elements
The DNA binding domain of the nuclear receptor superfamily recognizes specific DNA sequences known as hormone-response elernents (HRE). These sequences are specific for the type or subfamily of receptor and are made up of a 15 nucleotide-long partially palindromic sequence, found at the 5'-flanking region of the gene. Glucocorticoid-type receptors (including progesterone and androgen receptors) and estrogen receptors discriminate between each others HREs in vivo. This is accomplished through consensus elements that differ at two bases in each half site. region is folded into two zinc "fingers", Zn coordination sites made up of two zinc ions each, tetrahedrally coordinated by 4 cysteines to stabilize two peptide loops and cap arnino termini of two arnphipathic a-helices. The first finger specifies the receptor's
DNA recognition sequence, and the second allows for dirnerization of two receptor molecules during their binding with DNA (Wahli et al. 1991 ).
Glucocorticoid-type receptors (including progesterone (PR), and androgen receptors (AR)) and estrogen receptors (ER) discriminate between each others HREs in vivo. This is accomplished through consensus elernents that differ at two bases in each half site (Mader et al. 1989; Danielson et al. 1989; Umesono et al. 1989) (shaded areas in fig. 1.2). The HRE responsible for transcription of the PSA gene recognizes and binds only to the GRE motif. Hence, stimulation of any or al1 of these receptors increase transcription of this gene, leading to production of PSA. Moreover, as ER does not have this motif, PSA is unaffected directly by estrogen stimulation.
1.2.5.3 Hormone bindinq Domain: E Reaion
This region consists of 250 amino acids at the C-terminus of the steroid receptor and is responsible for ligand binding. Once bound by a com2atible hormone, conformational changes are induced that rearrange important amino acid residues to a proper tridimensional position, activating the transcriptional activity function of the receptor. Other transcriptional factors may interact with the activated receptor, leading to RNA synthesis. When unbound, this region folds over the DNA-binding domain (C region), preventing any association between unbound hormone and DNA (Jensen et al. 1991). This process is necessary for proper binding to DNA.
1.2.5.4 Mechanism of action
In the absence of ligand, the latent receptor resides as part of a macromolecular complex containing heat shock protein (hsp) 90, hsp 70, p59, and other proteins, located in the nucleus of the cell (Smith et al. 1993). If a ligand for a steroid receptor
(progestin or androgen) is present within the cytoplasrn of the cell, it can passively diffuse into the nucleus and bind with its respective receptor(s). Once bound by ligand, alterations in the receptor structure occur. Steroid receptor agonists and antagonists induce distinct conformational changes within their specific receptors (Allan et al. 1992;
Beekman et al. 1993). This change promotes the dissociation of the heat shock proteins, thereby permitting the interaction of the receptor with specific DNA sequences in the regulatory regions of target gene promoters (Martinez et al. 1989; Tsai et al.
1989; Kumar et al. 1988). After activation of the complex though phosphorylation, subsequent binding occurs with its HRE on the PSA gene. This stimulates and facilitates binding of transcription factors with their regions on the PSA gene, which results in transcription of the gene. Translation occurs soon after, and measurable amounts of PSA protein are produced. The estrogen receptor does not bind to the HRE of the PSA gene. Therefore, the presence of estrogen cannot contribute to the production of PSA. However, estrogen, in relatively large amounts, may contribute to inaccessibility (through competitively binding with androgen and progestin receptors, or hormonal balance manipulations) of ligands to these steroid receptors, which may ultimately result in PSA not being produced (fig. 1.3).
1.2.6 Plant-derived Com~ounds
Flavonoids are a class of compounds possessing a diphenolic structure (fig. 1.4).
They are found ubiquitously in plants, and are in highest concentrations in soy, vegetables, fruits, tea, and red wine (Hertog et al. 1993; Hertog et al. 1995). The class includes isoflavonoids, flavans, fiavones, fiavonones, flavonols, anthocyanins, and catechins (Miksicek 1995). Flavonoids are found in nature as glycosides. Upon consurnption, they are conjugated in the gut, deconjugated in the blood, and reconjugated in the liver and kidney for excretion. The relative arnounts of free to conjugated forms are unknown, but the total half-life of these compounds in the body is approximately 7 hours. These conjugates can be rneasured in urine, blood, bile and feces (Adlercreutz et al. 1995).
Many compounds with diphenolic structure are able to function as phytoestrogens, with agonistic or antagonistic activity (Coward et al. 1993). Although potencies and affinities Vary tremendously from among groups within the phytoestrogen class, as well as within groups, on average their binding affinities are 100 to 10,000 times higher than diethystilbestrol (DES) or estradiol (Farmakalidis et al. 1985). Their potencies, as measured through experiments with genistein, approximate 10" to IO'= that of these estrogens (Kitts et al. 1980; Somjen et al. 1976; Mayr et al. 1992;
Welshons et al. IWO; Newsorne et al. 1980; Folman et al. 1969; Markiewicz et al. 1993;
Adlercreutz 1990), allowing them to act as partial agonists and partial antagonists of the Steroid
Nucleus A %%=Receptor
Hormone-Receptor complex \
Figure 1.3 PSA Production
In the absence of ligand, the nuclear receptor resides as part of a macromotecular complex in the nucleus of the cell. If a ligand for a steroid receptor (progestin or androgen) is present within the cytoplasm of the cell, it can passively diffuse into the nucleus and bind with its respective receptor(s). Once bound by ligand, the complex is phosphorylated and binds to its HRE on the
PSA gene. Transcription factors bind to the gene, and transcription occurs. The mRNA is translated in the endoplasmic reticulum, producing PSA protein. Through blocking of receptor, phosphorylating enzymes, or transcription factors, PSA production may be blocked. il O O Isofiavone FIavone Fiavanone
Flavonol Anthocyanin Flavan
Figure 1.4 Structures of Flavonoids
Flavonoids are a class of compounds possessing a diphenolic structure. The class includes isoflavonoids, flavans, fiavones, flavanones, flavonols, anthocyanins, and catechins. estrogen receptor. These structures are also quite similar to those of androgens, and hence may also function on these and other nuclear subfamily receptors.
Flavonoids have been demonstrated to have various activities in vitro and in vivo.
Their antioxidant capacities have been a major focus of research, in terms of prevention of coronary heart disease (CHD). The "French paradox" may be partially explained by the high flavonoid concentration in red wine, and several epidemiological studies have shown that populations consuming high amounts of these polyphenols in their diets
(e.g. Mediterranean diet) have lower incidence of CHD than populations who consume low amounts (Hertog et al. 1995).
In the cancer arena, flavonoids have been demonstrated to block many enzyme systems responsible for cell proliferation including aromatase and tyrosine kinase, arrest the cell cycle in various phases, cause apoptosis, inhibit tumorigenesis in vivo, scavenge free radicals. and manipulate estrogen concentrations in various ways.
1.2.7 Corn~onentsof Various Plant Foods
The original work in the field of flavonoids arose through the discovery of isoflavones in soybeans and soya products. Estrogenic effects of these compounds were discovered by evaluating what was causing reproductive failure in female animals such as cheetahs and ewes fed clover or soy feed, later termed "clover disease"
(Setchell et al. 1987; Adams et al. 1979a; Adams et al. 1979b; Bennetts et al. 1946).
This occurred due to impaired transport of spermatazoa through the cervix of these animals (Lightfoot et al. 1967) caused by rnorphological changes of the reproductive tract induced by isoflavones (Nwannenna et al. 1995). Such changes occur in rnany other species as well, including some strains of rats and mice treated with fiavonoids during neonatal life (McEwen et al. 1972). Isoflavones rnay also function to defeminize females and demasculize males of some species, e.g. rats, by decreasing luteinizing hormone surges in the fernales, and retaining capacity of this surge in males (Herbst et al. 1972; Arai et al. 1968). Such problerns do not occur among al1 species, including cattle, and do not occur in humans. It is believed that nonsusceptible populations convert isoflavones relatively more rapidly to inactive metabolites than those susceptible to reproductive and sexual effects, or lack metabolic pathways for activation of highly estrogenic rnetabolites (Cassidy et al. 1995).
1.2.7.1 Soya
Soya is a very rich source of flavonoids, containing 2 mg of biochanin A, genistein, and daidzein, per gram (defatted) (Coward et al. 1993). These compounds are not destroyed or removed by processing, except for concentrates prepared by alcohol extraction (Anderson et al. 1985). In prospective and case-controlled studies significant negative correlations were found behveen intake cf isoflavones (from miso or tofu consurnption) and breast cancer risk (Hirohata et al. 4985; Nomura et al. 1978;
Hirayarna et al. 1986; Lee et al. 1991 ). In at least one of these studies (Lee et al. 1991) the ratio of soya to total protein remained significant after control for other dietary variables.
The anti-estrogenic effects of isoflavones have also been demonstrated in relation to reproductive stages in humans, namely menopause and menstrual cycle.
Studies have been conducted to determine whether or not soya supplementation can reduce menopausal symptoms and increase cycle length (Cassidy et al. 1994).
Japanese women have been shown to have follicular phases 2-3 days longer than
American women. Moreover, unlike in Western societies, where menopause is associated with physiological, physicat and emotional stresses, in rural Asian areas, where tofu consumption is abundant, this natural transition of life stage occurs without trauma (Murkies et al. 1995).
In one study on the effects of soya on rnenstrual cycle, it was found that 60 g per day (equivalent to 45 mg of isoflavones) given for 1 month significantly (p Supplernentation of soy flour also significantly decreased the number of hot flashes in menopausal women over a 12-week period, with a significant response after 6 weeks. The negative control, wheat flour also resulted in decreases of these episodes, though not as significantly. This choice of control was obviously a faulty one, as wheat has been shown to contain other phyto-sex steroids, namely enterolactones (lignans). Therefore, these compounds were both shown to have estrogenic activity (Murkies et al. 1995). More studies, using a truly negative control, are necessary to confirm these results. Phyto-sex steroids may prevent or be used to treat hormone-dependent cancers through different mechanisms. These include their effects on hormone metabolism and sex hormone receptors. Diets high in flavonoids, e.g. Asian-type diets have been associated with increased levels of SHBGand decreased levels of free estradiol. These compounds stimulate SHBG synthesis by the liver, resulting in less binding of estrogen receptors. This process is believed to be related to lower breast cancer risk (Mousavi et al. 1993; Murphy 1982). These compounds also competitively bind to ERS in breast cancer cells or to hormone-metabolizing enzymes. Their antiestrogenic effects can inhibit the growth and proliferation of hormone-dependent cancer cells. Many phyto-sex steroids are structurally similar to tamoxifen, which lowers risk of contralateral breast cancer in women with breast cancer and is currently under investigation as a primas, preventive agent for high-risk women. Many in vitro and animal studies have been conducted with soy isoflavones to determine their anticarcinogenic effects. In vitro effects of genistein at low concentrations (100-200 nM) in the absence of estrogenic steroids include increased growth of MCFJ breast cancer cells (Martin et al. 1978; Zava et al. 1997). At higher concentrations (>2 PM), found often in human sera. genistein has been demonstrated in the MCF-7 breast cancer cell line to bind to the estrogen receptor, thus preventing binding by estradiol. Thereby this receptor cannot be translocated to the nucleus. Inhibition of cellular growth has also been seen (Zava et al. 1997; Peterson et al. 1996) in both ER positive and negative cell lines of breast and prostate cancer origin. This suggests more than one mechanisrn by which these compounds act, one that is similar to estradiol's effects on signal transduction (Migliaccio et al. 1993). Genistein, along with the other flavonoids, has also been demonstrated to inhibit tyrosine phosphorylation by tyrosine protein kinases, which may regulate phosphorylation of estrogen receptors, essential for estrogen receptor activation (Ogawara et al. 1989). Genistein can also act additively or synergistically with other antitumor agents (e.g. tamoxifen) to inhibit cancer cell growth or to induce differentiation. Furthermore, daidzein and equol, an isoflavan produced by intestinal microfloral activity on soy isoflavones, have been shown to have estrogenic and anticarcinogenic activity (Tang et al. 1980). Equol accounts for approximately 30% of total isoflavones in biological fluids (Axelson et al. 1984; Setchell et al. 1984), and hence may play a major role in hormone- dependent cancer resistance. Genistein has also been demonstrated to arrest human gastric cancer (HGC-27) cells at the G2-M phase (Matsukawa et al. 1993) (fig. 1.5). Dose-dependent inhibition of growth is observed between 10 and 60 PM, with an 50% inhibition concentration (ICsO) of genistein of 20 pM on day 4 in the HGC-27 cell line. This flavonoid may also cause a weak block in late S phase. A concentration of greater than 25 pM genistein was needed to alter cell cycle progression, with a maximum accumulation of cells at G2-M observed at 40 FM or more. Genistein is also cytostatic against human leukemic (HL- 60) cells (Hirano et al. 1994). This rnay be achieved through blocking membrane hJa'K'-~~Paseof tumor cells (Suolinna et al. 1975; Kuriki et al. 1976; Belt et al. 1979). Genistein has been shown to inhibit DNA topoisornerases 1 and II in a manner similar to anticancer agents and plant lignan etoposides (Marcovits et al. 1989). This I- I- Growing ce11 1 Commits ta division: + passes START Newbom 1 cell 4 Enters Go (quiescent cetl) Figure 1.5 Cell Cycle Many flavonoids have been dernonstrated to be cytostatic at various phases of the cell cycle. Genistein arrests human gastric cancer cells at the G2-M, blocking division of the parent cell to Wo daughter cetls. Apigenin has similar activity in neuronal cells. Quercetin has been shown to inhibit gastric and colon cancer cells at the G1-S boundary, thus preventing DNA replication. isoflavone also inhibits ribosomal S6 kinase activity and phosphatidylinositol breakdown. These resülts can be observed at concentrations as low as 1 to 1O pmol/ml, and are seen in other cancer lines of human and rodent origins (Robinson et al. 1993). These inhibitions of activities act separately or in combination to induce DNA damage and apoptosis (Devasagayam et al. 1995). Animal models have demonstrated the antitumorigenicity of soy isoflavones on mammary cancer. Genistein and daidzein have been shown to reduce tumor numbers in rats (Molteni et al. 1995; Coward et al. 1993). Female Sprague-Dawley rats treated on days 2,4 and 6 postpartum with genistein and initiated with dimethylbenz(a)anthracene (DMBA) had mammary tumor development delayed by 37 days (p 1995). Female rats treated neonatally with genistein had an incidence of mammary tumors 12% lower than controls, and had almost 50% fewer tumors (p Martiniere et al. 1995). Pretreatment with genistein and/or daidzein of ICR female rats initiated with DMBA or DMSO-reduced DNA adduct formation at least marginally, as well as reduced proliferation of bone marrow cells (Giri et al. 1995). 1.2.7.2 Coffee In the past, the relationship between coffee and hormone-dependent cancers was quite a controversial subject (Minton et al. 1979; Minton et al. 1981). However, recent studies have shown no association between the two (Schairer et al. 1987; Hsing et al. 1990b), and even a protective effect in some cases (Franceschi et al, 1995). The compounds present within this "hot" beverage are the xanthines, including caffeine, theobromine, and theophylline. Caffeine is the chernical within coffee most studied in the literature. Retrospective studies in both premenopausal and postmenopausal women found no correlation between caffeine intake and breast cancer (Folsom et al. 1993; Smith et al. 1994). However, another study on postmenopausal wornen showed inverse associations between caffeine intake and bioavailable testosterone, and a direct association with plasma estrone. SHBG levels, binding free estrogen were also increased (Ferrini et al. 1996). These results suggest that caffeine may influence sex hormone concentrations, but with increased SHBG, should not increase breast cancer incidence. Caffeine was not associated with prostate cancer in a retrospective study either (Slattery et al. 1993). Theobromine, another xanthine found in coffee presented a different result to this study. It was found that men consuming the higher 2 tertiles of this compound had an increased risk of prostate cancer, caused by a biological mechanism sirnilar to that of caffeine in women. The study concluded that further exploration into theobromine was necessary (Slattery et al. 1993). 1.2.7.3 Tea and Red Wine Epidemiological studies have shown an association between tea and red wine consumption and protection against human cancers. Case-control studies have pointed to reductions in at least gastric, if not other types of cancer (Wang et al. 1992; You et al. 1989; Buiatti et al. 1989; Kono et al. 1988). Various tea and red wine fiavonoids have been demonstrated in vitro to be cytostatic in several different cell lines, arresting the cell cycte at different phases. Quercetin has been shown to inhibit gastric and colon cancer cells at the G1-S boundary of the cell cycle (Yoshida et al. 1990; Hosokawa et al. 1990) (fig.l.5), and produces dose-dependent growth inhibition of MCF-7 breast cancer cells (Scambia et al. 1994). Apigenin is cytostatic in the neuronal cell line BIO4 (Sato 1994). At 15-60 pM it inhibited proliferation in a dose-dependent manner, with its lCsobeing 12.5 pM on day 4. lt arrests the cell cycle in the G2-M phase. This action was seen in BI04 with other flavonoids as well, and such growth inhibition is believed to be independent of structural groups. The use of apigenin as a chemopreventive and antitumor drug is presently being investigated (Sato et al. 1994). As phytoestrogens, quercetin and kaempferol have also been shown to decrease estrogen receptor concentration, and induce progesterone receptor-negative cells to express progesterone receptors (Scambia et al. 1993). This effect may optimize hormonal therapy in breast cancer treatment. Much animal work has also been conducted using tea or its flavonoids (also found in red wine). Treatment with black or green tea water extracts reduced lung and gastric papilloma incidence and tumor size in female AIJ mice treated with N- nitrosodiethylarnine (NDEA) (Yang et al. 1994). 0.6% decaffeinated green tea or black tea given during treatment with nitrosamine 4-(methylnitrosamino)-1-(3-pyridy1)-1- butanone (NNK) as the sole source of drinking water, tumor multiplicity was reduced by 67% or 65%, respectively. When given after NNK treatment until the end of the experiment, 0.3% and 0.6% decaffeinated green tea reduced tumor multiplicity by 74% and 85%, respectively, and tumor incidence by 14% and 30%, respectively. Decaffeinated black tea decreased tumor multiplicity by 63%, but did not significantiy reduce tumor incidence (Yang et al. 1994). These results showed that black tea, which contains much lower amounts of catechins than green tea, was just as effective as green tea in inhibiting tumorigenesis when given to mice during the carcinogen treatment. This is in line with Wang et ai (Wang et al. 1988) who reported that green and black tea polyphenols inhibited benzo(a)pyrene metabolisrn in rat liver microsornes. Thus tea preparations may have the ability to inhibit activation of a variety of carcinogens. Other mechanisms suggested include induction of phase II enzymes (Sparnins et al. 1982; Khan et al. 1992) and trapping of ultimate carcinogens (Wang et al. 1989). Administration of 0.6% decaffeinated green tea or black tea extract as sole source of drinking water to Sprague-Dawley rats treated by subcutaneous injection with NMBA (2.5 mglkg) twice weekly for 5 weeks resulted in less esophageal tumor incidence and 70% less multiplicity. Decreases in esophageal papilloma sizes by 89% and 97% in the green and black tea groups, respectively, were seen (Han et al. 1990). These tea infusions also inhibited esophageal tumorigenesis caused by NMBA precursors (methylbenzylamine plus sodium nitrite) in rats, reducing tumor incidence by 80% to 95% (Xu et al. 1990). Quercetin and luteolin have been demonstrated to be antitumorigenic agents. It was observed that administration of quercetin or luteolin to 20-methylcholanthrene (MC)- treated mice reduced tumor incidence 47% and 40%, respectively. Turnor volume was also significantly lower in these groups, as well as rutin- and (+)-catechin- treated animals than in those treated with carcinogen alone (Elangovan et al. 1994). There was also a significant delay in growth, and reduced death rate in the flavonoid- treated animals. Furthermore, inhibition of (3~)thyrnidineincorporation was found to be highest in animais treated with luteolin, followed by quercetin, rutin and (+)satechin. This is important because incorporation of (3~)thymidineis reflective of cell division, and cancer is a disease of uncontrollable replication. Therefvre one mechanism by which luteolin and quercetin reduce tumorigenesis is through their effect on DNA synthesis (Elangovan et al. 1994). Another mechanism is through inhibition or stimulation of enzyme systems. Animals treated with these quercetin and luteolin also showed a reduction in lipid peroxide and cytochrome P450, and an increase in g1utathione-S- transferase (Elangovan et al. 1994). Compounds such as quercetin with 3',4'dihydroxy substituents in the 6 ring and conjugation between the A and B rings, have antioxidant potentials four times that of Trolox, a vitarnin E analogue. Removing the ortho-dihydroxy substitution, as in kaempferol, or the potential for electron delocalization by reducing the 2,3 double bond in the C ring, as in catechin and epicatechin, decreases the antioxidative potential by more than 50%, but these structures are still more effective than ascorbic acid or a- tocopherol (Morel et al. 1994). The iron-chelating ability of catechin and quercetin has been investigated on iron-loaded hepatocyte cultures, measuring the prevention of iron- increased lipid peroxidation and inhibition of intracellular enzyme release. Catechin was more effective than quercetin in protecting these cells from damage. The lCsoswere related to structural characteristics, and showed that these compounds function not only as antioxidants, but as chelators as well, to be cytoprotective in normal cells (Liu et al. l99l). Antioxidant properties of tea and red wine components may act as cytoprotective agents by various mechanisms. These include guarding cells against lipid peroxidation, DNA single-strand breakage, and formation of 8-hydroxydeoxyguanosine. These insults rnay contribute to carcinogenesis at both initiation and post-initiation stages. Catechin was reported to inhibit NNK-induced DNA single-strand breaks in rat hepatocytes in vitro and in vivo (Bhimani et al. 1991), and (-)epigallocatechin-3-gallate was shown to inhibit TPA-induced 8-hydroxydeoxyguanosine formation in human epithelial HeLa cells (Xu et al. 1992). Oral administration of green tea was also shown to inhibit formation of 8-hydroxydeoxyguanosine in mice (Verdeal et al. 1980). Such mechanisms rnay help to explain the epidemiological evidence of red wine and tea being inversely correlated with chronic diseases including CVD and cancer. 1.2.7.4 Fruits and Veaetables The Canada Food Guide recomrnends that Canadians eat between 5 and IO servings of fruits and vegetables a day. This recommendation is derived from studies showing lower mortality rates in groups with high fruit and vegetable consumption (Key et al. 1996), especially in regards to CVD and cancer. Many cornpounds are found in these foods attributing to the decreased morbidity and mortality rates, including fiber, antioxidant and other vitamins, and flavonoids. In case-control studies of two Chinese populations, it was found that green vegeta ble intake had a statistically sig nificant inverse association with breast cancer risk (Yuan et al. 1995). A cohort study conducted over 6 years found tomato products, containing lycopene, inversely associated with risk of prostate cancer when consumed greater than 10 versus 1.5 times per week (Giovannuci et al. 1995). Moreover, another case-control study found a strong inverse association between total vegetable intake and breast cancer risk in premenopausal women, independent of vitamin C, a-tocopherol, folic acid, dietary fiber, and a-carotene components (Freudenheim et al. 1996). The Netherlands Cohort Study, a large prospective cohort study, found a non- significant inverse relationship between fruit consumption and breast cancer risk (Verhoeven et al. 1997). Animal and in vitro studies have demonstrated antitumorigenesis and cytostasis by flavonoids found in fruits and vegetables. The breast carcinoma cell line MDA-MB-435 showed 50% inhibition of proliferation after treatment with hesperetin, naringenin, or quercetin, and even greater inhibition in 1:1 combinations. IC50sobserved were from 4.7 pg/rnL quercetin plus hesperetin, and quercetin plus naringenin, to 22.5 pg/mL naringenin plus hesperetin. Tumor development was delayed in rats given orange juice or fed naringin-supplernented diets, compared with controls. Those given orange juice also had smaller tumors. The studies were concluded to demonstrate that citrus flavonoids, and other fiavonoids found in fruits and vegetables are effective inhibitors of breast cancer cell proliferation, especially when combined with quercetin (So et al. 1996). Citrus flavonoids (naringin, hesperidin, tangeritin) have also been shown to elevate levels of glutathione transferase in animals treated with them. This effect may be due to antioxidant effects and rnay be one of the factors for their anticarcinogenic activity (Elangovan et al. 1994; Oganivara et al. 1989). Flavonoids and related tannic acid also act to significantly inhibit single-strand breaks in plasmid pBR322 DNA induced by singlet molecular oxygen. Myricetin showed highest protective ability, followed by tannic acid, (+)-catechin, rutin, fisetin, luteolin and apigenin, after 90 minute incubation. Protective effects were time and concentration dependent. At equimolar concentrations (100 FM) antioxidative activity of myricetin was better than that of the antioxidant vitamins a-tocopherol and p-carotene (Rice-Evans et al. 1995). Through these mechanisms and others, flavonoids found in fruits and vegetables rnay act individually or in combination with other properties of these plant foods to reduce cancer initiation and progression. 1.2.8 Methods of Assessina Sex Hormone Activitv in Vi-tro In order to assess the activity of flavonoids on androgen and progesterone receptors, two breast cancer cell lines, and a marker protein were chosen. 1.2.8.1 Breast Cancer Cell Lines 1.2.8.1.1 T47-D This breast carcinoma cell line was isolated by Keydar from a pleural effusion obtained from a 54-year-old fernale patient with an infiltrating ductal carcinoma of the breast. This differentiated epithelial substrain contains receptors to 17 P-estradiol, androgens, progestins, and other steroids (Freake et al. 1981; Sher et al. 1981; Larnp et al. 1981). 1.2.8.1.2 BT-474 This line was isolated by Lasfargues and Coutinho (Lasfargues et al. 1978) from a solid, invasive ductal carcinoma of the breast obtained from a 60-year-old fernale patient. The cells Vary greatly in size. and have large nuclei with one or more nucleoli. It has androgen, progesterone, and estrogen receptors. 1.2.8.2 Prostate-S~ecificAntiaen Prostate-Specific antigen (PSA) is a 33 kDa monomeric glycoprotein widely used today in the monitoring of prostate cancer (Oesterling 1993). It was first identified in seminal plasma by Hara et al in 1971 (Hara et al. 1971), and isolated from prostatic tissue in 1979 (Wang et al. 1979). It was first found in human serum by Papsidero et al in 1980 (Papsidero et al. 1980). Its half-life in serum is 2.2 to 3.2 days (Stamey et al. 1987; Oesterling et ai. 1988). PSA is a kallikrien-like serine protease, produced primarily by epithelial cells lining the acini and ducts of the prostate gland. Its function is to lyse seminal coagulum (Lilja 1985). PSA has trypsin and chymotrypsin-like activity (Oesterling 1993). PSA is found in the serum of normal males at levels of less than 4 nglml. The clinical cut-off point of PSA concentrations as non-cancerous prostatic carcinoma (including benign prostatic hypertrophy, prostatitis, and prostatic infarction) is unclear. Some authorities consider it to be as high as 15 nglml (Mettlin 1993). Patients with biopsy-proven prostate cancer are unlikely to have extracapsular disease or metastasis if PSA is less than 20 nglml. PSA values of 40 nglml and above are usually indicative of metastatic prostate cancer, however, non-metastatic disease has been seen with PSA values of greater than 80 nglml. PSA is a helpful negative predictor of prostate cancer, with an accuracy rate of 94 to 96% (Mettlin 1993). Scientists are now focussing on proteins produced by breast tumors to aid in diagnosis, prognosis and treatment. One of these proteins is PSA. It is now believed that prostate-specific antigen is a favorable prognostic indicator in breast cancer. PSA immunoreactivity was found in 30% of over 1200 female breast tumor cytosols at levels greater than 0.03 ng PSNmg total protein. Al1 of these tumors were estrogen and progesterone receptor-positive, whereas ER and PR-negative tumors were found not to produce this protein. PSA is associated with early clinical stage of the disease, and smaller tumor sizes. Furthermore, PSA-positive patients have been found to be less likely to relapse (greater than 60% less) or die than PSA-negative patients (Yu et al. 1995a). PSA is measured in tumors and in vitro systems in supernatant or ceIl lysate as a 33 kDa protein. It can also be detected in serum, as a 100 kDa rnolecule, corresponding to PSA-bound al-antichyrnotrypsin (Yu et al. 1994; Yu et al. 1995a). PSA is also measurable in normal breasts of subjects taking progesterone-containing oral contraceptives (Yu et al. 1995b), in postpregnant women, and in the milk of lactating rnothers (Yu et al. 1995~).It has also been detected in breast cyst and amniotic fluids (Yu et al. 19956). PSA has been suggested to be in high arnounts in fetuses with congenital abnormalities (Yu et al. 1995~).It is also found in other tumors including ovarian, liver, kidney, adrenal, colon parotid and lung (Levesque et al. 1995). Such observations have led researchers to hypothesize that PSA may act similarly to other proteinases of the kallikrein family, and rnay play a role as a local regulator of uterine function (Yu et al. 1995a). In vitro studies have shown that breast cancer cell lines MCF-7 and T47D can be stimulated by androgens and progestins to produce PSA. Maximum production occurs in response to 1 to 1O-' mol/L of these cornpounds. Estrogen can suppress PSA production by these steroids (Yu et al. 1994). Therefore, PSA rnay be used as a model and an indicator of hormone balance within the tumor or cellular system between estrogen and androgenlprogestin. This model will allow us to assess the effects of plant-derived compounds as agonists and antagonists of the androgen and progesterone receptors. Agonisrn will be assessed by measuring amounts of PSA produced upon incubation of the cells with candidate compound alone. Antagonisrn will be determined by measuring PSA production upon incubation of the cells with candidate compound plus steroid, relative to PSA produced by steroid alone. 1.3 Research Hypothesis and Objectives I hypothesize that certain natural products, which are components of plant foods or beverages, may have steroid hormone agonist or antagonist activity by interacting with progesterone and/or androgen receptors. This hypothesis will be initially tested by using a tissue culture system consisting of a steroid hormone receptor-positive breast carcinoma cell line. The activity of the candidate compounds will be assessed by monitoring the expression of the prostate- specific antigen gene which is known to be regulated by steroid hormones (Yu et al. 1994). The main objectives are: 1. To determine whether the PSA assay system can be used to assess sex hormone activity by using a set of synthetic antiprogestins synthesized by the Southwestern Research Foundation. 2. To determine the agonistic activity of 45 plant-derived compounds on progesterone and androgen receptors by measuring PSA produced upon stimulation of Bi474 breast cancer cells with the plant-derived compounds. 3. To determine the antagonistic activity of 45 plant-derived compounds on PR and AR by assessing PSA production upon stimulation of T 47D breast cancer cells with plant-derived and steroid, relative to PSA production upon stimulation with steroid atone. CHAPTER TWO SYNTHETIC ANTIPROGESTINS Rask, then detached by trypsin-EDTA treatment to be split or subcultured in 48-well microtiter plates for experimentation. The subculture medium was the same as above, except charcoal stripped fetal calf serum was used instead of the regular fetal calf serum, as it is devoid of any steroid hormones. Cell clumps were minimized by passing cells and media through an 18-gauge syringe. Cells were grown as rnonolayers until confluency, with approximately 2 x lo5cells per well, and each well contained 1 ml of medium. 2.1.2 Harvestina of Supernatant Supernatant was separated from the cells through aspiration, using plastic pipets. Supernatant was irnmediately transferred into labeled Eppendorf tubes, and either irnmediately used for PSA assay or stored at -20°C until analysis. PSA is a very stable molecule, able to be stored for long periods of time without degradation (Hoffman et al. 1996). 2.1.3 Ex~erimentalDesian 2.1.3.1 Screenina for Potential Aaonistic Activitv BT-474 and T47-D breast cancer cells were grown to confluency in 48-well and 24-well plates, respectively, under standard conditions, and stimulated by seven novel antiprogestin compounds (table 2.1, encoded O, 1, 2, 3, 5, 6, and 9) at final concentrations of 1 and IO" M. AH compounds were diluted with pure anhydrous ethyl alcohol. Positive controls were norgestrel (a progestin), testosterone (an androgen), and norgestimate (a progestin) (Sigma Chemical Co., St Louis, MO 631 78). Alcohol was the negative control. Mifepristone (Roussel-UCLAF, Romainville, France) was used as a known antiprogestin with weak progesterogenic activity. 2.1.3.2 Dose-Response of Aeionistic Activitv BT-474 cells were incubated with 1PL of synthetic antiprogestin encoded #O, 1, 2, 3, 5, 6, or 9 at final concentrations of 1 to 1o4 M in 1ml media. Norgestrel at same concentrations was used as a positive control. Alcohol was a negative control. 2.1.3.3 Assessment of Antaaonistic Activitv T47-D cells were grown to confluency under standard conditions in 48-well microtiter plates. The cells were incubated with 1 pL in 1 mL media of cornpound #O, 1, 2, 3, 5, 6, or 9 for 1 hour at final concentrations of 1o-~ or 1O-' M. Dihydrotestosterone, or norgestrel were added at final concentrations of 10" or 10'" M. or norgestimate at 10-8 M to create triads of progestin blocker alone, blocker plus agonistic steroid, and steroid alone (fig. 2.1). Alcohol was a negative control. Incubation time was 7 days. 2.1.4 PSA Assav PSA protein was measured with a modified highly sensitive immunofluorometric procedure that can measure PSA at levels of 1 ng/L or higher (up to 10,000 nglL) with a precision of 4 0% (Ferguson et al. 1996). This assay uses a mouse monoclonal anti- PSA capture antibody coated to polystyrene rnicrotiter wells, a biotinylated mouse monoclonal detection antibody, and alkaline phosphatase-labeled streptavidin. blocker blocker + norgestrel blocker blocker + DHT norgestrel DHT blocker #1 #2 #6 #7 alcohol alcohol + norgestrel alcohol alcohol + DHT norgestrel DHT Figure 2.1 Creation of Triads for Assessment of Antagonistic Activity Triads were created for the assessrnent of antagonistic activity for both synthetic antiprogestins and plant-derived compounds. The triads were blocker alone, blocker plus agonistic steroid, and steroid alone. Alcohof was used as the negative control. 100 pl of sample were incubated in polystyrene microtiter wells with 50 pL of biotinylated anti-PSA antibody diluted 1:IO00 in PSA assay buffer and shaken for 1.5 hours. After 6 washings, 100 ~Lalkalinephosphatase-labeled streptavidin conjugate, diluted 1:1000 in 6% BSA, was added and shaken for 15 minutes, followed by another 6 washings. 100 pL5-ff uorosalicylate phosphate diluted 1:i O in substrate buffer was added and shaken for 10 minutes, then 100 pl ~b~'-EDTA ("developing solution") was added and the plate was shaken for a further 2 minutes. If PSA was present in the samples, a ternary fluorescent complex was formed between the released 5- fluorosalicylate, ~b~',and EDTA. The amount of fluorescence was quantified through the-resolved fluorescence using a pulse nitrogen laser excitation source (Cyberiiuor Inc., Toronto, ON), and correlated to PSA concentration in ng/L. 2.1.5 Statistics All studies were conducted four tirnes each, and expressed as mean + SEM. For analysis of antagonisrn studies, percent blocking was calculated as the percentage of PSA produced compared to the rneans of steroid (DHT or norgestrel) alone for each separate plate. Coefficients of variation were calculated for al1 compounds. Analysis between groups was conducted using Student-Newman-Keuls multiple range test. Pearson correlation was used to assess the strength of association between the receptors. 2.2 Results 2.2.1 Screenina for Potential Agonistic Activity Three of the seven compounds tested at 1oq7 M final concentration in both BT- 474 and T47-D cells were found to have agonistic activity. Thase were compounds #2, 5 and 9. Compound #5 was found to have strong activity, followed by compound #9, and finally cornpound #2 with moderate activity. Compound #O was found to have weak activity in the BT-474 cell line, but was negative in the T47-D line. Alt other cornpounds (#Il 3, and 6) were found to be negative for agonistic activity in Our system (table 2.1). These compounds at Iom9M final concentration in the BT-474 line were found to have no agonistic activity. However, in the T47-D line, weak activity was seen for compounds #5, 2, and 9 at this concentration (table 2.2). This was attributed to the use of 24-well microtiter plates used in this experiment, allowing for a greater number (approximately double) of cells to be stirnulated. 2.2.2 Dose-Response of Aaonistic Activity Dose-Response experirnents for compounds #2, 5 and 9 revealed strong activity at the highest concentrations (1O" M) for al1 three cornpounds. Moderate activity for compound #2 and #9 at 1oe8 M. NOactivity was seen at this concentration for compound #5. At final concentrations of 1O" to 1O-" M for al1 three compounds, no agonistic activity was observed (fig. 2.2). Table 2.1 Structures of Synthetic Antiprogestins Code 1 1p-(3- (Dirnethy1snino)pnenyl)- 19-norprepa4,94iene- 3-one 17P-czrboxylate rnethyl ester 11 p44- (Dimethy1amino)pheny1)- 19-ncr-l7a-preg na3,9- diene-3-one 21 ,17- carbolsctone Seven synthetic antiprogestins were tested for agonistic and antagonistic activity on androgen and progesterone receptors. These are the structures, codes, and chemical names of these compounds. Table 2.2 Agonistic Activity of Synthetic Antiprogestins in the BT 474 and T 47D Breast Cancer Cell c in es' 1 Compound 1 Agonistic Activity 1 Agonistic Activity 10-7 M BT 474 T 47~ .mifepristone 7 + 1 4545 0 512 OkO 1 III .4I1 6 IYl OkO 9 118k28 191 I38 Norgestrel 843 k 27 697 k 24 1 alcohol 1211 1 mifepristone 010 2kI O II1 O+O 6 lkl III 9 O+O 2kl Norgestrel 417 I80 9212 alcohol l+l 2 k 2 ' Mean values + standard error, N=4. Seven synthetic antiprogestins were first tested for agonistic activity. Norgestrel was used as a positive control and alcohol as a negative control. Mifepristone was used as an antiprogestin with weak agonistic activity. All compounds were tested four times. Compounds #O, 2, 5, and 9 were found to have agonistic activity at 1O" M. By 1O" M. this activity was no longer seen. 101-9 Concentration molli. Figure 2.2 Dose-Response of Synthetic Antiprogestins #2,5, and 9 Synthetic antiprogestins encoded #2, 5, and 9 were found to have agonistic activity on androgen and progesterone receptors. This was seen at IO-^ M final concentration, and dropped precipitously to O by IO-^ M. This activity was weaker than that seen with norgestrel, a progestin. 2.2.3 Assessrnent of Antaaonistic Activitv Compounds #O, 1, 2, 3, 5.6, and 9 were first tested for antiprogestin activity. This was done using both norgestrel, at 10-8 M and 10'" M, and norgestimate at 1o9 M final concentrations. For the 1O-~II o6 pair (blockerlprogestin), both experiments demonçtrated that compounds #O and 5 were the strongest blockers, with 98 _+ 2% and 100% blockade, respectively, followed closely by compound #2 at 93 f 3% blockade. Compound #9 had a 100% blockade using norgestimate, but 80% with norgestrel. Cornpound #6 had the weakest antiprogestin activity (~50%).Compound #3 was also weak in its antagonistic activity on the progesterone receptor. The 10~9110~10M pairs (blockerlnorgestrel) showed a similar pattern. Compound #O showed complete (100%) antiprogestin activity, followed by #5 with 97 + 4% blockade. Compound #2 showed 95 + 6% antagonistic activity. Compound #6 was the least effective in blocking the progesterone receptor (27% blockade) at this concentration as well (table 2.3). Antiandrogenic activity was tested using the synthetic antiprogestins with dihydrotestosterone in IO-~II and 1O'~/I O-'' M pairs (table 2.3). Similar results to antiprogesterogenic activity were seen. Least blocking was seen with compound #6 at both. This was followed by compounds #3 (77 + 1 and 37 + 12%) and #1 (80 f 6% and 41 I 15%). Greatest blocking of the AR was seen with compounds #5, 2, and #9, with percentage blocks being 95 + 4%, 91 f 5 and 90 f 3 at IO-^ M. and 73 + 9,83 I4 and 66 I12% at IO" M, respectively. Table 2.3 Antagonistic Activity of Synthetic ~ntiprogestins' Compound %Block of %Block of %Block of IO-~IIO-~M Pair Norgestrel Norgestimate Dihydrotestosterone O 98 + 2 1O0 9024 1 71 + 12 87 80 I 6 2 93 I3 1O0 91 15 Mean values k standard error, N=4. The antiprogesterogenic and antiandrogenic activities of the synthetic antiprogestins were tested. Compounds #O, 2, 5, and 9 were shown to have the greatest blocking effects. This was true for both the PR, demonstrated with norgestrel and norgestimate (a progestin with no cross- reactivity for the AR), and the AR, demonstrated with DHT. CHAPTER THREE PLANT-DERIVED COMPOUNDS 3. PLANT-DERlVED COMPOUNDS Once the breast cancer assay system was found to be appropriate for measuring sex hormone receptor activity, we proceeded with the assessrnent of agonistic and antagonistic activity of plant-derived compounds. 3.1 Materials and Methods The breast cancer ceIl lines, procedures of growth and maintenance of cells, harvesting of supernatant, and PSA assay are the same as outlined in chapter 2. 3.1 1 Ex~erimentalDesian A wide range of flavonoids, antioxidants, vitamins (Sigma Chemical Co., St Louis, MO 63178; lndofine Chernical Co., Sommerville, NJ 08876), teas (health food store) and wine (Liquor Control Board of Ontario) were tested for agonistic and antagonistic activities (table 3.1, fig. 3.1 ). These compounds were chosen because of the epidemiological research around the foodslbeverages in which they are found. The categories of foods that contain these compounds include soya, tea, wine, fruits, vegetables, and coffee. For some of these compounds, elaborate work has been conducted already to determine their rnechanism of action in cancer prevention and treatment. However, for the great majority, little to no literature exists. Table 3.1 Plant-derived Cornpounds 1 Food Source 1 Com~ound Concentrations 1 1 coffee 1 caffeine 1 10"- 104M 1 1 coffee 1 caffeic acid 1 ?O'~-IO~M 1 coffee theobromine 40"- 104 M coffee theophylline 10"- lo4 M coffee chlorogenic acid 70'~- lo4 M antioxidant vitarnin ascorbic acid 1 IO"-IO~M antioxidant vitamin a-tocopherol 10"- lo4 M antioxidant vitarnin B-carotene 10" - lo4 M folic acid ridoxine al1 areen ~lantsand veaetables chioro~hviiine 1 tea and red wine ne morin 40"- IO+ M ne gallic acid IO-' - IO+ M ne catechin 10" - lo4 M ne quercetin i0" - IO+ M ne taxifolin q0"- IO* M ne m-coumaric acid 10" - lo4 M 1 tea and red w ne D-cournaric acid 1 IO"-IO~M 1 tea and red wine 1 ferrulic acid 1 IO-'- lo4 M --..- tea and red wine vanillic acid 10"- lo4 M tea and red wine genistic 10'' - IO+ M tea and red wine syringic acid 10" - lo4 M tea and red wine rutin trihydrate 10'~- IO* M sova biochanin A 10" - lo4 M I soya daidzein 10" - lo4 M soya genistein 10" - lo4 M fruits, vegetables and leaves apigenin IO" - IO+ M fruits, vegetables and leaves myricetin 10" - IO+ M fruits, vegetables and leaves Iuteolin 10" - lo4 M fruits, vegetables and leaves kaempferol IO-^ - IO+ M berries ellagic acid 10'~- 1o4 M berries salicylic acid IO-'- lo4 M citrus fruits hesperetin IO-' - lo4 M citrus fruits hesperidin 1O" - lo4 M citrus fruits naringin IO-' - 104 M citrus fruits naringenin 10"- lo4 M red wine gamay- noir dilutions XI00, XI000, x?O O00 vollvol red wine cabernet franc dilutions XI00, XI000, XIO O00 vollvol tea oolong tea dilutions XIO, XI 00, XIO00 tea green tea dilutions XIO, XI 00, xiO00 tea Earl Grey dilutions XIO, XI 00, XI000 fruit extract used in prostate disease saw palrnetto dilutions xi00, XI000, XIO O00 vol/vol Forty-five plant-derived compounds and beverages were tested for agonistic and antagonistic activity on progesterone and androgen receptors. These were picked on the basis of plant-food group in which they are present. The soy isoflavones (Indofine Chemical Co.) and the other pure compounds (Sigma Chemical Co.) were diluted with absolute ethanol on a molar basis. The wines were diluted with ethanol on a vol/vol basis. The teas were steeped for 5 minutes with boiling distilled water, and the water extract was collected. This water extract was diluted with ethanol. Caffeine Theobromine 11 011 Chlorogenic acid a-tocopherol P-carotene OH Pyridoxine Gallic acid Apigenin &??:00?-7t~1 Chlorophyll p-coumaric acid Luteolin Salicylic acid Hesperetin Naringenin Figure 3.1 Structures of Plant-derived Compounds 21 compounds were found to have agonistic or antagonistic activity on progesterone or androgen receptors. These compounds have vey different structures. Many have hydroxyl groups. Chlorophyll is a very large molecule, with a magnesium core. 3.1 .1.1 Screenina for Potential Aaonistic Activity BT-474 breast cancer cells were grown to confluency as above. For the pure fiavonoids, antioxidants, methylxanthines, chlorophylline, and hornocysteine, final concentrations of 1o-~, 1 oa, and 1O-' M were used., with anhydrous ethanol being used for dilutions. The teas were prepared by boiling 300 mL distilled water, adding it to 3.2 g of tea (equivalent to two cups of tea per tea bag) and steeping for 5 minutes. Then dilutions of 1Ox, 1OOx, and 1000x were made. For the wines and saw palrnetto, dilutions were made of IOOx, IOOOx, and 10 OOOx vlv. Cells were incubated 1 pL plant- derived compound in 1 mL media. Alcohol was used as a negative control throughout the study, and norgestrel was used as a positive control. Incubation time was 7 days. 3.1.1.2 Dose-Response of Aaonistic Activity BT-474 cells were incubated with 1 pL of plant-derived compound and 1 ml media in the 48-well microtiter plates. The concentrations of cornpounds used ranged from 1om2 to 1o4 M, with a final concentration of 10'~to 1O-' M. All compounds tested were with anhydrous ethyl alcohol. lncubation tirne was 7 days. The compounds tested are listed in table 3.1. 3.1.1.3 Assessrnent of Antaaonistic Activitv T47-D cells were grown to confluency under standard conditions in 48-well microtiter plates. The cells were incubated with plant-derived cornpound at IO-^ M final concentration for one hour. DHT or norgestrel, at final concentration of 10" M was added to form triads of flavonoid atone, flavonoid plus androgen or progestin, and androgen or progestin atone. Alcohol was used as a negative control. Incubation tirne was 7 days. Statistics were performed as outlined in chapter 2. 3.2 Results 3.2.1 Screenina for Potential Aaonistic Activitv Forty-five compounds consisting of flavonoids, antioxidants, vitamins, and beverages were assessed for their agonistic effects on androgen and progesterone receptors in the BT-474 breast cancer cell line (table 3.1). The compounds were preliminarily assessed at 1 and 1u8 M final concentrations. Compounds that were positive at either concentration were then studied for dose-response at final concentrations of 1 to 1 M. 3.2.2 Dose-Res~onsefor Aaonistic Activity After repeated rounds of analyses, the only compound found to have agonistic activity was apigenin. Apigenin had a strong effect at the two highest concentrations, and produced lower, but detectable amounts of PSA at the lowest concentration (fig. 3.2). IOA-7 1OA-6 IOA-5 Concentration M Figure 3.2 Dose-Response of Apigenin Apigenin was the only plant-derived compound found to have agonistic activity on progesterone and androgen receptors. Androgen produced 900 nglL PSA at 10'~M concentration, and dropped precipitously by IO-' M. This compound was found to have a linear dose-response. 3.2.3 Assessrnent of Antaaonistic Activitv Chlorophylline was found to have the strongest antagonistic effect on both hormone receptors of al1 the plant-derived cornpounds (87 I 7% for PR, 94 k 3% for AR). This was followed for the progesterone receptor by the antioxidant vitamin P- carotene (82 + 3%),taxifolin (80 _+ 5%), homocysteine (75 + 15%), and a-tocopherol(65 I 10%). Other cornpounds with antiprogesterogenic activity included chlorogenic acid, theobromine, theophylline, saw palmette, luteolin, rutin trihydrate, and the red wines (table 3.2, fig. 3.3). After chlorophylline, the greatest antiandrogenic plant-derived compounds included hesperetin (79 + 11%), syringic acid (72 k 11), a-tocopherol (65 + 5%), taxifolin (58 -t. 11%), gallic acid (55 I 7%), and m-coumaric acid (53 + 11%). Other blockers of AR included caffeine, salicylic acid, pyridoxine, gamay noir, p-carotene, and Earl Grey tea (table 3.2, fig. 3.4). Plant-derived compounds were divided into 7 food groups: coffee, pigments, antioxidant vitarnins, vitamins and related cornpounds, tea and red wine, soya, and fruits, vegetables and leaves. Differences between groups were assessed. For the androgen receptor, antagonistic activity by pigments (chlorophyll) was significantly greater than blocking by vitamins, coffee, tea and red wine, soya, and fruits, vegetables and leaves (p<0.05). Antioxidant vitamins did not show statistical difference frorn any other group. The progesterone receptor showed much the same result. Chlorophyll had a greater blocking activity from al1 other groups (~~0.05)(fig 3.5). Table 3.2 Antagonistic Activity of Plantderived ~ompounds' Antiprogesterogenic Antiandrogenic Cornpound Activity (% block) Activity (% block) Coffee caffeine 2219 35+7 caffeic acid OkO 16 k 16 theobrornine 38h4 14I8 theophylline 36 t 7 30+31 chlorogenic acid 61 +2 24k 12 Antioxidant Vitarnins ascorbic acid 29+ 12 27 + 8 a-toco~herol 65 k 10 16515 1 Vitamins L folic acid 43k 19 52 + 20 homocysteine 751 15 67 + 20 methylcobalamin 24 2 8 4k4 pyridoxine 28k 10 48 + 5 Pigments chlorophylline 87 -t- 7 94 i 3 Tea and Red Wine 1 Earl Grev morin gallic acid catech in quercetin taxifolin m-coumaric acid p-cou maric acid 144 +24 1 ferulic acid I 1 vanillic acid t38+16.- genistic acid 1381:12 1 svringic acid 120+12 rutin trihydrate 47 + IO 19 & 12 Soya biochanin A 22 19 17I8 gen istein 21 18 715 daidzein 41 k16 16I9 Fruits and Vegetables apigenin 16 r4 11 36 +17 myricetin 28k 17 6+6 luteolin 35+4 7k3 ellagic acid 30&18 3rt3 salicylic acid 24&10 32 + 7 hesperitin 15 I 13 79+11 hesperidin OkO 16k9 naringin 16k9 14211 naringenin 15 & 12 616 kaempferol 25i15 21 +'Il saw palmetto 25 + 5 24+ 14 1 Mean values I standard error, N-4. 45 plant-derived cornpounds were tested for antagonistic activity on PR and AR. These are the percentage blocking of norgestrel and DHT, respectively. Chlorophylline Homocysteine T Figure 3.3 Antiprogesterogenic Activity of Plant-derived Compounds Many plant-derived compounds were found to have antiprogesterogenic activity. The greatest effect was sren by chlorophylline, followed by the antioxidant vitarnins (a-tocopherol and pcarotene), tea and red wine compounds (taxifolin), and rnethylxanthines from coffee (theobromine, theophylline, and chlorogenic acid). Chlorophylline 4-ffeine Figure 3.4 Antiandrogenic Activity of Plant-derived Compounds Many plant-derived compounds and beverages were found to have antiandrogenic activity. The greatest effect was seen by chlorophylline, followed by hesperetin, a citrus flavonoid, red wine and tea compounds (syringic acid, taxifolin), and antioxidant vitamins (a-tocopherol, p-carotene). Figure 3.5 Antagonistic Activity of Plant-derived Compounds by Groups We compared antagonistic activity of plant-derived compounds by groups. Grey bars represent the progesterone receptor, black bars the androgen receptor. Pigments (chlorophylline) was found to have significantly different activity from al1 other groups for boi receptors (p<0.05). No other group was statistically different from another. Correlations were made between the two receptors. A significant correlation was found between AR and PR for ail compounds tested (r=0.47, p=0.001). CHAPTER FOUR GENERAL DISCUSSION AND CONCLUSIONS 4. GENERAL DISCUSSION AND CONCLUSIONS 4.1 General Discussion Our study shows that several plant-derived cornpounds do indeed interact with progesterone and androgen receptors with agonistic and antagonistic results (fig. 3.3, 3.4). Strong agonistic activity was seen with apigenin, and compounds from many plant sources had significant blocking activity of PR, AR or both. Flavonoids comprise a field with much scope, but very little in-depth knowledge and literature. Categorizing compounds according to plant-food groups was very difficult for many reasons including ubiquitousness of flavonoids within the plant kingdom, very little work done in assaying plant samples, and no database being in existence. Therefore, we have separated plant-derived cornpounds into different groups, according to the Merck Index and articles in Medline. Hopefully in the near future fiavonoids will be entered into the database of foods (e.g. United States Department of Agriculture), with concentrations evaluated and specified. The primary purpose of this study was to assess plant-derived compounds for their activity as agonists and antagonists of the androgen and progesterone receptors, in order to determine their use in prevention and treatrnent of hormone-dependent cancers. Breast and prostate cancers, as hormone-dependent diseases, have been demonstrated through historical treatment as well as investigations and regiments used today, to be prevented, treated, and manipulated through three sex-hormone systerns, singly, or in combination. How this can be done through either dietary manipulation or through utilization of "neutraceuticals" is the ultimate goal we wish to pursue. We believe that this study has provided a necessary step in this long plan. In order to examine the usability of our tissue culture system to determine hormone agonism and antagonism, we decided to incorporate compounds for which some blocking studies had been conducted. These synthetic progestin antagonists were made available to us for the purpose of measuring progesterogenic agonistic activity by Southwest Research Foundation for Biomedical Research in San Antonio, Texas. We chose to extend this work to look at the antiprogestin and antiandrogen activity of these compounds as well, and compare Our results with those seen by their researchers (table 4.1). As our results generally correlated with theirs, we were able to conclude that Our assay system could accurately determine receptor stimulation and inhibition. Positive results were conclusive, with tight standard errors. This allowed us to proceed with the flavonoid study, with the necessary confidence that Our results could be interpretable. The blocking ability of the synthetic antiprogestins varied greatly from O to 100% of both the androgen and progesterone receptors. Such variation was seen at both the higher (10" M) and lower (IO" M) concentrations. By using norgestimate, a pure progesterone agonist (i.e. has no affinity for the androgen receptor) we were able to observe progesterone receptor blockade without androgen receptor cross-activity. The progesterone antagonist activity seen with norgestrel as stimulant corresponded with these results. More interesting to us was the conclusion that three of these synthetic antiprogestins (#2, 5 and #9) were in fact progesterone and androgen agonists as well. The activity demonstrated was quite strong at the higher concentration tested (>IO0 Tabfe 4.1 Collaborative Results of Antiprogesterogenic Activity Compound Code Relative Binding Anti-McGinty PSA Assay Assay (PR) (Antiprogestin Assay) (Antiprogestin Assay) O 114 1.O 96,l O0 1 42 0.15 83,47 2 43 0.332 95.89 We received information on various tests the Southwest Foundation for Biomedical Research conducted on the 7 synthetic antiprogestins studied. These included relative binding assay results for the progesterone receptor and the anti-McGinty assay, which determines antiprogestational activity. In general, these results were in accordance with ours. ng/L of PSA for 1O-' MM), and dropped precipitously at lower concentrations (10~~M). This knowledge could be beneficial for determining pharmacological concentrations to use as antagonists, i.e. for only blocking activity, one would use the lower concentration. Agonistic activity has been observed for other progesterone blockers, namely mifepristone (RU486), but the activity of the synthetic blockers measured here was much greater than the one demonstrated for mifepristone, indicating that in some situations these cornpounds could be beneficial as partial agonists. During the initial few months of the plant-derived compound study, we determined the best techniques to use, through much trial and error, encompassing the growing and maintenance of the cells, concentrations of compounds to use, and the number of days to incubate the plates. After several attempts, we found that in order to keep the nurnber of cells consistent, growing thern as rnonolayers was a necessity. This was accomplished by utilizing a syringe with an 18-gauge needle to separate the individual cells irnmediatety before plating thern. Concentrations of dry compounds were determined through past protocols of blocking of steroids, where steroids are used at 1O-' M final concentration, and potential blockers are at 1O-fold higher concentrations than the steroids. Determining dilutions for the teas and wines took more consideration. After looking at several possibilities, we decided to steep the tea as would be done in a natural situation. It was then injected into the media at full strength, as well as IOx, 1OOx, and 1000x dilutions. This would allow us to see if any activity would be seen at lower concentrations as well as full strength. The wines and saw palmetto were diluted on a volume per volume basis at dilutions of 1OOx, IOOOx, and 1O 000x. These were equivalent to the dry cornpound concentrations. The plant-derived compounds we tested were in their pure forms, i.e. they were not metabolized prior to injecting into cellular media. Hence, we were unsure as to whether or not the molecules would enter the cell, necessary for any potential receptor binding to occur. As other studies have been conducted using these and/or similar compounds, we did not believe this would be a problem. Our results are indicative of the compounds diffusing easily through the cell and nuclear membranes into the nucleus for possible interaction with the nuclear receptors (AR and/or PR). The alternate possibility is that cellular metabolism of these compounds occurs once in the cytoplasm of the cells, conducted by enzymes already present. The large majority of the plant-derived compounds we tested did not have agonistic activity. The only compound that gave a linear and significant dose-response was apigenin, a flavonol form of quercetin. This cornpound is found ubiquitously in fruit, vegetables, leaves, and other plant foods. Other cornpounds with some agonistic activity were naringenin, the non-conjugated form of naringin, derived from citrus fruit, and found in the fruit itself, as well as the peel and seeds. Several plant-derived compounds were found to have antagonistic activity. These included the methylxanthines from coffee, pigment, antioxidant vitamins, other vitamins and related compounds, tea and red wine flavonoids, and compounds found in fruits, vegetables and berries. The soy isoflavones were not found to have any significant blocking activity on either hormone receptor. Perhaps their estrogenic andlor antiestrogenic effects accounted for these results. It has been found that estrogens can have blocking actions on androgen and progesterone receptors (Yu et al. 1994). Hence, this may be true for strong phytoestrogens as well. Many of the compounds found to be either agonists or antagonists of the PR or AR have been documented in the literature as antioxidants. Many of these compounds are known for their ability, as antioxidants, to reduce cardiovascular risks including blood cholesterol and LDL oxidation. The "French paradox", the revelation that although the French eat a diet high in saturated fat do not seem to die from cardiovascular diseases, has been attributed, at least pattly, to red wine phenolics. In vitro experiments have concluded that the higher the polyphenolic content of the wine, the greater its antioxidative effect measured as change in lag time in different oxidation systems. Moreover. upon stripping the polyphenols from the beverage, the lag time returns to control levels in isolated LDL (Abu-Amsha et al. 1996). Hot water extracts from green tea, black tea, and various wines, also have been documented to scavenge ascorbyl radicals (Satoh et al. 1996). The relationship between antioxidant activity and hormones, namely estrogen, is an interesting one, though not completely understood. Estrogens have been associated with decreased risk of heart disease, and are used as preventive medicine for post-menopausal women in the form of hormone replacement therapy (HRT). Perhaps the relationship can be further explained by Our results; since estrogen and progesterone and androgen seem to have an inverse relationship in some situations, perhaps the antagonistic activity of some plant-derived compounds on PR and AR play a role in this cardioprotection. This may either be through a higher estrogen:androgen activity ratio, or as a direct result of androgen and progesterone blockade. Such theories must be further investigated. Chlorophyll, the pigment responsible for the color of al1 green plants, and the necessary component for photosynthesis, is rernarkably unstudied at this point. It seems rather surprising that the molecule found ubiquitously in all green plants and vegetables would not be investigated for antioxidative potential. but this is what the lack of literature seems to indicate. Necessary for photosynthesis, a process based on ultraviolet and other forms of radiation, this compound must be extremely stable, and therefore a supremely good antioxidant. Chlorophyll was found to have exceptional blocking ability for both the PR and AR, significantly greater than al1 other plant-derived compound groups studied. This compound is an extrernely large one (MW s 900), and is not absorbed as such from the gut. However, chlorophyll has a magnesium ion at its core. This trace minerai has gained interest at present, and is being examined for its positive effects on chronic health, especially cancer and heart disease (Singh et al. 1997; Gawaz et al. 1996). We hope this revelation, along with the results of other fruit and vegetables flavonoids presented here, will lead to more investigation in Rs many roles on chronic health, disease, and aging. ln summary, the data we have obtained indicate that some flavonoids and other organic compounds do function as agonists or antagonists of the androgen and progesterone receptors. The observation that compounds are responsive in such a system, are consistant with the epidemiological and prospective literature demonstrating an association between high vegetable and other plant-food consumption and lower risk of breast and prostate cancers. How we may manipulate this knowiedge to increase health and extend longevity is a possibility that needs further investigation. 4.2 Conclusions These results convey two major findings. First, the antiprogestin work allows us to deduce that this tissue culture systern is a good assay for determining the agonistic and antagonistic activity of compounds on progestin and androgen receptors in at least two breast cancer cell lines. The data are consistent and reproducible, allowing the results to be reliable and interpretable. Secondly, and more importantly, some purified polyphenolic and related compounds were found to have agonistic andlor antagonistic activity in this breast cancer tissue system. This demonstrates that some of these compounds do enter the cell, and have hormonal or antihormonal consequences on nuclear receptors. Whether the compounds are metabolized by the cell or not is yet to be determined. However, this is a good start in determining the effects of flavonoids on hormonal systerns within human breast cancer cell. Tea and wine polyphenols were found to have weak but significant antagonistic activity in our system. We have also demonstrated that some antioxidant vitamins, other vitamins, and other plant-derived compounds have antiandrogen or antiprogestin activity. These compounds have gained much attention epidemiologically, and Our in vitro work confirms data found in the literature. We have potentially determined one mechanism by which they function. However, much more work is needed to what other mechanisms exist, and how these pathways interact and interrelate. Finally, this study has focussed our attention on a constituent of plant foods not typically examined: chlorophyll. This extremely important compound may function to decrease cancer initiation and promotion by many mechanisms. At its core is a magnesium ion, which is now being looked at in terms of reducing oxidative darnage and CHD. Now we observe a potent anti-hormonal effect of this vital compound. Hopefully this project will lead towards more studies on its beneficial effects on health and chronic disease. 4.2.1 Future Studies Our preliminary observations of dong quai, a root used in Eastern medicine for treatment of premenstrual and menopausal symptoms indicate that this extract has both strong agonistic and antagonistic activity in both the PR and AR systems. Our observations of yam extract, also used in women's health show a strong blocking ability on these receptors. Further study, with purer and more standardized forms of these compounds is necessary for conclusions to be made. Experiments are already being conducted to determine the effects of the metabolites produced upon consumption of large amounts of flavonoids and lignans on PR and AR in the T47-D system. Our preliminary results indicate that the metabolites produced after consumption of flaxseed (enterodiol and enterolactone) block PSA production after stimulation with norgestrel or DHT. Once deterrnined which sera have such effects, metabolites may be identified using GC-mass spectrometry (Adlercreutz et al. 1993), and cornparisons rnay be made of which diets and/or metabolites are most beneficial in yielding such results. Dissecting out compounds from beverages found to have blocking activity (e.g. red wine and tea) will also be helpful to determine what components of these sources produce the effects seen. Resveratrol is a polyphenol which has received a lot of attention regarding its antitumorigenic and antioxidant activities (Soleas et al. 1997; Bonard 1997). This is one compound we intend to study in the future. Other studies looking into the mechanism of hormone receptor blockade must be undertaken. Furthermore, we hope to investigate effects on the estrogen receptor as well as understand the bioavailability and biotransformation of plant-derived compounds in the human body. Ultirnately, the rnechanism by which plant foods exert their anti- tumor potential may be elucidated, step by step. 4.2.2 Summary By measuring agonistic and antagonistic activity of synthetic antiprogestins on androgen and progesterone receptors, the PSA assay was found to be useful in determining such activity of the plant-derived compounds chosen. The agonistic activity of plant-derived cornpounds was deterrnined. The flavonoid apigenin was found to have significant agonistic activity on progesterone and androgen receptors, with a linear dose-response. At 1û5 M final concentration, 900 nglL PSA were produced. At IO-^ M apigenin, 500 ng PSA was produced. This dropped precipitously; by IO-' M no PSA was detected. Naringenin was also found to have some agonistic activity. 3. The antiprogesterogenic and antiandrogenic activity of plant-derived compounds was determined. Chlorophyll had the strongest effect (87 t 7% for PR, 94 + 3% for AR), followed by the antioxidant vitamins (59 k 16% and 47 + 11%, respectively). Methylxanthines from coffee, other vitamins, and fruit and vegetable compounds were also found to be blockers of these hormone receptors. Tea and red wine components were also found to be antagonists both in pure forms and as diluted beverage forms. Soy isoflavones were had no activity on progesterone or androgen receptors in oui study. The implications of this study are that plant-derived compounds could possibly be used in hormonal manipulation necessary in prevention and treatment of hormone-dependent cancers. Whether antiprogestins and agonists would be beneficial in breast cancer treatment, and antiandrogens in prostate cancer treatment, will have to be studied further. CHAPTER FlVE REFERENCES 5. REFERENCES Abu-Amsha R, Croft KD, Puddey 16, Proudfoot JM, Beilin LJ. Phenolic content of various beverages determines the extent of inhibition of human serum and low- density lipoprotein oxidation in vitro: identification and mechanism of action of some cinnamic derivatives from red wine. Clin Sci 1996;91:449-58. Adams NR, Oldham CM, Heydon RA. Ovulation rate and oocyte numbers in ewes after protonged exposure to oestrogenic pasture. J Reprod Fert 1979a;55:87-9. Adams NR. Depressed incidence of oestrus in ewes with clover disease at the beginning of the breeding season. Aust Vet J I979b;55:481-4. Adlercreutz H. Western diet and western diseases: some hormonal and biochernical mechanisrns and associations. Scan J Clin Lab lnvest 1990;50:3- 23. AdIercreutz H, Fotsis T, Lampe J, Wahala K, Makela T, Brunow G, Hase T. Quantitative determination of lignans and isofiavonoids in plasma of ornnivorous and vegetarian women by isotope dilution gas chromatography-mass spectrometry. Scand J Clin Lab lnvest l993;53:5-l8. Adlercreutz, H, Mousavi, Y, Clark, J, Hockerstedt, K, Hamalainen, E, Wahala, K, Makela, T, Hase, T. Dietary Phytoestrogens and Cancer: In Vitro and In Vivo Studies. J Steroid Biochern Molec Biol l992;4l :BI-7. Adlercreutz H, Bannwart C, Wahala K, Makela T, Brunow G, Hase T, Arosemena PJ, Kellis JT Jr, Vickery LE. Inhibition of' hurnan arornatase by rnammalian lignans and isoflavonoid phytoestrogens. J Steroid Biochern Molec Bi01 1993;44:147-53. Adlercreutz H, van der Wildt J, Kinzel J, Attalla H, Wahala K, Makela T, Hase T, Fotsis T. Lignan and isoflavonoid conjugates in human urine. J Steroid Biochem Molec Biol l995;52:97-103. Allan GF, Leng X, Tsai S-T, Weigel NL, Edwards DP, Tsai M-J, O'Mallery BW. Hormone and antihormone induce distinct conformational changes which are central to steroid receptor activation. J Bi01 Chem l992;267: 19513-20. Allegra JC, Barlock A, Huff KK, Lippman ME. Changes in multiple or sequential oestrogen receptor determinations in breast cancer. Cancer 1980;45:792-4. Amero SA, Kretsinger RH, Moncrief ND, Yamamoto KR, Pearson WR. The origin of nuclear receptor proteins: a single precursor distinct from other transcription factors. Mol Cell Endocrinol 1992;6:3-7. Anderson, RL, Wolf, WJ. Cornpositional Changes in Trypsin Inhibitors, Phytic Acid, Saponins and Isoflavones Related to Soybean Processing. J Nutr l985;l25:58l S-8s. Arai Y, Gorski RA. Critical exposure time for androgenization of the developing hypothalamus in the female rat. Endocrinol l968;82:1OlO-4. Axelson M, Sjorvall J, Gustafsson BE, Setchell KDR. Soya - a dietary source of the non-steroidal oestrogen equol in humans and animals. J Endocrinol 1984 ;1 02 :49-56. Beato M. Gene regulation by steroid hormones. Celt 1989;56:335-44. Beekman JM, Allan GF, Tsai S-Y, Tsai M-J, O'Malley BW. Transcriptional activation by the estrogen receptor requires a conformational change in the ligand binding domain. Mol Endocrinol 1993;7: 1266-74. Belt JA, Thomas JA, Buchsbaum RN, Racker E. Inhibition of lactate transport and glycolysis in Ehrlich ascites tumor cells by bioflavonoids. Biochem 1979; l8:3506-Il. Bennetts HW, Undewood EJ, Shier FL. A specific breeding problem of sheep on subterranean clover pastures in Western Australia. Aust Vet J l946;22:2-12. Berger FG, Watson G. Androgen-regulated gene expression. Annu Rev Physiol 4 989151 151 -65. Bhimani R, Frankel K. Suppression of H202production and oxidative DNA damage in HeLa cells by (-)-epigallocatechin gallate (EGCG). Proc Amer Assoc Canc Res 1991;32:126. Block G, Patterson B, Subtar A. Fruit, vegetables and cancer prevention: a review of the epidemiological evidence. Nutrition and Cancer l992;18:1-29. Bonard EC. The virtues of grapes. Rev Med Suisse Romande 1997;l l7:357-8. Buell P. Dunn JF Jr. Cancer mortality among Japanese Issei and Nisei of California. Cancer 1965; 18:656-64. Buiatti E, Palli D, Amadori D, Marubini E, Puntoni R, Avellini C, Bianchi S, Cipriani F, Cocco Pl Decarli A, et al. Methodological issues in a multicentric study of gastric cancer and diet in Italy: study design, data sources and quality controls. Tumori 1989;75:41O-9. Canadian Pharmaceutical Association. Krogh CME, Gilles MC, Shave DG, Bisson R, Blais Dl eds. Compendium of Phamaceuficals and Specialities. Toronto: CK Productions, 1992. Canobbio L, Galligioni El Gasparini G, Fassio T, Crivellari T, Villalta DlSantini G, Monfardini S, Boccardo F. Alternating tamoxifen and medroxyprogesterone acetate in postmenopausal advanced breast cancer patients -- short and long term endocrine effects. Breast Cancer Res Treat l987;lO:2Ol. Cassidy A, Bingham S, Setchell KDR. Biological effects of a diet of soy protein rich in isoflavones on the menstrual cycle of premenopausal women. Am J Clin Nutr 1994;60:333-40. Cassidy A, Bingharn S, Setchell K. Biological effects of isoflavones in young women: importance of the chemical composition of soyabean products. Br J Nutr 1995;74:587-601. Cato ACB, Skroch Pl Weinmann JI Butkeraitis Pl Ponta H. DNA sequences outside the receptor-binding sites differently modulate the responsiveness of the mouse mammary tumor promoter to various steroid hormones. EMBO J 1988;7:1403-IO. Chang A, Yeap BI Davis Tl Blum R, Hahn R, Khanna O, Fisher HlRosenthal J, Witte R, Schinella R, Trump D. Double-blind, randomized study of prirnary hormonal treatment of stage D2 prostate carcinoma: flutamide versus diethylstilbestrol. J Clin Oncol 1996; 14:2250-7. Clark J, Chu M, Celebresi P. Growth inhibition of colon cells by genistein and suramin. Proc Am Assoc Cancer l989;30:559. Co-operative Breast Cancer Group. Testosterone propionate therapy of breast cancer. J Amer Med Assoc l964;188:1069-72. Coward L, Barnes NC, Setchell KDR, Barnes S. Genistein and daidzein, and their p-glycoside conjugates: anti-tumor isoflavones in soybean foods of the American and Asian diets. J Agric Food Sci l993;4l:? 961-7. Danielson M, Hinck L, Ringold GM. Two amino acids within the knuckle of the first zinc finger specify response element activation by the glucocorticoid receptor. Cell l989;57:ll3l-8. Devasagayam TP, Subrarnanian M, Singh BB, Ramanathan R, Das NP. Protection of plasmid pBR322,DNA by flavonoids against single-stranded breaks induced by singlet molecular oxygen. J Photochem Photobiol B - Biol 1995;30:97-103. Elangovan V, Sekar N, Govindasarny S. Chemopreventive potential of dietary bioflavonoids against 20-rnethylcholanthrene-induced tumorigenesis. Cancer Lett 1994;87: 107-13. Evans RM. The steroid and thyroid hormone receptor superfamily. Science 1988;240:889-95. Farmakalidis E, Hathcock JN, Murphy PA. Oestrogenic potency of genistin and daidzin in mice. Food Chem Toxicol 1985;23:74l-5. Ferguson RA, Yu H, Kalyvas M, Zammit S, Diamandis EP. Ultrasensitive detection of prostate specific antigen by a tirne-resolved immunofluorornetric assay and the Immulite8 lmrnunochernilurninescent third generation assay: potential applications in prostate and breast cancers. Clin Chern l996;42:675- 84. Ferrini RL, Barrett-Connor E. Caffeine intake and endogenouç sex steroid levels in postmenopausal women. Am J Epidemiol 1996;144:642-4. Fisher B, Redrnond C, Poisson R, Margolese R, Wolrnark N, Wickerrnan L, Fisher El Deutsch M, Caplan H,Pilch Y, Glass A, Shibata H, Lerner H, Terz J, Sidorovich L. Eight-year results of a randomized clinical trial comparing total mastectomy and lumpectomy with or without radiation in the treatment of breast cancer. N Engl J Med 1989;320:822-8. Folman Y, Pope GS. Effect of norethisterone acetate, dimethylstilbestrol, genistein and coumestrol on uptake of (3~)oestradiolby uterus, vagina and skeletal muscle of immature mice. J Endocrinol 1969;44:213-8. Folsom AR, McKenzie DR, Bisgard KM, Kushi LH, Sellers TA. No association between caffeine intake and postmenopausal breast cancer incidence in the Iowa Wornen's Health Study. Am J Epidemiol 1993;138:380-3. Franceschi S, Favero A, La Vecchia C, Negri E, Dai Maso L, Salvini S, Decarli A, Giacosa A. Influence of food groups and food diversity on breast cancer in Italy. In J Cancer 1995;63:785-9. Franceschi S, Favero A, Decarli A, Negri E, La Vecchia C, Ferraroni M, Russo A, Salvini S, Amadori DlConti E, et al. lntake of macronutrients and risk of breast cancer. Lancet IW6;347: 1351 -6. Freake HC, Marcocci Cl Iwasaki J, Maclntyre 1. 1,25-dihydroxyvitamin D3 specifically binds to a human breast cancer cell line (T47D) and stimulates growth. Biochem Biophys Res Commun 1981;101:1131-8. Freudenheim JL, Marshall JR, Vena JE, Laughlin , Brasure JR, Swanson MK, Nemoto T, Graham S. Premenopausal breast cancer risk and intake of vegetables, fruits, and related nutrients. J Natl Cancer lnst 1996;88:340-8. Fuller PJ. The steroid receptor superfamily: mechanism for diversity. FASEB J 1991;5:3092-9. Garland Ml Willett WC, Manson JE, Hunter DJ. Antioxidant micronutrients and breast cancer. J Am Coll Nutr l993;I2:4OO-l?. Gawaz M, Reininger A, Neumann FJ. Platelet function and platelet-leukocyte adhesion in symptomatic Coronary Heart Disease. Effects of intravenous magnesium. Thromb Res l996;83:341-9. Giovannucci E, Ascherio A, Rimm EB, Stampfer MJ, Colditz GA, Willett WC. lntake of carotenoids and retinol in relation to risk of prostate cancer. J Natl Cancer lnst l995;87:1767-76. Giovannucci ElRirnm ES,Colditz GA et al. A prospective study of dietary fat and risk of prostate cancer. J Natl Cancer lnst 1993;85:1571-9. Giri AK, Lu L-JW. Genetic darnage and the inhibition of 7,12- dimethylbenz(a)anthracene-induced genetic darnage by the phytoestrogens, genistein and daidzein in female ICR rnice. Cancer Lett 1995;95: 125-33. Gordon G,Halden A, Horn Y, Fuery J, Parsons R, Walter R. Calusterone (7,17P-dimethyltestosterone) as prirnary and secondary therapy of advanced breast cancer. Oncol 1973;28: 138-46. Granner DK. Hormones of the Gonads. In: Dolan J, Langan, eds. Harper's Biochemistry. Stamford: Appleton and Lange 1996566-80. Gregoire RC, Stern HS, Yeung KS, Stadler J, Langley S, Furrer R, Bruce WR. Effect of calcium supplementation on mucosal cell proliferation in high risk patients for colon cancer. Gut 1989;30:376-82. Gregoire R, Yeung KS, Stadler J, Stern HS, Kashtan Hl Neil G, Bruce WR. Effect of high-fat and low-fat meals on the cell proliferation activity of colorectal mucosa. Nutr Cancer 1991;15:21-6. Gundersen S, Kvinnsland S, Lundgren S, Klepp O, Lund E, Bormer O, Host H. Cyclical use of tamoxifen and high-dose medroxyprogesterone acetate in advanced estrogen receptor postive breast cancer. Breast Cancer Res Treat 1990;1 7:45. Haenszel W. Cancer mortality among the foreign born in the United States. J Natl Cancer lnst 1961;26:37-132. Haenszel W, Kurihara M. Studies of Japanese migrants. 1. Mortality from cancer and other diseases among Japanese in the United States. J Natl Cancer lnst 1968;69:83-92. Halsted WS. Operations for carcinorna of the breast. J Hopkins Hosp Rep 1890-1;2:277-80. Ham J, Thomson A, Needham MyWebb P, Parker M. Characterization of response elements for androgens, glucocorticoids and progestins in mouse marnrnary tumor virus. Nucl Acids Res 1988;16:5263-76. Han C, Xu Y. The effect of Chinese tea on occurrence of esophageal tumors induced by N-nitrosornethylbenzylamine in rats. Biomed Environ Sci 1990;3:35- 42. Hara, M, Innore, T, Fukuyama, T. Some physico-chemical characteristics of gamma-seminoprotein, an antigenic component specific for human seminal plasma. Jpn J Leg Med 1971;25:322-4. Henderson BE, Ross RK, Judd HL, Frailo MD, Pike MC. Do regulatory ovulatory cycles increase breast cancer risk? Cancer 1985;56:1206-8. Henderson IC, Harris JR, Kinne DW, Hellman S. Cancer of the breast. In: DeVita VT. Hellman S. Rosenberg SA, eds. Cancer Principles and Practice of Oncology. Philadelphia: JB Lippincott Company l989:ll97. Herbst AL, Kurman RJ, Scully RE, Poskaner DC. Clear-cell adenocarcinorna of the genital tract in young fernales. Registry report. New Engl J Med 1972;287: 1259-64. Hertog MG, Kromhout D, Aravanis C, Blackburn H, Buzina Nedeljkovic S, et al. Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study. Arch lntern Med 1W5;I 55:381-6. Hertog MGL, Feskens EJM, Hollman PCH, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancett l993b;342:lOO7-ll. Hirano T, Gotoh Ml Oka K. Natural flavonoids and lignans are potent cytostatic agents against human leukemic HL-60 cells. Life Sci 1994;55: 1O61 -9. Hirayama T. A large scale cohort study on cancer risks by diet - with special reference to the risk reducing effects of green-yellow vegetable consumption. In: Hayashi Y, et al, eds. Diet, Nutrition and Cancer. The Netherlands: Uetrecht, Tokyo:VNU Sci Press 1986:41-53. Hirohata T, Shigematsu Tl Nmura AMY, Nomura Y, Hordie A, et al. Occurrence of breast cancer in relation to diet and reproductive history: a case-control study in Fukuoka, Japan. Natl Cancer lnst Monogr 1985;69:l87-90. Hoffman BR, Yu H, Diamandis EP. Assay of prostate-specific antigen from whole blood spotted on filter paper and application to prostate cancer screening. Clin Chem l996;42:536-44. Horowitz KB. The molecular biology of RU486. 1s there a role for antiprogestins in the treatment of breast cancer? Endocrine Rev 1992; 13: 146-63. Hosokawa N, Hosokawa Y, Sakai Tl Yoshida Ml Marui NI Nishino Hl Kawai K, Aoike A. lnhibitory effect of quercetin on the synthesis cf a possibiy cell-cycle- related 17 kDa protein, in human colon cancer cells. Int J Cancer 1990;45:1119- 24. Howell A, Harland RNL, Banes DM, Baildam AD, Wilkinson MJS, Hayward El Swindell R, Sellwood RA. Endocrine therapy for advanced carcinoma of the breast: relationship between the effect of tamoxifen upon concentrations of progesterone receptor and subsequent response to treatment. Cancer Res 1987;47:300. Hsing AW, Cornstock GW, Abbey H, Polk BF. Serologic precursors of cancer. Retinol, carotenoids and tocopherol and risk of prostate cancer. J Natl Cancer Inst I99Oa;82:941-6. Hsing AW, McLaughlin JK, Schuman LM, Bjelke E, Gridley G, Wacholder SI Chien HT, Blot WJ. Diet, tobacco use, and fatal prostate cancer: results from the Lutheran Brotherhood Cohort Study. Cancer Res 1WOb;50:6836-40. Hunter DJ, Manson JE, Colditz GA, Starnpfer MJ, Rosner B, Hennekens CH, Speizer FE, Willett WC. A prospective study of the intake of vitamins C, El and A and the risk of breast cancer. N Engl J Med 1993;329:234-40. lnstitute of Cancer Research and the Royal Marsden Hospital. Prostate Cancer. Lancet 1993;342:9OI -5. lshimoto H, Nakamura H, Miyoshi T. Epiderniological study on relationship between breast cancer mortality and dietary factors. Tokushima J Exper Med l994;4l:lO3-l4. Jenster G, van der Korput HA, van Vroonhoven C, van der Kwast TH, Trapman J, Brinkman AO. Domains of the human androgen receptor involved in steroid binding transcriptional activation and subcellular localization. Molec Endocrinol 1991;5:1396-404. Kennedy B. Fluoxymesterone therapy in treatment of advanced breast cancer. N Engl J rned 1958;259:673-5. Key TJ, Thorogood M, Appleby PN, Burr ML. Dietary habits and rnortality in 1 1,000 vegetarians and health conscious people: results of a 17 year follow up. BMJ l996;313:775-9. Khan SG, Katiyar SK, Agatwal R, Mukhtar H. Enhancement of antioxidant and phase Il enzymes by oral feeding of green tea polyphenols in drinking water to SKH-1 hairless mice: possible role in cancer chemoprevention. Cancer Res 1992;52:4050-2. Kitts DD, Krishnamurti CR, Kitts WD. Uterine weight changes and ridine di ne uptake in rats treated with phytoestrogens. Can J Animal Sci 1980;60:531-4. Kohlmeier L, Sirnonsen N, Mottus K. Dietary modifiers of carcinogenesis. Environ Health Perspectives l995;lO3:i??-84. Kono SIlkeda M, Tokudorne SIKuratsune M. A case-control study of gastric cancer and diet in northern Kyushy, Japan. Jap J Cancer Res 1988;79:1067-74. Kumar V, Chambon P. The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell l988;55: 145-56. Kuriki Y, Racker E. Inhibition of (Na+, K+) adenosine triphosphatase and its partial reactions by quercetin. Biochem 1976;15:4951-6. La Vecchia C, De Carli A, Negri E, Parauini F. Epidemiological aspects of diet and cancer: a summary review of case-control studies from Northern Italy. Oncol 1988;45:374-70. La Martiniere CA, Moore J, Holland Ml Barnes S. Neonatal genistein chemoprevents marnmary cancer. Proc Soc Exper Biol Med l995;2O8: 120-3. Lamp SJ, Findlay DM, Moseley JM, Martin TJ. Calcitonin induction of a persistent activated state of adenylate cyclase in hurnan breast cancer cells (T47D). J Biol Chem 1981;256:12269-74. Lasfargues EY, Coutinho WG, Redfield ES. Isolation of two human tumor epithelial cell lines from solid breast carcinomas. J Natl Cancer lnst 1978;61:967-8. Laudet V, Hanni C, Coll J, Catzeflis F, Stehelin D. Evolution of the nuclear receptor gene superfamily. EMBO J l992;ll :i003-1 3. Le Marchand L, Kolonel L, Wilkens LR, Myers BC, Hirohata T. Animal fat consumption and prostate cancer: a prospective study in Hawaii. Epiderniol 1994;5:276-82. Lee HP. Gourley L, Duffy SW, Esteve J, Day NE. Dietary effects on breast- cancer risk in Singapore. Lancet 1991;337: 1 197-200. Levesque Ml Yu H, D'Costa M, Diamandis EP. Prostate specific antigen expression by various tumors. J Clin Lab Anal i995;9:123-8. Lightfoot JR, Croker KP, Neil HG. Failure of sperm transport in relation to ewe infertility following prolonged grazing on oestrogenic pastures. Aust J Agric Res 1967;18:755-65. Lilja H. A kallikrien-like serine protease in prostatic fîuid cleaves the predominant seminal vesicle protein. J Clin lnvest l985;76:1899. Liu L, Castonguay A. Inhibition of the rnetabolism and genotoxicity of 4- (methy1nitrosamino)-1-(3-pyridy1)-1 -butanone (NNK) in rat hepatocytes by (+)- catechin. Carcinogenesis 1991;12: 1203-8. Lundgren S, Gundersen S, Klepp R, Lonning PE, Lund E, Kvinssland S. Megestrol acetate versus aminoglutethirnide for metastatic breast cancer. Breast Cancer Res Treat 1989; 1MOI. Mader S, Kumar V, devereneuil H, Chambon P. Three amino acids of the oestrogen receptor are essential to its ability to distinguish an oestrogen from a glucocorticoid-responsive receptor. Nature 1989;338:271-4. Makela S, Santti R, Salo L, McLachlan JA. Phytoestrogens are partial estrogen agonists in the adult male rnouse. Environ Health Perspectives i995;lO3: 123-7. Marcovits JI Linassier C, Fosse P, Couprie J, Pierre J, Jacquemin-Sablon A, Saucier JM, Le Pecq JB, Larsen AK. Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisornerase II. Cancer Res l989;49:5lll-7. Markiewicz L, Garey J, Adlercreutz H, Gurpide E. ln vitro bioassays of non- steroidal phytoestrogens. J Steroid Biochem Molec Bi01 l993;45:399-405. Martin MB, Saceda M, Lindsey RK. Estrogen and progesterone receptors. In: Martin ME, Haourigui M, Pelissero C, Benassayag C, Nunez EA. Interactions between phytoestrogens and human sex steroid binding protein. Life Sciences 1996;58:429-36, Martin PM, Horowitz KB, Ryan DS, McGuire WL. Phytoestrogen interaction with estrogen receptors in human breast cancer cells. Endocrinol l978;l O3:l860-7. Martinez E, Wahli W. Cooperative binding of estrogen receptor to imperfect estrogen-responsive DNA elements correlates with their synergistic hormone- dependent enhancer activity. EMBO J l989;8:3781-91. Matsukawa Y, Marui N, Sakai T, Satorni Y, Yoshida M, Matsumoto K, Nishino H, Aoike A. Genistein arrests cell cycle progession at G2-M. Cancer Res I993;53:l328-31. Mayr U, Butsch A, Schneider S. Validation of two in vitro test systems for estrogenic activities with zearalenone, phytoestrogens and cereal extracts. Toxicol 1992;74:135-49. McEwen BS, Plapinger L, Chaptal C, Gerlach J, Wal!ach G. Role of fetoneonatal estrogen binding proteins in associations of estrogen with neonatal brain ceIl nuclear receptors. Brain Res 1975;96:40O-6. McGuire WL, Cavalli FI Bonomi P, Alexieva-Figusch J. Progestin therapy for breast cancer. Breast Cancer Res Treat 1985;6:2l3. McGuire WL, Johnson PA, Muss HB, Osborne CK. Megestrol acetate in breast cancer - a panel discussion. Breast Cancer Res Treat 1989;14:33. McKeown-Eyssen G, Holloway C, Jazmaji V, Bright-See E, Dion P, Bruce WR. A randomized clinical trial of vitarnins C and E in the prevention of recurrence of colorectal polyps. Cancer Res l988;48:4701-5. Messina, MJ, Persky, V, Setchell, KDR, Barnes, S. Soy lntake and Cancer Risk: A Review of the In Vitro and In Vivo Data. Nutr Cancer 1994;21 :113-31. Mettlin C. The status of prostate cancer early detection. Cancer l993;72: 1050- 5. Migliaccio A, Pagano M, Auricchio F. lmmediate and transient stimulation of protein tyrosine phosphorylation by estradiol in MCF-7 cells. Oncogene I993;8:2183-9l. Miksicek RJ. Estrogenic flavonoids: structural requirements for biological activity. Froc Soc Exper Biol Med 1995;208:44-50. Miller EC, Miller JA. Mechanisms of chernical carcinogenesis. Cancer 1981;47: 1055-64. Miller MA, Sheen YY, Mullick A, Katzenellenbogen BS. Antiestrogen binding to estrogen receptors and additional antiestrogen binding sites in human breast cancer cells. In: Jordan VC, ed. Estrogen/Antiestrogen Action and Breast Cancer Therapy. Madison: University of Wisconsin Press. 1986: 127-48. Mills PK, Beeson WL, Phillips RL, Frazer GE. Cohort study of diet, lifestyle and prostate cancer in Adventist men. Cancer 1989;64:598-604. Minton JP, Foecking MK, Webster DJ, Matthews RH. Response of fibrocystic disease to caffeine withdrawal and correlation of cyclic nucleotides with breast disease. Am J O bstet Gynecol 1979; 135: 157-8. Minton JP, Abou-Issa H, Reiches N, Roseman JM. Clinical and biochemical studies on methylxanthine-relatedfibrocystic breast disease. Surgery 1981;90:299-304. Molteni, A, Brizio-Molteni, L, Persky, V. In Vitro Hormonal Effects of Soybean Isoflavones. J Nutr I995;125:75lS-6S. Morel 1, Lescoat G, Cillard P, Cillard J. Role of flavonoids and iron chetation in antioxidant action. Meth Enzyrnol 1994;234:437-43. Mousavi Y, Adlercreutz H. Genistein is an effective stimulator of sex hormone- binding globulin production in hepatocarcinoma human liver cancer cells and suppresses proliferation of these cells in culture. Steroids l993;58:301-4. Murkies AL, Lombard C, Strauss BJG, Wilcox G, Burger HG, Morton MS. Dietary flour supplementation decreases post-rnenopausal hot flushes: effects of soy and wheat. Maturitas 1995;21:189-95. Murphy PA. Phytoestrogen content of processed soybean products. Food Tech 1982; 1:60-4. National Cancer Institute of Canada: Canadian Cancer Statistics 7997. Toronto, Canada, 1997. Negri E, La Vecchia C, Franceschi S, DIAvanzo B, Parauini F. The role of vegetables and fruit in cancer risk. In: Hill MJ, Giacosa A, Caygill CPJ, eds. Epidemiology of Diet and Cancer. Chichester: Ellis Horwood, 1994:327-34. Newsome FE, Kitts WD. Action of phyto-estrogens cournestrol and genistein on cytosolic and nuclear oestradiol-17P receptors in immature rat uterus. Animal Reprod Sci l980;3:233-45. Nomura A, Henderson BE, Lee J. Breast cancer and diet arnong the Japanese in Hawaii. Am J Clin Nutr l978;3l:2O2O-5. Nwannenna Al, Lundh TJ, Madej A, Fredriksson G, Bjornhag G. Clinical changes in ovariectornized ewes exposed to phytoestrogens and 17 beta- estradiol implants. Proc Soc Exper Biol Med 1995;208:92-7. Oesterling JE, Chan DW, Epstein JI, Kimball AW Jr, Bruzek DJ, Rock RC, Brendler CB, Walsh PC. Prostate specific antigen in the preoperative and postoperative evaluation of localized prostate cancer treated with prostatectomy. J Ur01 l988;139:766-72. Ogawara H, Akiyama T, Watanabe S, Ito N, Kobori M, Seoda Y. Inhibition of tyrosine protein kinase activity by synthetic isoflavones and flavones. J Antibiotics 1989;42:340-3. O'Malley B. The steroid receptor superfamily: more excitement predicted for the future. Mol Endocrinol 1990;4:363-9. Paganini-Hill A, Chao A, Ross R, Henderson BE. Vitamin A, beta-carotene and risk of prostate cancer: a prospective study. J Natl Cancer lnst 1987;79:443-8. Papsidero, LD, Wang, MC, Valenzuela, LA, Murphy, GP, Chu, TM. A prostate antigen in sera of prostatic cancer patients. Cancer Res 1980;40:2428-2432. Parnes HL, Abrams JS, Tchekmedyian NS, Tait N, Aisner J. A phase 1/11 study of high-dose megestrol acetate in the treatment of metastatic breast cancer. Breast Cancer Res Treat 1991;l8:l7l. Pelissero C, Lenczowski MJ, Chinzi D, Davail-Cuisset B. Sumpter JF, Fostier A. Effects of flavonoids on aromatase activity, an in vitro study. J Steroid Biochem Molec Biol l996;57:215-23. Peterson TG, Coward L, Kirk M, Falany CN, Barnes S. The role of rnetabolism in mammary epithelial cell growth inhibition by the isoflavones genistein and biochanin A. Carcinogenesis 1996; 17: 1861-9. Pronzato P, Brema F, Amoroso D, Bertelli G, Conte PF, Martini MC, Pastorino G, Rosso R. Megestrol acetate: phase II study of a single daily administration in advanced breast cancer. Breast Cancer Res Treat 1990;17:51. Resnick MI, Grayhack JT. Treatment of stage IV carcinoma of the prostate. Urol Clin North America 1975;2: 141-61 . Rice-Evans CA, Miller NJ, Bolwell PG, Bramley PM, Pridham JB. The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Rad Res 1995;22:375-83. Robinson MJ, Corbett AH, Osheroff N. Effects of topoisomerase Il-targeted drugs on enzyme-rnediated DNA cleavage and ATP hydrolysis: evidence for distinct drug interaction domains on topoisomerase II. Biochem 1993;32:3638- 43. Rochefort H. Oestrogens, proteases and breast cancer. From cell lines to clinical applications. Eur J Cancer 1994;3OA:I583-6. Rohan TE, Howe GR, Friedenreich CM, Jain M, Miller AB. Dietary fiber, vitarnins A, C, and E, and risk of breast cancer: a cohort study. Cancer Causes and Control l993;4:29-37. Rutqvist LE, Johansson H. Mortality by laterality of the prirnary tumor among 55 000 breast cancer patients from the Swedish Cancer Registry. Br J Cancer I990;6'l:866-8. Rutqvist LE, Pettersson D, Johansson H. Adjuvant radiation therapy versus surgery alone in operable breast cancer: long-term follow-up of a randomized clinical trial. Radiother Oncol l993;26:104-1 IO. Sato F, Matsukawa Y, Matsumoto K, Nishino H, Sakai T. Apigenin induces morphological differentiation and G2-M arrest in rat neuronal cells. Biochem Biophys Res Comm l994;204:578-84. Samaan NA, Buzdar AV, Aldinger KA, Schultz PN, Yang K, Romsdahl MM, Martin R. Estrogen receptor - a prognostic factor in breast cancer. Cancer 198 1;47:554-60. Satoh K, Sakagami H. Ascorbyl radical scavenging activity of polyphenols. Anticancer Res 1996;'i 6:2885-90. Scambia G, Ranelletti FO, Panici PB, Piantelli M, de Vincenzo R, Ferrandina G, Bonanno G, Capelli A, Mancuso S. Quercetin induces type-Il estrogen binding sites in estrogen-receptor-negative (M DA-MB231) and estrogen-receptor-positive (MCF-7) human breast-cancer cell lines. Int J Cancer 1993;54:462-6. Schairer C, Brinton LA, Hoover RN. Methylxanthines and breast cancer. Int J Cancer 1987;40:469-73. Sedlack SM, Horowitz KB. The role of progestins and progesterone receptors in the treatment of breast cancer. Steroids 1984;44:467. Setchell KD, Gosselin SJ, Welsh MB, Johnston JO, Balistereri WF, Kramer LW, Dresser BL, Tarr MJ. Dietary estrogens - a probably cause of infertility and liver disease in captive cheetahs. Gastroenterol 1987;93:225-33. Setchell KDR, Adlercreutz H. Mammalian lignans and phytooestrogens. Recent studies on their formation, metabolism and biological role in health and disease. ln: Roland IR, ed. Role of Gut Flora in Toxicityand Cancer. London: Academic Press, I988:U 5-45. Setchell KDR, Borriello SP, Hulme P, Kirk DN, Axelson M. Nonsteroidal estrogens of dietary origin: possible roles in hormone-dependent disease. Am J Clin Nutr l984;40:569-78. Sher E, Eisman JA, Moseley JM, Martin TJ. Whole-ce11 1,25- dihydroxycholecalciferol by breast cancer cells (T47D) in culture. Biochem J 'i 981 ;200:315-20. Sirnopoulos AP. Nutritional cancer risks derived from energy and fat. Med Onco and Tumor Pharm I987;9:199-ZV. Singh RB, Gupta UC, Mittal N,Niaz MA, Ghosh S, Rastogi V. Epidemiologic study of trace elements and magnesium on risk of coronary artery disease in rural and urban lndian populations. J Am Col1 Nutr l997;l6:62-7. Skolnick MH, Frank T, Shattuck-Eidens D, Tavtigian S. Genetic susceptibility to breast and ovarian cancer. Pathol Bi01 1997;45:245-9. Slattery ML, West DW. Smoking, alcohoi, coffee, tea, caffeine, and theobromine: risk of prostate cancer in Utah (United States). Cancer Causes Control 1993;4:559-63. Smith DF, Toft DO. Steroid receptors and their associated proteins. Mol Endocrinol 1993;7:4-II. Smith SJ, Deacon JM, Chilvers CE. Alcohol, smoking, passive smoking and caffeine in relation to breast cancer risk in young wornen. UK National Case- Control Study Group. Br J Cancer l994;70:112-9. So FV, Guthrie N, Chambers AF, Moussa M, Carroll KK. Inhibition of human breast cancer cell proliferation and delay of mammary turnorigenesis by flavonoids and citrus juices. Nutr Cancer l996;26:167-81. Soleas GJ, Diarnandis EP, Goldberg DM. Resveratrol: a molecule whose time has corne? And gone? Clin Biochem 1997;30:91-113. Somjen D, Kaye AM. Specificities in the synthesis of a cytoplasmic estrogen- induced uterine protein. Mol Cell Endocrinol 1976;4:353-8. Sparnins VL, Venegas PL, Wattenberg LW. Glutathione S-transferase activity: enhancement by compounds inhibiting chernical carcinogenesis and by dietary constituents. J Natl Cancer lnst 1982;68:493-6. Spicer DV, Pike MC. The prevention of breast cancer through reduced ovarian steroid exposure. Acta Oncol l992;3l: 167-74. Stamey TA, Yang N, Hay AR, McNeal JE, Freiha FS, Redwine E. Prostate specific antigen as a serum rnarker for adenocarcinonia of the prostate. N Engl J Med l987;3I7:9O9-l6. Suolinna EM, Buchsbaum RN, Racker E. The effect of flavonoids on aerobic glycolysis and growth of tumor cells. Cancer Res 1975;35: 1865-72. Tang BY, Adams NR. The effect of equol on oestrogen receptors and on synthesis of DNA and protein in the immature rat uterus. J Endocrinol I980;85:29l-7. Thomas DB, Karagas MR. Cancer in first and second generation Arnericans. Cancer Res l987;47:5?'71-6. Tominaga S, Kuroishi T. Epiderniology of breast cancer in Japan. Cancer Letters 1995;90:75-9. Tominaga S. Cancer incidence in Japanese in Japan, Hawaii, and Western United States. Natl Cancer Inst Monogr 1985;69:83-92. Tonioli P, Riboli E, Shore RE, Pasternack 8s. Consumption of meat, animal products, protein and fat and risk of breast cancer: a prospective cohort study in New York. Epiderniol 1994;5:387-8. Tormey D, Lippman M, Cassidy J. Evaluation of tamoxifen doses with and without fluoxymesterone in advanced breast cancer. Ann lntern Med 1983398: 139-43. Tsai S-Y, Tsai M-J, O'Malley BW. Cooperative binding of steroid hormone receptors contributes to transcriptional synergism at target enhancer elements. CeIl 1989;57:443-8. Umesono K, Evans RM. Determinants of target gene specificity for steroidlthyroid hormone receptors. Cell l989;sï: 1139-46. Uppsala-Orebro. Breast Cancer Study Group. Sector resection with or without postoperative radiotherapy for stage I breast cancer: a randornized trial. J Natl Cancer lnst 1990;82:277-82. Verdeal K, Brown RR, Richardson T. Ryan DS. Affinity of phytoestrogens for estradiol-binding proteins and effect of coumestrol on growth of 7,12- dimethylbenz(a)anthracene-induced rat mammary turnors. JNCI 1980;60:285- 90. Verhoeven DT, Assen N, Goldbohm RA, Dorant E, Van't Veer P, Surmans F, Herrnus RJ, Van den Brandt PA. Vitamins C and E, retinol, betal-carotene and dietary fiber in relation to breast cancer risk: a prospective cohort study. Br J Cancer 1997;75: 149-55. Wahli W, Mertinez E. Superfamily of steroid nuclear receptors: positive and negative regulators of gene expression. FASEB J 1991;5:2243-9. Wall ME, Wani MC, Fullas F, Oswald JB, Brown DM, Santisuk T, Reutrakul V, McPhail AT, Farnsworth NR, Pezzuto JM, et al. Plant antitumor agents. 31. The calycopterones, a new class of biflavonoids with novel cytotoxicity in a diverse panel of human turnor cell lines. J Med Chem 1994;37:1465-70. Walsh PC. Physiologic basis for hormonal therapy in carcinoma of the prostate. Urol Clin North America 3 975;2: 125-40. Wang, MC, Valenzuela, LA, Murphy, GP, Chu, TM. Purification of a human prostate specific antigen. lnvest Urol l979;17:159-63. Wang ZY, Bickers DR, Mukhtar H. Interaction of epicatechins derived from green tea with rat hepatic cytochrome P-450. Drug Metab Dispos 1988;16:93- 103. Wang ZY, Cheng SJ, Zhou ZC, Athar M, Khan WA, Bickers DR, Mukhtar H. Antimutagenicity of green tea polyphenols. Mutat Res l989;223:273-85. Wang ZY, Agarwal R, Khan WA, Mukhtar H. Protection against benzo(a)pyrene- and N-nitrosodiethylamine-induced lung and forestornach tumorigenesis in AIJ mice by water extracts of green tea and licorice. Carcinogenesis l992;13: 1491- 4. Weiss BD. RU 486. The progesterone antagonist. Archiv Fam Med 1993;2:63-9 Welshons WV,Rottinghaus E, Nonneman DJ, Dolan-Timpe M, Ross PF. A sensitive bioassay for detection of dietary estrogens in animal feeds. J Vet Diagn lnvest 1990;2:268-73. Williams MR, Todd JH, Ellis 10, Dowle CS, Haybittle JL, Elston CW, Nicholson RI, Griffiths K, Blamey RW. Oestrogen receptors in primary and advanced breast cancer: An eight year review of 704 cases. Br J Cancer 1987;55:67-73. Xu Y, Han C. The effect of Chinese tea on the occurrence of esophageal tumors induced by N-nitrosomethylbenzylamine formed in vivo. Biomed Environ Sci 1WO;3:406-I 2. Xu Y, Ho C-T, Amin SG, Han C, Chung F-L. Inhibition of tobacco-specific nitrosamine-induced lung tumorigenesis in AIJ mice by green tea and its major polyphenol as antioxidants. Cancer Res 1992;52:3875-9. Yang CS, Wang 2-Y, Hong J-Y. Inhibition of tumorigenesis by chernicals frorn garlic and tea. In: Jacobs MM. Diet and Cancer Markers, Prevention, and Treatment. New York: Plenum Press 1994:113-22. Yoshida M, Sakai T, Hosokawa N, Marui N, Matsumoto K, Fujioka A, Nishino H, Aoike A. The effect of quercetin of cell cycle progression and growth of human gastric cancer cells. FEBS Lett I990;26O: 10-3. You WC, Blot WJ, Chang YS, Ershow A, Yang ZT, An Q, Henderson BE, Fraumeni JF Jr, Wang TG. Allium vegetables and reduced risk of stomach cancer. J Natl Cancer lnst 1989;8I:l62-4. Yu, H, Diamandis, EP, Zarghami, N, Grass, L. Induction of Prostate-specific Antigen Production by Steroids and Tamoxifen in Breast Cancer Cell Lines. Breast Cancer Research and Treatment l994;32:291-300. Yu, H, Giai, M, Diamandis, EP, Katsaros, D, Sutherland, DJA, Levesque, M, Roagna, R, Ponzone, R, Sismondi, P. Prostate-specific Antigen is a New Favorable Prognostic lndicator for Women with Breast Cancer. Cancer Research 1995a;55:2104-10. Yu H, Diamandis EP, Monne Ml Croce CM. Oral contraceptive-induced expression of prostate specific antigen in the female breast. J Biol Chemistry 1995b;270:6615-8. Yu H, Diamandis EP. Prostate specific antigen in milk of lactating women. Clin Chem 1995c;41:54-60. Yu H, Diamandis EP. Prostate specific antigen imrnunoreactivity in arnniotic fluid. Clin chem l995d;4I:204-lO. Yuan JM, Wang QS, Ross RK, Henderson BE, Yu MC. Diet and breast cancer in Shanghai and Tianjin, China. Br J Cancer l995;7l:l353-8. Zava DT, Duwe G. Estrogenic and antiproliferative properties of genistein and other flavonoids in human breast cancer cells in vitro. Nutr Cancer 1997;27:31- 40. APPLIED- IMAGE, tnc -= 1653 East Main Street ----. , Rochester, NY 14609 USA ---- Phone: 7161482-0300 ------Fax: 71 61288-5989 Q 1993. Applied Image. Inc., All Righls Resewed